How Charge, Size and Protein Corona Modulate the Specific Activity of Nanostructured Lipid Carriers (NLC) against Helicobacter pylori

The major risk factor associated with the development of gastric cancer is chronic infection with Helicobacter pylori. The available treatments, based on a cocktail of antibiotics, fail in up to 40% of patients and disrupt their gut microbiota. The potential of blank nanostructured lipid carriers (NLC) for H. pylori eradication was previously demonstrated by us. However, the effect of NLC charge, size and protein corona on H. pylori-specific bactericidal activity herein studied was unknown at that time. All developed NLC formulations proved bactericidal against H. pylori. Although cationic NLC had 10-fold higher bactericidal activity than anionic NLC, they lacked specificity, since Lactobacillus acidophilus was also affected. Anionic NLC achieved complete clearance in both H. pylori morphologies (rod- and coccoid-shape) by inducing alterations in bacteria membranes and the cytoplasm, as visualized by transmission electron microscopy (TEM). The presence of an NLC protein corona, composed of 93% albumin, was confirmed by mass spectrometry. This protein corona delayed the bactericidal activity of anionic NLC against H. pylori and hindered NLC activity against Escherichia coli. Overall, these results sustain the use of NLC as a promising antibiotic-free strategy targeting H. pylori.


Introduction
Since the rediscovery of Helicobacter pylori in 1982 by Marshall and Warren, it is known that this bacterium colonizes the human stomach and is responsible for several gastric disorders [1][2][3]. It is now estimated that H. pylori colonizes around half of the world population and is responsible for 90% of the gastric cancer burden [4,5], its eradication is recommended for all infected patients, even if asymptomatic [6]. Although 40 years have passed, the therapeutic regimen recommended for H. pylori eradication still relies on a combination of antibiotics [6,7]. However, antibiotic treatments are progressively failing (20-40%), mainly due to the increase in antibiotic resistance, but also fueled by low patient compliance to the complex treatment schemes with adverse side effects [6,8,9]. Antibiotic therapy also affects gut microbiota, causing dysbiosis, which increases susceptibility to infections by opportunistic bacteria (such as Clostridium difficile) and the development of inflammatory and autoimmune disorders [6,7,10].
It was recently reported that H. pylori can form biofilms in the stomach, where bacteria are protected by a complex biofilm matrix [11,12]. Moreover, in adverse conditions and due to its morphological plasticity, H. pylori can enter a low metabolically active state ("viable but non-culturable" (VBNC)), by changing its morphology from rod-to 2. Materials and Methods 2.1. Nanostructured Lipid Carriers (NLC) 2.1.1. NLC Production and Optimization NLC were produced by hot homogenization and ultrasonication (adapted from Seabra et al.) [19]. NLC were composed of a solid lipid (Precirol ® ATO5; Gattefossé, Saint-Priest, France), a liquid lipid (Miglyol ® 812; Acofarma, Madrid, Spain) and one of the following surfactants: (i) Tween ® 60 (NLC60); (ii) Tween ® 80 (NLC80); and (iii) different ratios of the surfactants Tween ® 60 and Cetyltrimethylammonium bromide-CTAB (NLC CTAB) in concentrations detailed in Table 1. Tween ® 60 and Tween ® 80 were obtained from Merck, Germany and CTAB from Sigma-Aldrich, St. Louis, MO, USA. All the components were weighted and melted together at 65 • C. Then, Type I water (ultrapure water with resistivity > 18 MΩ-cm and a conductivity < 0.056 µS/cm, Milli-Q ® , Millipore, Burlington, MA, USA), at the same temperature, was added to the blended components. Afterward, the mixture was sonicated (Vibra-Cell model VCX 130, Sonics and Materials Inc., Newtown, CT, USA) with a ∅ 6 mm tip. To obtain NLC with different sizes, the sonication time was varied from 5 min up to 20 min and the amplitude from 40% up to 90% (the tested conditions are described in Table S1).

NLC Characterization
NLC hydrodynamic diameter and surface charge (ζ-potential) were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively, using a Malvern Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK). For sample measurement, NLC were diluted (1:50) in Type I water (Milli-Q ® ) and placed on a disposable capillary cell (DTS1070, Malvern, UK). Measurements were done at 37 • C with a backscattering angle of 173 • . Values were obtained by calculating the average of three runs (each with twelve cycles). NLC concentration was assessed by nanoparticle tracking analysis (NTA) using a NanoSight ® NS300 (Malvern Panalytical, Malvern, UK). The samples were diluted (1:200,000) in Type I water (Milli-Q ® ) and measurements were performed in triplicate. Additionally, NLC morphology and size were evaluated by transmission electron microscopy (TEM). For this, NLC were diluted (1:200) and 10 µL of each sample were mounted on Formvar/carbon film-coated mesh nickel grids (Electron Microscopy Sciences, Hatfield, PA, USA). After 2 min, the excess liquid was removed with filter paper. For negative staining, 2 µL of 1% uranyl acetate was added to the grids. After a short incubation (10 s), excess liquid was removed with filter paper. Visualization was carried out on a JEOL 100CXII (Tokyo, Japan) at 60 kV.

NLC Activity against H. pylori 2.2.1. Bacterial Growth Conditions
NLC were tested against the human-isolated H. pylori strain J99 (provided by the Department of Medical Biochemistry and Biophysics, Umeå University, Sweden) according to Seabra et al. [20]. Briefly, the bacterium was cultured in spots in specific H. pylori medium plates composed of blood agar base 2 (Oxoid, France) supplemented with 10% v/v defibrinated horse blood (Probiológica, Lisbon, Portugal) and 0.2% v/v of an antibiotic cocktail composed by Polymyxin B, Vancomycin, Amphotericin B and Trimethroprim (all from Sigma-Aldrich, St. Louis, MO, USA) [19]. The bacterium was grown in a microaerophilic environment (GenBox system, BioMérieux, Marcy-l'Étoile, France) for 48 h at 37 • C. Then, colonies were streaked in fresh medium plates and incubated for another 48 h under the same conditions as above. Finally, the bacteria were harvested from the solid medium, centrifuged 774× g, RT, 10 min (Eppendorf™ 5810R, Eppendorf, Germany) and resuspended in Müeller-Hinton broth medium (MHB, Merck, Germany) supplemented with 10% of heat-inactivated (30 min, 65 • C) Fetal Bovine Serum (FBS, Gibco, Waltham, MA, USA), prior to transfer to T-flasks with the same medium. Optical density (OD) was adjusted to 0.1 (λ = 600 nm; UV/VIS spectrophotometer, Lambda 45, Perkin Elmer, Waltham, MA, USA). To obtain rod-shaped H. pylori, incubation proceeded overnight (16-18 h) under microaerophilic conditions, at 37 • C and 150 revolutions per minute (rpm). For inducing H. pylori morphological conversion to coccoid-shaped, incubation in liquid media was done for 72 h [25].

NLC Antibacterial Performance
After incubation in liquid medium (as described in Section 2.2.1) for 16-18 h, the bacterial culture was adjusted to approximately 1 × 10 7 (OD ≈ 0.03) colony forming units (CFU)/mL in MHB + 10% FBS medium [26]. Different NLC concentrations (10 10 , 10 11 , 10 12 , 10 13 particles/mL) were incubated with the bacteria over 24 h, at 37 • C in microaerophilic conditions. After 24 h, 100 µL of each sample was collected, serially diluted 10-fold in To compare the NLC60 effect against different H. pylori morphologies, two cultures were prepared: one was incubated in liquid medium for 16-18 h and another for 72 h (as described in Section 2.2.1). Afterward, cultures were centrifuged (774× g, RT, 10 min) and the pellets were re-suspended in PBS, being then adjusted to approximately 1 × 10 7 CFU/mL (OD ≈ 0.03). Different NLC concentrations (10 12 and 10 13 particles/mL) were incubated with the bacteria for 6 h at 37 • C in microaerophilic conditions. Since this assay was performed in PBS, the time of incubation was shorter (6 h) to avoid bacterial death due to prolonged exposure to nutrient starvation (incubation in PBS). After 6 h, samples were plated as described above and incubated at 37 • C for 4-5 days in microaerophilic conditions. The number of viable bacteria/mL was calculated by CFU counting (CFU/mL = (nº colonies × dilution factor)/volume of culture plated). The NLC formulation was considered bactericidal if a reduction of at least 99.9% (≥3 log 10 ) of the total count of CFU/mL was observed when compared with the control (bacteria without NLC). Each condition was tested in triplicate.

Transmission Electron Microscopy (TEM)
H. pylori cultures were prepared as described in Section 2.2.1. Bacterial cultures of 16-18 h (rod) and 72 h (coccoid) incubation were centrifuged (774× g, RT, 10 min) and pellets were re-suspended in PBS and adjusted to approximately 1 × 10 7 CFU/mL (OD ≈ 0.03). Two NLC60 concentrations (10 12 and 10 13 particles/mL) were incubated with the bacteria for 6 h at 37 • C in microaerophilic conditions. To ensure enough bacterial content for posterior sample preparation, 10 replicates of each condition were prepared. Then, all the replicates per each condition were combined and centrifuged (3000× g, RT, 10 min). The bacterial pellets were fixed with a solution of 2% glutaraldehyde, 2.5% formaldehyde (both from Electron Microscopy Sciences, Hatfield, PA, USA) and 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h at room temperature. Then, samples were post-fixed with 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) diluted in 0.1 M sodium cacodylate buffer and re-suspended in Histogel TM (HG-4000-012, Thermo Fisher Scientific, Bremen, Germany). Afterward, they were stained with aqueous 1% uranyl acetate solution overnight, dehydrated with ethanol and propylene oxide and then embedded in Embed-812 resin (Electron Microscopy Sciences, Hatfield, PA, USA). Each resin was cut in ultra-thin sections of 50 nm thickness on an RMC Ultramicrotome (PowerTome, Moses Lake, WA, USA) using Diatome diamond knives (Delaware Diamond Knives, Wilmington, DE, USA) and mounted on mesh nickel grids (Electron Microscopy Sciences, Hatfield, PA, USA). Next, the sections were stained with uranyl acetate substitute and lead citrate (both from Electron Microscopy Sciences, Hatfield, PA, USA) for 5 min each. Ultra-thin sections were analyzed in a JEOL JEM 1400 transmission electron microscope (JEOL, Tokyo, Japan) and images were digitally recorded using a CCD digital camera Orius 1100W (Gatan, Tokyo, Japan).

NLC Antibacterial Performance
The bacterial cultures (prepared as described in Section 2.3.1) were centrifuged at 774× g, RT for 10 min. After, the supernatants were discarded, and the bacterial pellets were re-suspended in either MHB or MHB + 10% FBS medium. Bacterial concentration was adjusted to approximately 1 × 10 5 CFU/mL [27]. Different NLC concentrations (10 10 , 10 11 , 10 12 , 10 13 particles/mL) were incubated with the bacteria for 24 h at 37 • C (in microaerophilic conditions for L. acidophilus-01). Then, 100 µL of each sample was collected, and serially diluted 10-fold in PBS, and 10 µL of each dilution was plated in the appropriate medium plates. The plates were incubated for 48 h at 37 • C for L. acidophilus-01 and overnight (16-18 h) for E. coli ATCC ® 25922™. The number of bacteria/mL was calculated by CFU counting. Each condition was done in triplicate. Sample preparation for LC-MS/MS was done by solubilizing the NLC in 100 mM Tris(hydroxymethyl)aminomethane (Tris) pH 8.5, 1% sodium deoxycholate, 10 mM tris(2carboxyethyl)phosphine (TCEP), 40 mM chloroacetamide and 1 × complete TM protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) for 10 min at 95 • C and 1000 rpm (Thermomixer, Eppendorf, Germany). The samples were handled for proteomic analysis according to the solid-phase enhanced sample-preparation (SP3) protocol from Hughes et al. [28]. Enzymatic digestion was done by incubating the samples with 2 µg of Trypsin/LysC overnight at 37 • C and 1000 rpm. For protein analysis, 500 nanograms of each sample was introduced in a nanoLC-MS/MS system composed of an Ultimate 3000 liquid chromatography system attached to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany). Each sample was loaded onto a trapping cartridge (Acclaim PepMap C18 100Å, 5 mm × 300 µm i.d., 160,454, Thermo Fisher Scientific, Waltham, MA, USA) in a mobile phase of 2% acetonitrile and 0.1% formic acid at 10 µL/min and processed as described in Melo et al. [25]. Protein quantification was conducted by label-free quantification (LFQ). Raw data were processed using Proteome Discoverer 2.5 software (Thermo Scientific, Waltham, MA, USA) and searched against the Uniprot Bos taurus database. Proteins with two or more identified unique peptides were considered for protein analysis.

Effect of Protein Corona on NLC Bactericidal Activity
To test the effect of the protein corona on NLC bactericidal activity, NLC60 were prepared and incubated in different media as described in Section 2.4.1. H. pylori (grown as described in Section 2.2.1) concentration was adjusted to approximately 1 × 10 7 CFU/mL in PBS. Different NLC60 concentrations (10 11 , 10 12 , 10 13 particles/mL) were incubated with the bacteria for 6 h at 37 • C, in microaerophilic conditions. Afterward, samples were collected and plated as described in Section 2.2.2. The plates were incubated at 37 • C for 4-5 days in microaerophilic conditions and the number of viable bacteria/mL was calculated by CFU counting.

Statistical Analysis
Statistical analysis was performed using Graph Pad Prism 8.0 (Graph-Pad Software, San Diego, CA, USA). Data were expressed as mean ± standard deviation. Statistical significance was assessed using Two-way ANOVA followed by Tukey's multiple comparisons test. Statistically significant differences were considered for p < 0.05.

NLC with Different Surface Charge
Blank NLC previously developed by us had an average diameter of 211 ± 8 nm and negative surface charge (ζ-potential close to −28 mV) [19,20]. These NLC were produced with Precirol ATO5 ® , Miglyol-812 ® and Tween ® 60 using an amplitude of sonication of 60% for 5 min. For positively charged NLC, the same lipids and sonication parameters were used but a cationic surfactant (CTAB) was added. In the optimization process, CTAB was mixed with non-ionic Tween ® 60 in different ratios to access the effect on nanoparticle charge (ζ-potential) and hydrodynamic diameter ( Figure 1).

Statistical Analysis
Statistical analysis was performed using Graph Pad Prism 8.0 (Graph-Pad Software, San Diego, CA, USA). Data were expressed as mean ± standard deviation. Statistical significance was assessed using Two-way ANOVA followed by Tukey's multiple comparisons test. Statistically significant differences were considered for p < 0.05.

NLC with Different Surface Charge
Blank NLC previously developed by us had an average diameter of 211 ± 8 nm and negative surface charge (ζ-potential close to −28 mV) [19,20]. These NLC were produced with Precirol ATO5 ® , Miglyol-812 ® and Tween ® 60 using an amplitude of sonication of 60% for 5 min. For positively charged NLC, the same lipids and sonication parameters were used but a cationic surfactant (CTAB) was added. In the optimization process, CTAB was mixed with non-ionic Tween ® 60 in different ratios to access the effect on nanoparticle charge (ζ-potential) and hydrodynamic diameter ( Figure 1).  NLC composed of 100% CTAB had a mean size of 148 ± 7 nm and a surface charge of +62 ± 2 mV ( Figure 1). Reducing the CTAB percentage (increasing Tween ® 60), increased NLC size (from 148 ± 7 to 188 ± 4 nm), while the surface charge diminished (from +62 ± 2 to +30 ± 0.3 mV). NLC with 12.5% CTAB (NLC CTAB) were selected for further assays since this combination yielded NLC with a similar size and absolute ζ-potential (around |30|mV) to the blank NLC.
The effect of the amplitude of sonication on NLC size (5 min sonication time) varied according to the NLC formulation ( Figure 2a). NLC60 had the highest size variation with increasing amplitude of sonication. NLC60 size increased from 230 ± 3 to 300 ± 6 nm when the amplitude of sonication rose from 40 to 90%, respectively. NLC80 had more discrete changes with the increase in amplitude, with size significantly increasing only at 90% of amplitude (varied ≈ 30 nm). NLC CTAB size remained constant independently of the amplitudes tested. Since 90% of amplitude led to higher changes in NLC size, the influence of the sonication time on NLC size was studied at a fixed amplitude of 90% ( Figure 2b).
NLC80 and NLC CTAB were not considerably affected by the sonication time: for NLC80 the size remained constant (≈230 nm), while for NLC CTAB a slight increase was observed (180 ± 8 to 205 ± 9 nm) up to 10 min of sonication, plateauing after this ( Figure 2b). NLC60 had a higher size variation with different sonication times, increasing up to 486 ± 15 nm at the longest time of sonication (20 min) (Figure 2b).
Overall, only the NLC60 formulation could be modulated by changing the amplitude and time of sonication to create nanoparticles with different sizes. Therefore, NLC60 with three different sizes were obtained: smaller (NLC60 S ), medium (NLC60 M ; control) and larger (NLC60 L ). The conditions selected to prepare these NLC60 are described in Table 2.

NLC for In Vitro Assays
The optimized sonication parameters and the features of all NLC formulations selected for in vitro assays are described in Table 2.
NLC sizes were evaluated by TEM and by DLS. As expected, NLC analyzed by TEM had a smaller size than NLC analyzed by DLS (hydrodynamic size). This difference is associated with the liquid layer that is formed around the nanoparticles when measured in DLS, which is not present when NLC were observed by TEM (dry NLC). However, hydrodynamic sizes are representative in the context of the in vitro assays since the effect of NLC was evaluated in media.
All the NLC had similar, low polydispersity indexes (PdI), with values close to 0.2 ( Table 2), which indicates a monodispersed distribution. Regarding the surface charge, NLC60 (independently of size) and NLC80 were anionic, with similar negative ζ-potential (−26 to −30 mV), while NLC CTAB were cationic with a positive ζ-potential close to +38 mV.
TEM assays were also used to evaluate NLC morphology ( Figure 3). The images revealed that the produced NLC had a smooth spherical or spheroidal morphology. This spheroidal morphology could be related to the drying process and is more pronounced in larger NLC (Figure 3b-d).

NLC Antibacterial Performance
The antibacterial performance of the NLC formulations (Table 2) was tested against the H. pylori J99 strain. Also, their safety towards gut microbiota was assessed by screening against E. coli ATCC ® 25922 TM and L. acidophilus-01, bacteria representative of the normal gut microbiota.
As shown in Figure 4a, all NLC formulations were bactericidal against H. pylori. NLC CTAB (positively charged) had the highest bactericidal effect, with complete eradication at 10 11 particles/mL, whereas NLC60 and NLC80 only achieved the same effect at 10 12 particles/mL. Moreover, no statistically significant differences were found between NLC60 and NLC80 formulations.
For E. coli ATCC ® 25922™ (Figure 4b) and L. acidophilus-01 (Figure 4c), no bactericidal activity was observed for either NLC60 or NLC80 at the range of concentrations tested. However, NLC CTAB were bactericidal at 10 11 particles/mL against L. acidophilus-01 (Figure 4c). Thus, despite presenting the highest bactericidal effect against H. pylori J99, the cationic NLC CTAB emerged as non-gut microbiota friendly, due to its bactericidal effect on L. acidophilus-01.

NLC Antibacterial Performance
The antibacterial performance of the NLC formulations (Table 2) was tested against the H. pylori J99 strain. Also, their safety towards gut microbiota was assessed by screening against E. coli ATCC ® 25922 TM and L. acidophilus-01, bacteria representative of the normal gut microbiota.

Effect of NLC Charge (Surfactant Composition)
NLC with different ζ-potential and surfactant compositions but similar sizes (NLC60M, NLC80 and NLC CTAB) were tested against H. pylori J99, E. coli ATCC ® 25922™ and L. acidophilus-01 (Figure 4).  As shown in Figure 4a, all NLC formulations were bactericidal against H. pylori. NLC CTAB (positively charged) had the highest bactericidal effect, with complete eradication at 10 11 particles/mL, whereas NLC60 and NLC80 only achieved the same effect at 10 12
Results demonstrated that size influenced NLC60 activity against H. pylori ( Figure 5). NLC60 L (443 ± 11 nm) achieved a bactericidal effect at 10 11 particles/mL, while NLC60 S and NLC60 M required 10 12 particles/mL. Still, at this concentration, all formulations reached full H. pylori clearance.

Effect on Different H. pylori Morphologies
To assess the NLC bactericidal effect on different H. pylori morphologies, NLC60 M were tested against bacteria of rod and coccoid morphology. To achieve coccoid morphology, before the bactericidal assay, H. pylori was grown for 72 h instead of the usual 16-18 h to promote bacterial stress by nutrient depletion, consequently leading to the change in morphology. The morphological change was confirmed by optical microscopy.
NLC CTAB emerged as non-gut microbiota friendly, due to its bactericidal effect on L. acidophilus-01.

Effect on Different H. pylori Morphologies
To assess the NLC bactericidal effect on different H. pylori morphologies, NLC60M were tested against bacteria of rod and coccoid morphology. To achieve coccoid morphology, before the bactericidal assay, H. pylori was grown for 72 h instead of the usual 16-18 h to promote bacterial stress by nutrient depletion, consequently leading to the change in morphology. The morphological change was confirmed by optical microscopy.
Since complete H. pylori eradication was observed after 24 h incubation with NLC60M at a concentration of 10 12 particles/mL and above in the previous assays, the concentrations 10 12 and 10 13 particles/mL were chosen to test the effect of NLC60M on both H. pylori morphologies after 6 h in PBS. The results are shown in Figure 6. Since complete H. pylori eradication was observed after 24 h incubation with NLC60 M at a concentration of 10 12 particles/mL and above in the previous assays, the concentrations 10 12 and 10 13 particles/mL were chosen to test the effect of NLC60 M on both H. pylori morphologies after 6 h in PBS. The results are shown in Figure 6. H. pylori was grown in rod-and coccoid-shape and both initial cultures were adjusted to start at 1 × 10 7 CFU/mL. Since the coccoid-shaped H. pylori have a slower metabolism, the bacterium grew less than the rod-shaped H. pylori [16]. As expected, a difference in growth was observed in the controls for both morphologies, with rod-shaped bacteria growing about 1.5 logs CFU/mL more than the coccoid-shaped H. pylori. Nonetheless, it was observed that NLC60M were effective at the same concentration (10 12 particle/mL) for both morphologies (Figure 6).
The effect of NLC60M on the H. pylori membrane in both morphologies was also studied by transmission electron microscopy (TEM). Figure 7 shows the TEM images of rodand coccoid-shaped H. pylori with (H. pylori + NLC60M) and without (H. pylori) exposure to NLC60M for 6 h in PBS. H. pylori was grown in rod-and coccoid-shape and both initial cultures were adjusted to start at 1 × 10 7 CFU/mL. Since the coccoid-shaped H. pylori have a slower metabolism, the bacterium grew less than the rod-shaped H. pylori [16]. As expected, a difference in growth was observed in the controls for both morphologies, with rod-shaped bacteria growing about 1.5 logs CFU/mL more than the coccoid-shaped H. pylori. Nonetheless, it was observed that NLC60 M were effective at the same concentration (10 12 particle/mL) for both morphologies (Figure 6). The effect of NLC60 M on the H. pylori membrane in both morphologies was also studied by transmission electron microscopy (TEM). Figure 7 shows the TEM images of rodand coccoid-shaped H. pylori with (H. pylori + NLC60 M ) and without (H. pylori) exposure to NLC60 M for 6 h in PBS. shaped H. pylori exposed to NLC60M (10 12 particles/mL), (g,i,k) coccoid-shaped control and (h,j,l) coccoid-shaped exposed to NLC60M (10 12 particles/mL). Scale bars: (a,b, [29]. In the antibacterial assays, the medium is supplemented with FBS as a cholesterol source. However, E. coli and L. acidophilus do not require cholesterol. Therefore, to assess if FBS influences  , (b,d,f) rod-shaped H. pylori exposed to NLC60 M (10 12 particles/mL), (g,i,k) coccoid-shaped control and (h,j,l) coccoidshaped exposed to NLC60 M (10 12 particles/mL). Scale bars: (a,b,g,h)  TEM images showed that the rod-shaped control bacteria (H. pylori) (Figure 7a,c,e) had a healthy morphology, presenting intact cell membranes. Moreover, images of the coccoid-shaped H. pylori (Figure 7g,i,k), although in coccoid-shape, also show intact cell membranes. When exposed to NLC60 M (Figure 7b,d,f,h,j,l) changes in the cytoplasm (I) and cell membrane irregularities, such as the formation of vesicles (II) and membrane disruption (III), were observed in both morphologies. These results clearly show the effectiveness of NLC60 M against both rod and coccoid-shaped bacteria, explaining the bactericidal effect described in Figure 6. 3.2.4. Effect of FBS on NLC Antibacterial Performance against E. coli and L. acidophilus H. pylori is a fastidious bacterium that is auxotrophic for cholesterol [29]. In the antibacterial assays, the medium is supplemented with FBS as a cholesterol source. However, E. coli and L. acidophilus do not require cholesterol. Therefore, to assess if FBS influences NLC performance, NLC60 M were incubated with E. coli and L. acidophilus-01 in MHB medium with and without FBS (Figure 8).  NLC60M had a bactericidal effect against E. coli when FBS was removed from the medium and complete eradication was reached at 10 12 particles/mL (Figure 8a). The differences in NLC60 activity observed in E. coli suggest that the proteins of the FBS used for the in vitro bacterial assays influence NLC activity.
For L. acidophilus-01, a difference in growth was observed when comparing the controls of the different media. With FBS in the medium, the bacterium grows 2 logs CFU/mL more than without FBS. However, when exposed to the NLC, no differences were observed either with or without FBS, since NLC60M was not bactericidal (Figure 8b). NLC60 M had a bactericidal effect against E. coli when FBS was removed from the medium and complete eradication was reached at 10 12 particles/mL (Figure 8a). The differences in NLC60 activity observed in E. coli suggest that the proteins of the FBS used for the in vitro bacterial assays influence NLC activity.
For L. acidophilus-01, a difference in growth was observed when comparing the controls of the different media. With FBS in the medium, the bacterium grows 2 logs CFU/mL more than without FBS. However, when exposed to the NLC, no differences were observed either with or without FBS, since NLC60 M was not bactericidal (Figure 8b).

NLC Protein Corona
The presence of FBS in the in vitro assay medium affected the NLC activity towards E. coli, demonstrating that media composition impacts NLC activity. This could be due to the interaction of proteins present in the medium with the nanoparticles, leading to the formation of a protein corona.

Characterization of NLC Protein Corona
Protein corona in NLC60 M was evaluated after the incubation of nanoparticles in PBS, MHB and MHB supplemented with 10% FBS. After being washed to remove any potential medium debris and to assure that only the adsorbed proteins would be present in the NLC, the nanoparticles were analyzed by LC-MS/MS (Figure 9).

NLC Protein Corona
The presence of FBS in the in vitro assay medium affected the NLC activity towards E. coli, demonstrating that media composition impacts NLC activity. This could be due to the interaction of proteins present in the medium with the nanoparticles, leading to the formation of a protein corona.

Characterization of NLC Protein Corona
Protein corona in NLC60M was evaluated after the incubation of nanoparticles in PBS, MHB and MHB supplemented with 10% FBS. After being washed to remove any potential medium debris and to assure that only the adsorbed proteins would be present in the NLC, the nanoparticles were analyzed by LC-MS/MS (Figure 9). According to the mass spectrometry results (Figure 9), NLC incubated in PBS had no adsorbed proteins on their surface, as was expected. For NLC incubated in MHB and MHB + 10%FBS, 4 and 70 different proteins were found, respectively, and 2 of them (Collagen alpha 1 and 2) were common to both samples. In MHB + 10%FBS, the predominant protein in the sample was serum albumin with an abundance of 93%. These results demonstrate that NLC60 adsorb proteins on their surface creating a protein corona, the composition of which depends on the media.

Effect of NLC Protein Corona on Antibacterial Performance against H. pylori
The presence of a protein corona composed of proteins present in the FBS was validated. So, the possibility that this protein corona impacts the NLC antibacterial activity against H. pylori was considered. To study the influence of the different media in NLC60M activity towards H. pylori, the nanoparticles were pre-immersed in different media as described in Section 2.4.1. The results are shown in Figure 10. According to the mass spectrometry results (Figure 9), NLC incubated in PBS had no adsorbed proteins on their surface, as was expected. For NLC incubated in MHB and MHB + 10% FBS, 4 and 70 different proteins were found, respectively, and 2 of them (Collagen alpha 1 and 2) were common to both samples. In MHB + 10% FBS, the predominant protein in the sample was serum albumin with an abundance of 93%. These results demonstrate that NLC60 adsorb proteins on their surface creating a protein corona, the composition of which depends on the media.

Effect of NLC Protein Corona on Antibacterial Performance against H. pylori
The presence of a protein corona composed of proteins present in the FBS was validated. So, the possibility that this protein corona impacts the NLC antibacterial activity against H. pylori was considered. To study the influence of the different media in NLC60 M activity towards H. pylori, the nanoparticles were pre-immersed in different media as described in Section 2.4.1. The results are shown in Figure 10. After 6 h of incubation with NLC60M exposed to MHB + 10% FBS, no bacter effect was reached in any of the tested concentrations ( Figure 10). Non-supplem MHB showed bactericidal activity at 10 13 particles/mL, but complete clearance w obtained. In PBS, NLC60M achieved complete H. pylori clearance at 10 12 particles/mL. it was observed that the NLC protein corona masks the nanoparticles and delays th tericidal activity. Additionally, when comparing these results with the MS analysis (Fig  it is observed that an increase in protein adsorption to the NLC surface directly corr with an increasing delay in NLC activity.

Discussion
H. pylori is classified by the International Agency for Research on Cancer (IARC group 1 carcinogen [5]. Eleven infectious pathogens have this classification, and, a them, H. pylori ranks first as an infectious cause of cancer worldwide [5]. Due to th of H. pylori resistance to available antibiotics, it is urgent to find alternative soluti fight this bacterium. Previously, we described that blank NLC per se have bacter activity against H. pylori, even if organized in biofilms [19,20,24]. Here, the effect of c size and protein corona on NLC antimicrobial specificity toward H. pylori was expl Since the NLC previously described prepared with Tween ® 60 (NLC60) were a (−28 mV) [19,20], this surfactant was substituted by the cationic surfactant CTAB t duce cationic nanoparticles (NLC CTAB). NLC prepared with 100% CTAB generat noparticles with +62 mV of ζ-potential, but this value was not comparable with the tively charged NLC60 (ζ-potential −28 mV). Optimization using dif CTAB/Tween ® 60 ratios demonstrated a decrease in NLC ζ-potential from +62 mV mV when the CTAB percentage was changed from 100% to 12.5% (Figure 1). Formul with two or more surfactants usually have smaller sizes because of the surfactant c nation effect [30]. However, the NLC CTAB size increased (from ~150 to ~200 nm the decrease in CTAB % (from 100 to 12.5%), which could be explained by the high ence in the molecular weight (MW) of the two surfactants (MW CTAB = 364.45 g/m MW Tween ® 60 = 1311.7 g/mol).
Both NLC formulations, the anionic NLC60 and the cationic NLC CTAB were ricidal against H. pylori since a reduction of more than 3 logs CFUs after 24 h incub After 6 h of incubation with NLC60 M exposed to MHB + 10% FBS, no bactericidal effect was reached in any of the tested concentrations ( Figure 10). Non-supplemented MHB showed bactericidal activity at 10 13 particles/mL, but complete clearance was not obtained. In PBS, NLC60 M achieved complete H. pylori clearance at 10 12 particles/mL. Thus, it was observed that the NLC protein corona masks the nanoparticles and delays the bactericidal activity. Additionally, when comparing these results with the MS analysis (Figure 9), it is observed that an increase in protein adsorption to the NLC surface directly correlates with an increasing delay in NLC activity.

Discussion
H. pylori is classified by the International Agency for Research on Cancer (IARC) as a group 1 carcinogen [5]. Eleven infectious pathogens have this classification, and, among them, H. pylori ranks first as an infectious cause of cancer worldwide [5]. Due to the rise of H. pylori resistance to available antibiotics, it is urgent to find alternative solutions to fight this bacterium. Previously, we described that blank NLC per se have bactericidal activity against H. pylori, even if organized in biofilms [19,20,24]. Here, the effect of charge, size and protein corona on NLC antimicrobial specificity toward H. pylori was explored.
Since the NLC previously described prepared with Tween ® 60 (NLC60) were anionic (−28 mV) [19,20], this surfactant was substituted by the cationic surfactant CTAB to produce cationic nanoparticles (NLC CTAB). NLC prepared with 100% CTAB generated nanoparticles with +62 mV of ζ-potential, but this value was not comparable with the negatively charged NLC60 (ζ-potential −28 mV). Optimization using different CTAB/Tween ® 60 ratios demonstrated a decrease in NLC ζ-potential from +62 mV to +30 mV when the CTAB percentage was changed from 100% to 12.5% (Figure 1). Formulations with two or more surfactants usually have smaller sizes because of the surfactant combination effect [30]. However, the NLC CTAB size increased (from~150 to~200 nm) with the decrease in CTAB % (from 100 to 12.5%), which could be explained by the high difference in the molecular weight (MW) of the two surfactants (MW CTAB = 364.45 g/mol and MW Tween ® 60 = 1311.7 g/mol).
Both NLC formulations, the anionic NLC60 and the cationic NLC CTAB were bactericidal against H. pylori since a reduction of more than 3 logs CFUs after 24 h incubation was attained ( Figure 4a). As expected, the cationic NLC CTAB had a higher bactericidal effect, with total H. pylori clearance reached at a concentration 10 times lower (10 11 particles/mL) than the one used for NLC60 (10 12 particles/mL). However, despite this promising performance, NLC CTAB were also bactericidal against L. acidophilus (one of the bacteria tested as representative of gut microbiota) at a similar concentration (10 11 particles/mL) demonstrating that they are not selective to H. pylori (Figure 4c). This is in accordance with what is described in the literature, since positively charged nanoparticles usually have higher antibacterial activity due to their interaction with the negatively charged bacterial membrane, both in Gram-positive and Gram-negative bacteria [31]. However, NLC CTAB did not affect E. coli (Figure 4b). This might be due to the stronger outer membrane of E. coli that acts as a selective physical barrier, protecting the bacterium from external threats [32,33].
NLC were also produced using Tween ® 80 (NLC80) since, unlike Tween ® 60, soluble Tween ® 80 has a bactericidal effect against H. pylori [24,34,35]. Moreover, it was also reported that Tween ® 80 improved H. pylori eradication in infected patients when combined with the usually prescribed antibiotics [35]. The different antimicrobial effects observed between Tween ® 60 (polyethylene glycol (20) [36]. This antimicrobial effect might be associated with the unsaturated aliphatic chain of Tween ® 80. However, our results demonstrated no advantages in H. pylori bactericidal activity and selectivity for NLC80 compared to NLC60 (Figure 4a). This may be related to the orientation of the surfactant on the NLC surface, since the aliphatic chains are facing the lipid content, exposing its similar polyethylene glycol (20) chain to the aqueous phase.
NLC60 with different sizes (NLC60 S~1 50 nm, NLC60 M~2 60 nm and NLC60 L~4 50 nm) were successfully obtained by the alteration of sonication parameters (time and amplitude). The size of NLC60 increased with an increase in sonication amplitude and time ( Figure 2). This was not observed in NLC prepared with the other surfactants, where alterations in sonication parameters did not significantly affect the size of NLC80 and NLC CTAB (Figure 2). The size of NLC CTAB was only changed by altering the ratio of CTAB/Tween ® 60 ( Figure 1). Therefore, the effect of size on NLC selective bactericidal activity against H. pylori was evaluated using NLC60 (NLC60 S NLC60 M and NLC60 L ). All NLC60 sizes were bactericidal against H. pylori after 24 h incubation, with complete H. pylori clearance at 10 12 particles/mL ( Figure 5). At the same time, improved bactericidal performance was observed with NLC60 L , since the bactericidal effect was reached at a lower NLC concentration (10 11 particles/mL). This result contradicts the usual conception that smaller nanoparticles have a higher bactericidal effect [37][38][39][40]. However, in most cases, the nanoparticles are designed as drug delivery systems, where the bactericidal effect is intrinsically associated with the drug and not with the nanoparticle per se. Moreover, in the case of metallic nanoparticles, their small size allows them to cross the bacterial membrane causing changes in the bacterial membrane and metabolism, which ultimately promotes bacterial death [38][39][40]. Since these NLC affect H. pylori membranes by contact [19], it would be expected that larger NLC, by having a larger contact area with the bacterial membrane, were more efficient.
To have relevance as a new therapeutic strategy against H. pylori, ideally, these NLC need to be effective against both H. pylori morphologies, since H. pylori in a coccoid morphology are more resistant to antibiotics [14]. Until now, few antibiotic-free nanoparticles showed an effect against the coccoid morphology, and the ones that did had compounds incorporated into the nanoparticles, such as linolenic acid into liposomes [41]. NLC60 M were bactericidal for both rod-and coccoid-shaped bacteria, achieving complete H. pylori eradication at 10 12 particles/mL ( Figure 6). Transmission electron microscopy (TEM) images clearly showed the effect of NLC60 M on both morphologies of H. pylori, where NLC60 M induced the formation of protrusions, vesicles and, in some cases, membrane disruption (Figure 7f,j,l). The release of the cytoplasm was confirmed by observation of free space inside H. pylori cells exposed to NLC (Figure 7d,h). However, no significant increase in periplasmic space was observed when compared with the control samples (Figure 7e,f,k,l). These observations corroborate the hypothesis that blank NLC60 destabilize the H. pylori bacterial membrane, although why this occurs is still not fully understood [20,24]. H. pylori is adapted to cross the gastric mucus layer, which contains highly hydrophilic regions, to reach the epithelial cell layer. As such, it can be speculated that, since these NLC are coated with a very hydrated surfactant (due to the exposed polyethylene glycol), they can be perceived by H. pylori as the gastric mucus layer. The unexpected high H. pylori adhesion to a polyethylene glycol (PEG) surface was already demonstrated by us [42]. This was not anticipated since PEG surfaces are well known for their "non-fouling" properties, being able to avoid protein adsorption and bacteria adhesion [43].
Therefore, high protein adsorption to NLC60 was not expected due to their hydrated coating (Tween ® 60). However, since bactericidal assays were performed in media supplemented with 10% FBS, the adsorbed protein layer, normally designated by protein corona [44][45][46], was analyzed by mass spectrometry. Protein corona can influence the characteristics of the nanoparticles (e.g., size and charge) as well as their activity, degradation and recognition by the immune system in physiologic conditions and in in vitro settings [47,48]. Results demonstrated that when NLC60 M were incubated for 24 h with MHB supplemented with 10% FBS, 6.58 µg of protein (13.2 ng of protein/ng of NLC) were observed, corresponding to the presence of 70 different proteins (Figure 9). Among these proteins, serum albumin was the most prevalent, with an abundance of 93% (6.14 µg of the total 6.58 µg of detected protein). These results are corroborated by FBS composition, which has serum albumin as a major constituent (60 to 67% of total protein composition) [49,50], and by what is documented relatively to protein corona from lipid nanoparticles [51][52][53]. For NLC60 M preincubated in MHB, 1.34 µg of protein (2.7 ng of protein/ng NLC) was detected due to the presence of dour proteins, where 94% are collagen ( Figure 9). As expected, no protein content was detected when NLC60 M were incubated in PBS.
Although only H. pylori require FBS in the culture media, FBS was maintained in the selectivity assays, using E. coli and L. acidophilus, to keep the in vitro assays comparable. In the presence of FBS (NLC with protein corona) NLC60 M did not affect E. coli and L. acidophilus ( Figure 8). However, without FBS in the media, NLC60 M was bactericidal against E. coli, reaching complete clearance at 10 12 particles/mL (same concentration as for H. pylori) (Figure 8a). This was not observed for L. acidophilus. Thus, the protein corona masks NLC60 M activity against E. coli., being responsible for NLC60 M selectivity.
Studies in PBS using NLC60 M pre-coated with PBS, MHB and MHB + 10% FBS demonstrated that the protein corona (93% albumin) only delayed the NLC bactericidal effect against H. pylori ( Figure 10). Nevertheless, this NLC protein corona observed in the in vitro assays would not correspond to an in vivo protein corona, which is a more dynamic process [51][52][53].

Conclusions
NLC with different sizes and charges were successfully developed. All the NLC formulations were effective against H. pylori, with the cationic NLC CTAB being 10 times more efficient than the anionic NLC60 and NLC80. However, NLC CTAB was not selective to H. pylori, having a similar effect against L. acidophilus. A protein corona composed of 93% albumin only delayed NLC60 bactericidal activity against H. pylori but hindered their bactericidal activity against E. coli. These NLC achieved complete H. pylori clearance in both morphologies (rod-and coccoid-shape). These new insights establish NLC60 M as bactericidal against the more resistant morphology of H. pylori and reveal the possible role of the protein corona in their in vitro selectivity towards H. pylori. Overall, these results sustain NLC as a promising antibiotic-free treatment against H. pylori.