Synthesis of Magnetic Luminescent Nanoparticle Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD, a Potential Carrier of Antimicrobial Drug Ciprofloxacin

Abstract

Fe₃O₄@LaF₃:Eu,Ag hybrid magnetic luminescent nanoparticles (NPs) were synthesized using a simple co-precipitation method and then functionalized with β-cyclodextrin (β-CD) using (3-aminopropyl)triethoxysilane (APTES). The chemical composition, crystalline nature, particle size, and surface morphology of the Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs were investigated, using powder X-ray diffraction, and high-resolution transmission electron microscopy. The uptake and release profiling of the LaF₃:Eu,Ag@Fe₃O₄@β-CD NPs for the hydrophilic drug ciprofloxacin, showed 40 and 85% efficiency, respectively. The results indicated that the NPs have a high drug loading yield and a sustained drug releasing profile of the NPs, indicating that they can be used as a drug carrier. The photoluminescence spectral analysis of the NPs revealed their potentiality for use in bioimaging. Further analysis of the drug-loaded NPs (Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD-ciprofloxacin) revealed, 100% microbial inhibition efficiency against Escherichia coli and Vibrio cholerae, and a minimum of 80% against Bacillus cereus.

Introduction

Nonspecific anticancer drugs, not only necessitate high dose administration, but also exhort harmful impact on nearby cells/tissue [1]. The crucial factors in resolving the issue is targeted drug delivery to the afflicted cells and effective drug uptake by them [2]. This is something that nanotechnology has been consistently striving for [3]. Because of their customizable size, and physicochemical properties, magnetic NPs (MNPs), have demonstrated substantial potential for use in MRI [4], biomedicines [5], hyperthermia treatment [6], catalysis [7, 8], and drug targeting [9]. Furthermore, rare earth or alkali rare earth mineral fluorides such as LaF₃ are used as excellent host matrix and for bioimaging due to their low vibrational energy and exceptional brightness [10, 11]. However, MNPs, get oxidized and lose their paramagnetism due to their surface energy [12]. To prevent this, they are functionalized with materials like lanthanides-doped materials [13–16] β-cyclodextrin (β-CD) and others. Apart from improving the solubility, bioavailability, β-CD enable the NPs to act as a drug carrier [17, 18].

Based on results of our earlier study [19], the present NP, Fe₃O₄@LaF₃:Eu,Ag@(3-aminopropyl)triethoxysilane@β-CD (Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD), is supposed to have high luminesce, high magnetic saturation, and effective drug delivery potentiality, especially for hydrophilic drugs. As a result, herein, we used ciprofloxacin, a broad antimicrobial and anticancer hydrophilic drug [20, 21] to assess the drug carrying capacity of the NPs.

Methodology

Materials and Reagents

All the chemicals used for carrying out the experiment were of analytical grade and used without further purification. Ethylene glycol (EG), sodium hydroxide (NaOH), hydrochloric acid (HCl), and ferric chloride hexahydrate (FeCl₃·6H₂O) was procured from Merck, India, while, europium (III) oxide (Eu₂O₃, 99.999%), ferrous chloride tetrahydrate (FeCl₂·4H₂O), sodium molybdate (Na₂MoO₄·2H₂O, 98%) and APTES (99%) were purchased from Sigma Aldrich. 1-(p-tosyl)-imidazole was procured from TCI, India. β-CD was purchased from HiMedia Chemicals, India. Double distilled water was used for the preparation of buffers, reagents and preparing solutions. Other routine chemicals such as ammonium hydroxide (NH₄OH), ethanol (C₂H₅OH), ammonium fluoride (NH₄F), N-methyl pyrrolidone, and potassium iodide (KI) were of analytical grade.

Some of the routine equipment used in this experiment include autoclave (SLEFA), Equitron India, weighing balance (PGB 200), Wensar India, laminar flow (MFI H 2 × 2), µfilt India, centrifuge (REMI R-24 and CM8 Plus), REMI, India. Ultraviolet/Visible (UV/Vis) spectra were recorded using Systronics double beam 202, India.

Preparation of Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD

Fe₃O₄ was prepared by following Swain et al. (2022) [19] with slight modifications. 20 ml of 50% NH₄OH was added dropwise into a mixture of FeCl₂·4H₂O and FeCl₃·6H₂O (1:2 molar ratio) in deionized water to a pH 10.0 ± 0.2. After 20 min, the Fe₃O₄ MNPs produced, were stabilized by adding EG followed by homogenization through continuous stirring for 6 h. The particles obtained thereof were separated magnetically and washed repeatedly, using deionized water and C₂H₅OH. Finally, the particles were dried at 45 °C for overnight before characterization.

The Fe₃O₄@LaF₃:Eu:Ag nanoparticles were prepare by three time digestion of a mixture of 0.5 g La₂O₃, 0.0296 g Eu₂O₃, and 0.0572 AgNO₃ in HCl in a round bottom (RB) flask in the presence of double distilled water. Subsequently, 25 ml each of water and EG were added followed by 0.2495 g of NH₄F. The pH of the mixture was adjusted to 8.0 ± 0.2 by adding NH₄OH. 0.1 g of the previously synthesized Fe₃O₄ NPs were added into the reaction mixture and autoclaved at 120 °C for 24 h. The precipitate was then separated magnetically and washed with distilled water and C₂H₅OH at 12,000 RPM.

Fe₃O₄@LaF₃:Eu,Ag@APTES NPs were prepared by stirring Fe₃O₄@LaF₃:Eu,Ag with 150 µl of APTES. This APTES-loaded NPs were reacted further with 6-O-(p-toluenesulfonyl)-β-CD (6-TsO-β-CD) in dry N-methyl pyrrolidone (NMP) in the presence of KI. The detailed procedure is in our previous work [19]. The stepwise synthesis of Fe₃O₄@LaF₃:Eu,Ag@APTES-β-CD has been represented in the Fig. 1.

Fig. 1.

Schematic presentation of the stepwise synthesis of Fe₃O₄@LaF₃:Eu,Ag @APTES@β-CD NPs

Characterization of the NPs

High-resolution transmission electron microscopy (HRTEM) was performed on a JEOL transmission electron microscope (JEM-2010) using an accelerating voltage of 200 keV to understand the structural and morphological characteristics of the synthesized NPs. Powder X-ray diffraction (XRD) study was carri ved out using a Proto Desktop with Cu Kα radiation (λ = 1.54 Å) over a 2θ range of 10–100° to illustrate the chemical composition and crystalline size of the NPs. The photoluminescence spectra were recorded using Spectro fluorophotometer, Shimadzu (RF-5301 PC) equipped with a source of a xenon discharge lamp.

Uptake and Release Profiling of Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs

For estimation of the drug uptake potentiality of Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs, a batch equilibrium technique was performed. For this, 2.2 mg of Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs were taken in different vials with different concentrations of ciprofloxacin in water and then sonicated at 40 kHz for 5 min followed by stirring at 800 RPM for 2 h till equilibrium. Subsequently, the NPs were magnetically separated out as described earlier in the study. The changes in the concentration of ciprofloxacin in the water, before and after treating with Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs were estimated using UV–Vis spectrophotometer at λ_max of 280 nm.

Similarly for release profiling, 2.2 mg of Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD-ciprofloxacin were taken in a 5 ml of PBS buffer, pH 7.4 ± 0.2 and stirred at 800 RPM for different time interval. The NPs were magnetically separated out as described earlier in the study and changes in the concentration of ciprofloxacin in the PBS, pH 7.4 ± 0.2, before and after stirring were estimated using UV–Vis spectrophotometer at λ_max of 280 nm.

Antibacterial Assay of Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD-Ciprofloxacin NPs

Before the discovery of anticancer properties, ciprofloxacin was known for its broad antimicrobial properties [22] and to understand the drug carrying capacity of the Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs further, an in vitro antibacterial assay was conducted. The assay was carried out for LaF₃:Eu, LaF₃:Eu,Ag, Fe₃O₄@LaF₃:Eu,Ag, Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD against five water-borne bacterial strains, Escherichia coli (MTCC No. 41), Pseudomonas aeruginosa (MTCC No. 647), Bacillus cereus (MTCC No. 1277), Streptococcus aureus (MTCC No. 87), and Vibrio cholerae (MTCC No. 1277). For carrying out the assay, disc diffusion method was used [23] with ciprofloxacin (10 µl of 0.5 μg/μl) as positive control (PC). The bacteria procured from MTCC Chandigarh were analyzed further through catalase, oxidase, carbohydrate metabolism tests, and Gram’s staining. Subsequently, each strain was cultured in MH broth, pH 7 ± 0.2 for 4 h at 37 °C and lawn cultured each onto five different MH agar plates. Onto each plate, sterile discs (6.0 ± 0.1 mm), soaked with 10 µl of NPs (0.5 μg/μl) from each type were placed in triplicate followed by incubation at 37 °C for 24 h. The diameter (D) for the zone of inhibition (ZI) for each of the disc was measured and the % of inhibition was calculated (Eq. 1).

$$Inhibition(\%)=\left(\frac{D}{\mathit{Dpc}}\right)\times 100$$ 1

Result and Discussion

Characterization of LaF₃:Eu,Ag@Fe₃O₄@APTES@β-CD NPs

The XRD patterns of LaF₃ and Fe₃O₄ NPs agreed well to the XRD pattern of their pure forms i.e. JCPDF-072–1435 and JCPDF-88–0315, respectively (Fig. 2). The diffraction pattern indicated towards a cubic and hexagonal lattice structure of Fe₃O₄ and LaF₃ NPs, respectively. The lattice parameters, cell volume, and average nanocrystalline size of the NPs as estimated from Scherer’s equation has been listed in Table 1. The crystalline size and shape of the NPs changed on adding Fe₃O₄ to LaF₃:Eu,Ag, indicating Fe₃O₄@LaF₃:Eu,Ag core–shell formation. However, the XRD pattern of Fe₃O₄@LaF₃:Eu,Ag showed small lumps of the plane (220) of Fe₃O₄, which indicates that some parts of the Fe₃O₄ were not covered by LaF₃ [24, 25]

Fig. 2.

XRD patterns of LaF₃:Eu, LaF₃:Eu,Ag, Fe₃O₄@LaF₃:Eu,Ag, Fe₃O₄@LaF₃:Eu,Ag @APTES@β-CD, and Fe₃O₄ NPs

Table 1.

Lattice parameters, cell volume and crystalline size

Sample	a (Å)	b (Å)	C (Å)	Cell volume (Å3)	Size (nm)
JCPDF-072–1435 (LaF3)	7.176	7.176	7.344	327.51	–
LaF3	7.191	7.191	7.347	329.03	18.08
LaF3:Eu	7.178	7.178	7.334	327.34	18.09
LaF3:Ag	7.211	7.211	7.285	328.07	17.64
LaF3:Eu,Ag	7.173	7.173	7.325	326.49	14.97
Fe3O4@ LaF3:Eu,Ag	7.165	7.165	7.345	326.64	14.03
Fe3O4@ LaF3:Eu,Ag@APTES@β-CD	7.164	7.164	7.336	326.13	12.96
JCPDF-088-0315 (Fe3O4)	8.375	8.375	8.375	587.43	–
Fe3O4	8.367	8.367	8.367	585.87	20.63

The SAED pattern of Fe₃O₄ and Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD (inset Fig. 3a, c) revealed the interplanar distance between (311) and (111). The TEM images of Fe₃O₄ (Fig. 3a) showed cubical-shaped Fe₃O₄ NPs with particle size of ~ 17 and 18 nm, whereas, LaF₃:Eu (Fig. 3b) revealed spherical-shaped particles of size ~ 18 nm. However, Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD particles (Fig. 3c) seemed slightly elongated rod-like with shorter side diameter of ~ 15 nm indicating a core–shell structure [26, 27].

Fig. 3.

TEM images of a Fe₃O₄ (inset SAED pattern), b LaF₃:Eu, and c Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD (inset SAED) and histogram of particle size for d Fe₃O₄, e LaF₃:Eu, and f Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs

The room temperature emission and excitation spectra of the synthesized NPs are shown in Fig. 4. The excitation peaks at less than 300 nm are in the range of excitation spectra of Eu⁺³, indicating towards the charge transfer band arising from the transition of 2p electrons of O²⁻ to the empty 4f orbitals of Eu³⁺ ions [28]. The excitation peak of Eu³⁺ at 393 nm (⁷F₀ → ⁵L₆ transition) was found lower than the peak at 230 nm, thus the emission peaks under 230 nm excitation wavelength gives higher emission intensity than that of excitation wavelength of 393 nm, indicating charge transfer from the host. The inset Fig. 4 shows the integrated area under the curve calculated for the emission peak at 615 nm (⁵D₀ → ⁷F₂) for different samples. It is clear that the emission intensity gradually decreased on subsequent addition of Ag, Fe₃O₄ and APTES@β-CD onto LaFe:Eu.

Fig. 4.

Emission and (inset) excitation spectra of Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs

The emission spectra showed sharp lines at 590, 615, 652, and 700 nm, corresponding to ⁵D₀ → ⁷F₁, ⁵D₀ → ⁷F₂, ⁵D₀ → ⁷F₃ and ⁵D₀ → ⁷F₄ electronic transition of Eu³⁺, respectively. The lines at ⁵D₀ –⁷F₁ (at 590 nm) and ⁵D₀-⁷F₂ (at 615 nm) transition corresponds to magnetic dipole transition and electric dipole transition, respectively. The allowed magnetic dipole transition is supported by the Judd–ofelt theory, and higher electric dipole transition represents that Eu³⁺ ion do not have center of inversion and are susceptible to local symmetry [29, 30]. After functionalizing the NPs by APTES and β-CD (inset figure), the emission intensity decreased many folds, might be due to strong quenching of Fe₃O₄ and NH₂ functional groups. However, the resulting luminescence is quite strong enough for bioimaging applications.

Ciprofloxacin Uptake and Release Profiling of the Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs

The absorption spectra for three concentrations of ciprofloxacin, clearly showed decrease in the absorption peak at λ_max of 280 (Fig. 5a) after mixing with NPs, indicating uptake of ciprofloxacin by the Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs. The equilibrium inclusion capacity of the drug onto the NPs was calculated using Eq. 2. Where, Q_e is the equilibrium inclusion capacity of NPs for the drugs, C_o and C_e are the initial and final concentration of drug in the solution. M is the dry mass of the NPs and V is the volume of solution. The plot of Q_e vs. C_e (Fig. 5b) shows 33.50 mg/g equilibrium inclusion capability.

$$Q_e=\left(c_o,-,c_e\right)\frac{V}{M}$$ 2

Fig. 5.

a UV/Vis spectra of ciprofloxacin in the solution at 0.0165, 0.0264, and 0.0331 mg of ciprofloxacin, before and after mixing with Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs, b Equilibrium isotherm calculated for ciprofloxacin uptake by Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs c Freundlich and d Langmuir plot for the study of adsorption behavior of ciprofloxacin on the Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs

The adsorption behavior of ciprofloxacin onto Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs were also studied using Langmuir and Freundlich adsorption isotherms (Eqs. 3 and 4) [19, 31].

$$C_e/Q_e=C_e/q_m+1/q_m{K}_L$$ 3

$$ln{Q}_e=ln{K}_F+1/n_{F}ln{C}_e$$ 4

where q_m and K_L represent maximum adsorption density and Langmuir constant relating to the intensity of adsorption, respectively, whereas, K_F and n_F represent Freundlich constant related to the adsorption capacity and the adsorption intensity, respectively. The linear fitted plot of Langmuir and Freundlich equation (Fig. 5c, d) shows a R² ≈ 0.97, indicating a monolayer adsorption of the ciprofloxacin. The q_m, K_F and other parameter from Langmuir and Freundlich equation are presented in the Table 2.

Table 2.

Langmuir and Freundlich constants and parameters

Compound	Langmuir equation			Freundlich equation
	qm mg/g	KL ml/mg	R2	Kf mg/g	nF	R2
Ciprofloxacin	32.78	0.016	0.9706	76.70	3.39	0.8779

The graph for ciprofloxacin release (%) vs. time (Fig. 6) showed that the release was rapid at the first, then slowed until it reached a steady state by 6 h. The maximum % of release has attained to ~ 85%.

Fig. 6.

The release (%) of ciprofloxacin from Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs in PBS pH 7.4 ± 0.2

Antimicrobial Assay of Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD-Ciprofloxacin NPs

The results of antimicrobial assay (Fig. 7) clearly indicates that LaF₃:Eu, LaF₃:Eu,Ag, and Fe₃O₄@LaF₃:Eu,Ag NPs didn’t show any significant antimicrobial properties except few mild activities of LaF₃:Eu against B. cereus and V. cholerae while LaF₃:Eu,Ag against V. cholerae. This may be due to the inherent mild antimicrobial properties of the Eu against specific bacteria [32]. Furthermore, although Ag is well known antimicrobial agent [33], but in our case it didn’t showed anything such may be due to very low % of it. The results also indicated that core–shell formation have no role to play regarding antimicrobial properties of the NPs. However, Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD-ciprofloxacin NPs showed remarkable antimicrobial properties against all the five S. aureus, B. cereus, E. coli, P. aeruginosa, and V. cholerae. The same was found with ciprofloxacin as PC (Table 3). The inhibition (%) for the aforementioned five bacteria by the Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD-ciprofloxacin NPs is presented in the Fig. 8. The result showed almost a 100% of inhibition for E. coli and V. cholerae, and a minimum of 80% for B. cereus.

Fig. 7.

Antimicrobial assay of LaF₃:Eu, LaF₃:Eu,Ag, Fe₃O₄@LaF₃:Eu,Ag, Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs, and PC with S. aureus, B. cereus, E. coli, P. aeruginosa, and V. cholerae

Table 3.

The average diameter of ZI of Fe₃O₄@LaF₃:Eu,Ag, Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD-ciprofloxacin, and PC against S. aureus, B. cereus, E. coli, P. aeruginosa, and V. cholerae

NPs	Diameter of zone of inhibition with different bacterial strains
	S. aureus	B. cereus	E. coli	P. aeruginosa	V. cholerae
Fe3O4@LaF3:Eu,Ag	0.0	0.0	0.0	0.0	0.0
Fe3O4@LaF3:Eu,Ag@APTES@β-CD-ciprofloxacin	20.0 ± 0.0	16.0 ± 0.0	22.0 ± 0.0	20.0 ± 1.0	17.0 ± 0.6
PC	24.0 ± 0.0	20.0 ± 0.0	22.0 ± 0.0	21.0 ± 0.0	17.0 ± 0.0

Fig. 8.

Bar chart presentation of % of inhibition by Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD NPs for S. aureus, B. cereus, E. coli, P. aeruginosa, and V. cholerae

Conclusions

Magnetic luminescent NPs Fe₃O₄@LaF₃:Eu,Ag@APTES@β-CD were successfully synthesized. The shape and size of the NPs under HRTEM showed core–shell formation. The NPs were loaded with ciprofloxacin as drugs molecule with equilibrium inclusion capacity at 33.5 mg/g and the loaded drugs can be released up to 85% within 6 h. The ciprofloxacin loaded NPs showed almost 100% antimicrobial efficiency toward E. coli and V. cholera. The present study successfully establishes the idea of antibiotic carrying capacity of the NPs. However, in future, the authors will carryout other antimicrobial studies as growth curve assay and minimum inhibitory concentration test. Besides, the antibiotic-conjugated NPs will be studied in vivo for their biocompatibility assessment. Furthermore, the antimicrobial efficacy of the NPs will be evaluated against other possible bacterial or fungal strains including antibiotic resistance strains. The drug carrying potentiality and bioimaging applicability of the NPs in MRI will study in vivo in future.

Author Contributions

SKT: investigation, data analysis, preparing original draft, editing: GP: conceptualization, funding arrangement, data analysis, review and editing, supervision; SKD: antimicrobial study, data analysis, review and editing; SKT: supervision, fellowship arrangement, review and editing.

Funding

The author G. Phaomei would like to thank OSHEC, Govt. of Odisha for providing seed fund under OURIIP to support the work.

Data Availability

Available upon request from the corresponding author.

Declarations

Conflict of interest

None.