1. bookVolumen 73 (2022): Heft 4 (December 2022)
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26 Mar 2007
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Ocjena apsorpcije, djelotvornosti protiv bakterije,i citotoksičnosti krutih lipidnih nanočestica s moksifloksacinom

Online veröffentlicht: 07 Jan 2023
Volumen & Heft: Volumen 73 (2022) - Heft 4 (December 2022)
Seitenbereich: 260 - 269
Eingereicht: 01 Jul 2022
Akzeptiert: 01 Nov 2022
Zeitschriftendaten
License
Format
Zeitschrift
eISSN
1848-6312
Erstveröffentlichung
26 Mar 2007
Erscheinungsweise
4 Hefte pro Jahr
Sprachen
Englisch

Resistance to antibiotics has become a serious healthcare issue as it erodes the efficiency of many currently used antibiotics (14). According to the World Health Organization (WHO), antimicrobial resistance is one of the most important dangers for people’s health in the world and may become the cause of 10 million deaths every year by 2050 (5). New antimicrobial strategies are therefore needed to address this issue. In the fight against antibiotic resistance, new studies are focused on discovering and developing alternative agents like synthetic active ingredients (6), plant metabolites (7), antimicrobial peptides (8), virulence inhibitors (9), phages (10), and nanosized drug delivery technologies (11, 12). These include nanoparticles, which are carrier systems with a size of 10–1,000 nm in diameter, usually of natural or synthetic and biodegradable or non-biodegradable materials (13). In these systems, a drug can be dissolved, trapped, and/or encapsulated (14, 15) to achieve higher efficacy through higher uptake at lower risk of adverse effects (16).

Among them, lipid nanoparticles (LNPs) have gained popularity over the last few decades. Solid lipid nanoparticles (SLNs) consist of a solid lipid core stabilised by surfactants. Nanostructured lipid carriers (NLCs), are a step further from SLNs (16, 17), as they contain a mixture of solid and liquid lipids. Lipid particles have many advantages over standard drug formulations because of their adaptability, low toxicity, high bioavailability, possibility to deliver both hydrophilic and lipophilic drugs, and easy scaling up that enables large-scale production (1820).

Due to their lipophilic nature, SLNs and NLCs can also easily cross barriers like the bacterial cell membrane (21), and entrap hydrophobic drugs. Although some studies (22, 23) reported encapsulation of hydrophilic drugs as well, loading of a high amount of hydrophilic drugs in these carriers is more challenging and involves careful selection of solid or liquid lipids and suitable surfactants used in lipophilic and aqueous phases (24).

One such hydrophilic drug is moxifloxacin (MOX), a water-soluble fluoroquinolone derivative with broad-spectrum antibiotic activity. It inhibits the bacterial DNA gyrase and topoisomerase IV enzymes to disrupt DNA replication and repair and kill susceptible bacterial cells (25). It is used in the treatment of skin (26) and respiratory tract infections like chronic bronchitis, pneumonia, and acute bacterial sinusitis. It is especially effective against recurrent Escherichia coli (E. coli) infections (27, 28).

Loading an antibiotic into colloidal carriers like LNPs could counteract the efflux mechanism, through which resistance is developed, and increase intracellular retention of the drug (29). Wong et al. (30) encapsulated ciprofloxacin, which belongs to the same antibiotic family as MOX, in multilamellar liposome vesicles to improve its in vivo activity against Francisella tularensis and Brucella melitensis. However, as far as we know, MOX has not been studied in this context so far. The aim of our study was to find a way to enhance its uptake into bacterial cell using these lipid carrier systems, as they structurally resemble the bacterial membrane. We also wanted to see if lower antibiotic concentration could maintain the efficiency of a standard treatment dose of MOX. We also characterised the nanoparticles in terms of their size, distribution, zeta potential, encapsulation efficiency, and in vitro release. To determine the biocompatibility of the newly developed formulations, we also investigated the cytotoxic effects of LNPs in RAW 264.7 monocyte/ macrophage-like cells.

Materials and methods

Glyceryl dibehenate (CAS Nos. 6916-74-1, 77538-19-3, 3023364-8; brand name Compritol® 888 ATO), serving as solid lipid, was a gift from Gattefossé-SAS (Saint-Priest, France). Poloxamer 407, Tween (polysorbate) 80, triethanolamine (TEA), and fluorescein were bought from Sigma (Munich, Germany). MOX was a gift from Koçak Farma (Istanbul, Turkey). Docosahexanoic acid (DHA) (CAS No. 6217-54-5) and oleic acid (CAS No. 112-80-1), serving as liquid lipids, were kindly provided by Croda Inc. (New Jersey, NJ, USA). In addition, bacterial media , namely tryptic soy agar (TSA; agar 15 g/L, casein peptone 15 g/L, sodium chloride 5 g/L, soy peptone 5 g/L) and tryptic soy broth (TSB; casein peptone 17 g/L, dipotassium hydrogen phosphate 2.5 g/L, glucose 2.5 g/L, sodium chloride 5 g/L, soya peptone 3 g/L) used for growing the bacteria and Mueller Hinton agar (MHA; agar 17 g/L, beef infusion solids 2 g/L, casein hydrolysate 17.5 g/L, starch 1.5 g/L) and cation-adjusted Mueller Hinton broth (MHB; acid hydrolysate of casein 17.5 g/L, beef extract 3 g/L, starch 1.5 g/L) used for antibacterial activity tests were purchased from Merck Life Science (Darmstadt, Germany). All the other chemicals were of analytical grade.

Chemicals used for cytotoxicity testing were purchased from the following suppliers: Dulbecco’s Modified Eagle Medium (DMEM) and Dulbecco’s Phosphate-Buffered Saline (PBS) from Wisent Bioproducts (Quebec, Canada), trypsin-EDTA from Biological Industries (Beit-Haemek, Israel), foetal bovine serum (FBS) and penicillin-streptomycin from Capricorn Scientific (Ebsdorfergrund, Germany), and dimethyl sulphoxide (DMSO) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) from Sigma-Aldrich Chemicals (Munich, Germany).

E. coli ATCC 25922 were obtained from the American Type Culture Collection (ATCC).

The RAW 264.7 monocyte/macrophage-like cells were kindly provided by Yeditepe University Faculty of Pharmacy, Department of Pharmaceutical Toxicology, İstanbul, Turkey.

Preparation of lipid nanoparticles

SLNs and NLCs were prepared by ultrasonication (31) using components reported in Table 1. For SLNs we used only solid lipids in the lipid phase. Lipids were first melted and heated to 70 °C with Tween 80 or poloxamer 407 used as emulsifiers. Then we heated 10 mL of water containing 10 mg of MOX to 70 °C and added to the lipid phase. The mixture was sonicated with a probe sonicator (Sonopuls, Bandelin, Germany) at 50 % amplitude for 1 min and let to cool down to room temperature (25 °C) for 2 h to allow formation of particles. A similar method was used to obtain the NLCs, except that solid and liquid lipids were combined as the lipid phase (see Table 1 for details). Moreover, blank nanoparticles (without MOX) were prepared to compare the effects.

Composition of lipid nanoparticles

Formulation codeSolid lipid (glyceryl dibehenate)Liquid lipid (docosahexaenoic acid, DHA)Liquid lipid (oleic acid)Tween 80Poloxamer 407Triethanolamine (TEA)
SLN1150 mg--100 mg--
SLN2150 mg---100 mg-
NLC1110 mg40 mg10 mg-100 mg10 mg
NLC2110 mg40 mg--100 mg10 mg

NLC – nanostructured lipid carrier; SLN – solid lipid nanoparticle

To label the nanoparticles, fluorescein (5 mg) was added instead of MOX at the lipid phase step and mixed with magnetic stirrer for 30 s. The water phase and the remaining steps were applied as described above.

Determination of particle size, polydispersity index, and zeta potential

Nanoparticle size and polydispersity index (PDI) were measured with photon correlation spectroscopy (aka dynamic light scattering) and zeta potential with laser Doppler velocimetry (both using the Nicomp Nano Z3000 system, PSS, Inc., New York, NY, USA. Each sample was measured in triplicate (32).

Encapsulation efficiency (EE)

To calculate loaded MOX we used an indirect method by measuring MOX content in supernatants. Samples were centrifuged (IEC Centra MP4R, Rockville, USA) at 9000 g for 20 min and MOX detected with a high performance liquid chromatograph (HPLC, Agilent 1260 Infinity, Agilent Technologies Inc., Waldbronn, Germany) using a C18 column (150 mm × 4.6 mm i.d., 5 μm; ACE, Reading, UK) at the wavelength of 302 nm. Column temperature was 30 °C. The mobile phase was a mix of methanol-distilled water-acetonitrile (60:45:5) (v/v/v). It was adjusted to pH 2.7 with o-phosphoric acid. The flow rate was 1 mL/min (33).

In vitro drug release studies

MOX release from the nanoparticles was determined with the dialysis bag method (34). One millilitre (1 mL) of each nanoparticle suspension was placed in a dialysis bag (12–14 kDa, Spectrum Labs, USA) and the bag immersed in 50 mL of PBS (pH 7.4) and held in a shaker (Nuve, Istanbul, Turkey) operating at 50 rpm at 37 °C. MOX release was determined in 1 mL of PBS taken out from the pool at 15 and 30 min, and 1, 2, 4, 6, 8, 12, and 24 h. After each reading the PBS buffer was replaced with the same volume of a fresh one. The amount of released MOX was measured with the Agilent HPLC mentioned above.

TEM analysis

To characterise the morphology of MOX-loaded SLNs and NLCs, the particles were observed under a FEI Tecnai G2 Spirit BioTwin (Thermo Fisher, Waltham, MA, USA) transmission electron microscope (TEM) operating at 40 kV and 80,000× magnification. Samples were mounted on copper grids with a mesh size of 200 (75 microns), stained with 2 % uranyl acetate for 2 min, and then the excess removed with filter paper. The remainder was dried in a Petri dish for 2 h before microscopy.

Stability tests

Vials with formulations were kept in a dark fridge at +4 °C for one month and their particle size, polydispersity index, and zeta potential rechecked as described above.

Bacteria

E. coli ATCC 25922 were cultivated in tryptic soy agar and tryptic soy broth at 35±1 °C, and the study was carried out with 24-hour fresh bacterial cultures.

Bacterial uptake of nanoparticles

To determine the bacterial uptake of SLNs and NLCs we used fluorescent microscopy and flow cytometry. Briefly, 0.1 mL of E. coli suspension at log phase was added to SLNs and NLCs and incubated at 37 °C for 2 h. After incubation, the bacterial pellet and culture supernatant were separated by centrifugation at 4137 g. The pellet was washed with sterile distilled water three times to discard free NLCs or SLNs and then resuspended in 1 mL of sterile distilled water. The fluorescent intensity of E. coli cells was observed with a CYTOFlex cytometer (Beckman Coulter, Suzhou, China). Blue fluorescence was collected through a 525/40 BP fluorescent channel with a 488 nm blocking filter. For each sample around ten thousand cells were analysed. Data were measured and histograms created and interpreted using the CytExpert 2.4 Software (Beckman Coulter, Indianapolis, IN, USA). In addition, to detect the position of MOX-loaded NLCs and SLNs in bacteria, the cells were observed under a DM IL inverted fluorescent microscope at 40× magnification and images taken (Leica, Munich, Germany).

Antibacterial activity

Antibacterial activity against E. coli was tested with the broth microdilution method for minimal inhibition concentration (MIC) and with the disc diffusion test for the inhibition zone according to the European Committee on Antimicrobial Susceptibility Testing standards (35). For the disc diffusion test we used 4.0±0.5 mm deep Mueller-Hinton agar plates (25 mL in a 90 mm circular Petri dish). Bacterial suspensions were prepared from fresh E. coli cultures in sterile saline (0.9 % NaCl) to the density of a 0.5 McFarland measured with a DEN-1B densitometer (Biosan SIA, Riga, Latvia). A sterile cotton swab was dipped into bacterial suspensions and spread on the agar surface in three directions. Discs with MOX-loaded SLNs and NLCs were then placed on the surface of inoculated agar plates and incubated at 35±1 °C for 18±2 h. After incubation, inhibition diameters around the discs were measured and compared to standard MOX as control.

For the broth microdilution (MIC) test 100 μL of cation-adjusted Mueller-Hinton broth added to all U-bottom microplate wells. We added 100 μL of MOX-loaded nanoparticle solution to the first well and diluted it further across 12 wells. Then the bacterial suspension (in saline, 0.9 % NaCl) with the density of the 0.5 McFarland (1x108 CFU/mL) was 1:100 diluted and added to all wells to obtain the final bacterial concentration of 5x105 CFU/mL. The microplates were then incubated at 35±1 °C for 18±2 h, and MIC determined as the lowest concentration of MOX-loaded SLN and NLC solutions that visibly inhibited the growth of E. coli.

Preparation of RAW 264.7 cell cultures

RAW 264.7 cells were seeded in 25 cm2 flasks filled with 7 mL of DMEM supplemented with 10 % FBS, and 1 % penicillin-streptomycin and then grown in an incubator (Sanyo, Osaka, Japan) at 37 °C in an atmosphere supplemented with 5 % CO2 for 24 h.

Cytotoxicity assay

Cytotoxicity was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described earlier by Bacanli et al. (36). Cells were detached with a cell scraper and a total of 105 cells/well seeded in 96-well tissue-culture plates. The cells were incubated in full medium with various concentrations (0.010, 0.025, 0.05, and 0.1 μg/mL) of MOX alone, MOX-free SLNs, MOX-loaded SLNs, MOX-free NLCs, and MOX-loaded NLCs at 37 °C in an atmosphere supplemented with 5 % CO2 for 24, 48, and 72 h. For negative control we used a medium containing 10 % FBS and 1 % penicillin-streptomycin. After exposure, the medium was aspirated, cells washed with PBS, 10 μL of MTT (5 mg/ mL of stock solution with PBS) added to 100 μL of cell suspension per well, and cells incubated for another 3 h. The MTT dye was then carefully removed and 100 μL of DMSO added to each well. The absorbance of each well was measured with a microplate reader (Epoch, BioTek Instruments, Winooski, VT, USA) at 570 nm.

Statistical analysis

For statistical analysis we used the SPSS for Windows v. 20.0 software (IBM, New York, NY USA). All data are expressed as means ± standard deviations of measurements in three biological replicates. As the distribution was normal, differences between the groups were compared using Student’s t test. Statistical significance was set to p<0.05.

Results and discussion
Lipid nanoparticle properties

The composition of lipid nanoparticles is given in Table 1. Visual inspection did not reveal any separation between the oil and water phase in the MOX-loaded SLNs and NLCs, that is, emulsification was successful.

We aimed for small particles for better uptake by bacteria. SLN1, which contains Tween 80 as emulsifier, had significantly lower particle size and PDI compared to SLN2, which contains poloxamer 407 (p<0.05, Table 2). These differences in particle size and PDI are likely owed to the lower molecular weight of Tween 80 than that of poloxamer 407 (37), as suggested by similar studies (38, 39). PDI values of SLNs were below 0.3 characteristic of a monodisperse system (40). SLN1 had lower encapsulation efficiency than SLN2, which may be related to its lower particle size. Zeta potentials were significantly different (p<0.05), possibly due to different emulsifiers used in the SLNs.

Lipid nanoparticle properties

LNPParticle size (nm)Polydispersity indexZeta potential (mV)Encapsulation efficiency (%)
SLN1181.6±1.270.25±0.02-14.6±0.3268.61±0.20
SLN2201.8±1.650.29±0.02-17.5±0.1474.53±0.34
NLC1176.8±1.900.57±0.23-11.6±0.6177.82±0.21
NLC2132.4±1.620.51±0.53-6.3±0.3877.17±0.21

Results are represented as means ± standard deviations

NLC1 had bigger particles and distribution than NLC2. As known, particle size and distribution can be modified with sonication, which breaks coarse emulsion droplets to form a nano-emulsion, so it is one of important parameters to bear in mind while preparing lipid nanoparticles (41). If sonication is less effective, nanoparticles containing more liquid lipids, like NLC1, tend to form bigger particles, as liquid lipids increase the viscosity of coarse formulations.

Zeta potential of formulations may differ as particles contain different liquid phases. For instance, oleic acid, which was used in NLC2 as liquid phase, could shift the zeta potential from -6.3±0.38 to -11.6±0.61. However, there was no significant difference in encapsulation efficiency between NLCs (Table 2).

Table 3 shows that there were no significant changes in particle size, polydispersity index, and zeta potential measured in the formulations after one month, save for the drop in zeta potential in the SLN1 formulation. However, it is clear that particles tend to aggregate during storage, which leads to a gain in size and polydispersity index and loss in zeta potential.

Stability of lipid nanoparticles stored for one month at +4 °C

Particle size (nm)Polydispersity indexZeta potential (mV)
Type of LNPsBaseline valueAfter one month storageBaseline valueAfter one month storageBaseline valueAfter one month storage
SLN1181.6±1.27186.1±2.890.25±0.020.36±0.25-14.6±0.32-10.0±0.51*
SLN2201.8±1.65200.5±3.560.29±0.020.39±0.12-17.5±0.14-11.5±0.45*
NLC1176.8±1.90180.3±1.640.57±0.230.60±0.46-11.6±0.61-8.30±0.78
NLC2132.4±1.62135.2±3.980.51±0.530.65±0.22-6.3±0.38-5.62±0.96

Results are represent as means ± standard deviations. * significant difference from baseline (p<0.05)

TEM images and photon correlation spectroscopy of LNPs show spherical particles of similar size (Figure 1).

Figure 1

TEM images of SLN1 (a), SLN2 (b), NLC1 (c), and NLC2 (d)

Drug release

Figure 2 shows MOX release from nanoparticles. The release of standard (not nanoparticle-loaded) MOX was high in the first two hours, after which it kept dropping by the end of hour 4, at which point 95 % of MOX was released, and no more drug was until hour 24. LNPs slowed down its release thanks to its even dispersion in the lipid matrix and diffusion from it, as previously proposed by other authors (42, 43).

Figure 2

MOX release from nanoparticles

Bacterial uptake of nanoparticles

Figure 3 shows flow cytometry findings of E. coli cells. There is no fluorescence signal in control cells (Figure 3a), which were incubated with unlabelled nanoparticles, whereas the rest shows a strong signal (Figure 3b-f). Bacterial uptake of SLN1 and SLN2 (57.29 % and 50.74 %, respectively) was significantly more efficient than that of fluorescein solution alone (21.47 %) or either NLC (39.26 % for NLC1 and 32.79 % for NLC2) (p<0.05).

Figure 3

Flow cytometry results (a: blank NPS; b: fluorescein solution; c: SLN1; d: SLN2; e: NLC1; f: NLC2)

Judging by higher uptake of lipid nanoparticles than the fluorescein solution alone, drug encapsulation in nanoparticles should enhances drug absorption owing to the small size, prolonged release, and hydrophobic nature of lipid nanoparticles, which is similar to the Gram-negative bacterial cell wall (44). Apparently, smaller size of SLN1 is also the reason for higher bacterial uptake than that of SLN2, which is in line with previous reports (45).

These findings are confirmed by fluorescence microscopy (Figure 4), as only a small number of bacterial cells absorbed fluorescein solution alone (Figure 4a), whereas lipid nanoparticles show better absorption (Figure 4b-e) and therefore confirm that they can better enter bacteria than the free drug, as suggested in a related study (14).

Figure 4

Photomicrographs show bacterial uptake of formulations (a: fluorescein solution; b: SLN1; c: SLN2; d: NLC1; e: NLC2)(40× magnification)

Antibacterial activity

Table 4 shows the antibacterial activity of nanoparticles in terms of inhibition zone (diameter) and MIC. Only the SLN1 formulation was significantly more effective against E. coli (MIC 0.020 μg/mL, 34 mm) than MOX delivered as a standard solution (p<0.05). The NLC1 formulation showed better inhibition diameter (36 mm) than standard MOX but not MIC. In fact, SLN2 and both NLC formulations showed the same MIC as the corresponding standard MOX.

Antibacterial activity of MOX-loaded SLNs and NLCs

FormulationsInhibition diameter (mm)Minimal inhibition concentration (μg/mL)
Standard MOX solution for SLNs * (0.68 mg)33±0.070.041
SLN134±0.10***0.020
SLN232±0.110.041
Standard MOX solution for NLCs** (0.78 mg)35±0.080.023
NLC136±0.120.023
NLC235±0.050.023

The concentration was equal to the amount of MOX loaded into SLNs.

The concentration was equal to the amount of MOX loaded into NLCs.

significant difference p<0.05

Cytotoxic effects of MOX, MOX-free, and MOX-loaded LNPs in RAW 264.7 cells

RAW 264.7 cell viability (%)
24 h48 h72 h
0.010 µg/mL0.025 µg/mL0.05 µg/mL0.1 µg/mL0.010 µg/mL0.025 µg/mL0.05 µg/mL0.1 µg/mL0.010 µg/mL0.025 µg/mL0.05 µg/mL0.1 µg/mL
MOX88.65±0.0969.05±0.3556.48±0.0143.90±0.0389.42±0.0273.98±0.3359.43±0.0240.01±0.1086.62±0.2383.49±0.0854.58±0.0231.93±0.11
SLN1+MOX81.56±0.2675.43±0.2975.92±0.2374.55±0.2394.62±0.0281.95±0.1171.44±0.0358.43±0.0692.13±0.0891.46±0.3489.06±0.2873.70±0.22
SLN195.30±0.0868.91±0.1262.78±0.2557.75±0.0195.75±0.2991.22±0.0585.44±0.0664.09±0.0387.12±0.2782.65±0.0973.35±0.1169.72±0.17
SLN2+MOX93.14±0.0880.42±0.1966.66±0.4662.10±0.2188.73±0.0677.09±0.1263.16±0.1256.05±0.0492.13±0.0891.46±0.3489.06±0.2873.71±0.22
SLN290.98±0.3086.81±0.1264.91±0.0960.60±0.3196.51±0.2294.42±0.1280.82±0.0868.49±0.2296.02±0.3387.31±0.2769.95±0.0955.88±0.06
NLC1+MOX80.46±0.0981.65±0.1885.84±0.2681.47±0.0988.72±0.0972.27±0.0462.23±0.0251.10±0.1585.24±0.0976.56±0.3073.78±0.0956.03±0.32
NLC189.19±0.1378.73±0.1770.83±0.3353.27±0.0492.75±0.1382.93±0.0463.82±0.0954.44±0.0589.70±0.0980.44±0.0863.51±0.3554.76±0.20
NLC2+MOX78.43±0.3165.99±0.0556.58±0.2950.72±0.0476.23±0.2759.40±0.0353.59±0.0653.05±0.0894.10±0.2779.09±0.0373.90±0.0651.68±0.17
NLC285.54±0.0760.58±0.1056.67±0.2650.07±0.1683.62±0.0662.94±0.1254.81±0.0552.27±0.0491.99±0.4282.25±0.1274.99±0.0554.11±0.09

Results were given as means ± standard deviations

We know that E. coli with its thinner peptidoglycan layer and an outer lipopolysaccharide membrane has a limited permeability to drugs (46). Nanostructure with controlled drug release weakens its membrane resistance and increases drug uptake over time. In this sense, our findings are consistent with the ceftriaxone study by Kumar et al. (47).

We believe that the higher antibacterial activity of MOX loaded into SLN1 is related to the smaller size of SLN1 and distinct lipid and surfactant features, as earlier studies suggest that lipid nanoparticles with Tween 80 can generate higher antibacterial activity against E. coli (42). Furthermore, SLN1 can carry MOX directly to the target within the bacterium and also act as efflux pump inhibitor, that is, inhibit drug clearance from the cell (14, 48).

Nanoparticle cytotoxicity

None of the nanoparticles, whether loaded with MOX or not, lowered RAW 264.7 cell viability below 50 % after 24, 48, and 72 h of exposure to all studied concentrations. The highest concentration of standard MOX (not loaded into nanoparticles), however did lower cell viability below 50 %. Our findings therefore suggest that lipid nanoparticles in addition to having low cytotoxicity improve lower the cytotoxicity of MOX and lower its biocompatibility.

Similar observations of nanocarriers lowering MOX cytotoxicity were reported by several studies (4951).

Conclusion

By combining emulsifiers and lipids we successfully prepared stable SLNs and NLCs with high encapsulation efficiency for MOX. These newly designed lipid carriers offered better delivery of the drug into E. coli ATCC 25922 compared to the standard MOX formulation, and showed acceptable cytotoxicity and even protection from cytotoxic effects of MOX on RAW 264.7 cells. This finding, however, calls for further investigation and verification in other cell models.

Our findings show that MOX-loaded nanoparticles hold promise as potent chemotherapeutic drugs against E. coli. The most promising formulation is SLN1, as it has shown the best results with bacterial uptake and comparable antibacterial activity to MOX at much lower concentration.

Figure 1

TEM images of SLN1 (a), SLN2 (b), NLC1 (c), and NLC2 (d)
TEM images of SLN1 (a), SLN2 (b), NLC1 (c), and NLC2 (d)

Figure 2

MOX release from nanoparticles
MOX release from nanoparticles

Figure 3

Flow cytometry results (a: blank NPS; b: fluorescein solution; c: SLN1; d: SLN2; e: NLC1; f: NLC2)
Flow cytometry results (a: blank NPS; b: fluorescein solution; c: SLN1; d: SLN2; e: NLC1; f: NLC2)

Figure 4

Photomicrographs show bacterial uptake of formulations (a: fluorescein solution; b: SLN1; c: SLN2; d: NLC1; e: NLC2)(40× magnification)
Photomicrographs show bacterial uptake of formulations (a: fluorescein solution; b: SLN1; c: SLN2; d: NLC1; e: NLC2)(40× magnification)

Cytotoxic effects of MOX, MOX-free, and MOX-loaded LNPs in RAW 264.7 cells

RAW 264.7 cell viability (%)
24 h 48 h 72 h
0.010 µg/mL 0.025 µg/mL 0.05 µg/mL 0.1 µg/mL 0.010 µg/mL 0.025 µg/mL 0.05 µg/mL 0.1 µg/mL 0.010 µg/mL 0.025 µg/mL 0.05 µg/mL 0.1 µg/mL
MOX 88.65±0.09 69.05±0.35 56.48±0.01 43.90±0.03 89.42±0.02 73.98±0.33 59.43±0.02 40.01±0.10 86.62±0.23 83.49±0.08 54.58±0.02 31.93±0.11
SLN1+MOX 81.56±0.26 75.43±0.29 75.92±0.23 74.55±0.23 94.62±0.02 81.95±0.11 71.44±0.03 58.43±0.06 92.13±0.08 91.46±0.34 89.06±0.28 73.70±0.22
SLN1 95.30±0.08 68.91±0.12 62.78±0.25 57.75±0.01 95.75±0.29 91.22±0.05 85.44±0.06 64.09±0.03 87.12±0.27 82.65±0.09 73.35±0.11 69.72±0.17
SLN2+MOX 93.14±0.08 80.42±0.19 66.66±0.46 62.10±0.21 88.73±0.06 77.09±0.12 63.16±0.12 56.05±0.04 92.13±0.08 91.46±0.34 89.06±0.28 73.71±0.22
SLN2 90.98±0.30 86.81±0.12 64.91±0.09 60.60±0.31 96.51±0.22 94.42±0.12 80.82±0.08 68.49±0.22 96.02±0.33 87.31±0.27 69.95±0.09 55.88±0.06
NLC1+MOX 80.46±0.09 81.65±0.18 85.84±0.26 81.47±0.09 88.72±0.09 72.27±0.04 62.23±0.02 51.10±0.15 85.24±0.09 76.56±0.30 73.78±0.09 56.03±0.32
NLC1 89.19±0.13 78.73±0.17 70.83±0.33 53.27±0.04 92.75±0.13 82.93±0.04 63.82±0.09 54.44±0.05 89.70±0.09 80.44±0.08 63.51±0.35 54.76±0.20
NLC2+MOX 78.43±0.31 65.99±0.05 56.58±0.29 50.72±0.04 76.23±0.27 59.40±0.03 53.59±0.06 53.05±0.08 94.10±0.27 79.09±0.03 73.90±0.06 51.68±0.17
NLC2 85.54±0.07 60.58±0.10 56.67±0.26 50.07±0.16 83.62±0.06 62.94±0.12 54.81±0.05 52.27±0.04 91.99±0.42 82.25±0.12 74.99±0.05 54.11±0.09

Lipid nanoparticle properties

LNP Particle size (nm) Polydispersity index Zeta potential (mV) Encapsulation efficiency (%)
SLN1 181.6±1.27 0.25±0.02 -14.6±0.32 68.61±0.20
SLN2 201.8±1.65 0.29±0.02 -17.5±0.14 74.53±0.34
NLC1 176.8±1.90 0.57±0.23 -11.6±0.61 77.82±0.21
NLC2 132.4±1.62 0.51±0.53 -6.3±0.38 77.17±0.21

Stability of lipid nanoparticles stored for one month at +4 °C

Particle size (nm) Polydispersity index Zeta potential (mV)
Type of LNPs Baseline value After one month storage Baseline value After one month storage Baseline value After one month storage
SLN1 181.6±1.27 186.1±2.89 0.25±0.02 0.36±0.25 -14.6±0.32 -10.0±0.51*
SLN2 201.8±1.65 200.5±3.56 0.29±0.02 0.39±0.12 -17.5±0.14 -11.5±0.45*
NLC1 176.8±1.90 180.3±1.64 0.57±0.23 0.60±0.46 -11.6±0.61 -8.30±0.78
NLC2 132.4±1.62 135.2±3.98 0.51±0.53 0.65±0.22 -6.3±0.38 -5.62±0.96

Composition of lipid nanoparticles

Formulation code Solid lipid (glyceryl dibehenate) Liquid lipid (docosahexaenoic acid, DHA) Liquid lipid (oleic acid) Tween 80 Poloxamer 407 Triethanolamine (TEA)
SLN1 150 mg - - 100 mg - -
SLN2 150 mg - - - 100 mg -
NLC1 110 mg 40 mg 10 mg - 100 mg 10 mg
NLC2 110 mg 40 mg - - 100 mg 10 mg

Antibacterial activity of MOX-loaded SLNs and NLCs

Formulations Inhibition diameter (mm) Minimal inhibition concentration (μg/mL)
Standard MOX solution for SLNs * (0.68 mg) 33±0.07 0.041
SLN1 34±0.10*** 0.020
SLN2 32±0.11 0.041
Standard MOX solution for NLCs** (0.78 mg) 35±0.08 0.023
NLC1 36±0.12 0.023
NLC2 35±0.05 0.023

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