Norfloxacin Nanocrystals Through Surfactant Assisted Grinding

Abstract

The innovative work shows a new strategy to modify the physicochemical properties of medicines and improve patients' lives. The mechanochemical synthesis and characterization of the first Norfloxacin nanocrystal are described. In recent decades, nanotechnology has gained significant attention in the scientific field through the study of the design, formation and application of materials at the nanoscale level. In the pharmaceutical sector, nanotechnology has been the focus of studies on systems that allow increased bioavailability. The present work investigated the formation of pharmaceutical nanocrystals. Nanocrystals are crystalline nanoparticles ranging in size from a few nanometers to 1,000 nm, composed of 100% of the drug and stabilized by surfactants or polymeric stabilizers. They have a safe formulation and are well tolerated compared to conventional medicines. The drug used in this study was Norfloxacin, a broad-spectrum antibiotic that has low bioavailability. Several nanocrystallization techniques are found in the literature, among which the mechanochemical method stands out. Mechanochemistry is an approach that allows the reduction of particle size through the mechanical energy provided in the reaction medium. Thus, it was possible to obtain nanomaterials and explore the influence of surfactants in controlling the particle size and morphology of the materials obtained.

Keywords:
Norfloxacin; nanocrystal; mechanochemical; surfactant assisted grinding

1. Introduction

Nanotechnology has received great prominence in the scientific field in recent decades in the study of the design, formation, and application of materials on a nanometric scale¹. In the pharmaceutical sector, nanotechnology has been the target of studies into systems allowing for increased bioavailability². The decrease in particle size increases the dissolution rate and, as a result, increases the bioavailability of the drug³. In this context, the present study seeks the formation of crystalline drug nanomaterials, known as pharmaceutical nanocrystals⁴. Nanocrystals were described in the early 1,990’s and by the 2,000’s they were already available on the market. They are crystalline nanoparticles between a few nanometers and 1,000 nm in size, made up of 100% of the drug and stabilized by surfactants or polymeric stabilizers⁵. It has a safe and well-tolerated formulation compared to conventional drugs⁵.

Several nanocrystallization techniques can be found in the literature, including the mechanochemical method. The mechanochemical method is a top-down approach that allows particles to be reduced using the mechanical energy provided in the reaction medium⁶,⁷. The surfactant assisted grinding (SAG) is a mechanochemical technique to obtain nanocrystals. This technique allows a particle size reduction through mill mechanical energy⁸. The use of surfactant in the formation of nanocrystals influences particle size, morphology, stability, and agglomeration potential. It is expected that in this technique, the surfactant creates a film on the particle that prevents it from nucleating, growing, and agglomerating⁹.

Norfloxacin (Figure 1) is a broad-spectrum antibiotic used in the treatment of various infectious diseases such as gonorrhea, prostate infections and other infections of the gastrointestinal tract and urinary tract¹⁰-¹². This drug can cause adverse gastrointestinal effects when administered continuously due to its low aqueous solubility (0.28 mg mL^-1) and permeability, classified in the Biopharmaceutical Classification System BCS as class IV¹⁰, ¹³-¹⁵.

Figure 1
Structural formula of Norfloxacin.

Based on this information, the size reduction of NOR particles through SAG technique may improve NOR's dissolution rate and consequently its bioavailability

2. Experimental section

2.1. Sample preparation

The samples were prepared through surfactant assisted grinding (SAG) using Norfloxacin (Sigma-Aldrich; purity > 98%) and liquid (2:1 m/m), as shown in Figure 2. Surfactant (Tween 80 U.S.P. by Synth) and Ethanol (Neon Commercial Analytical Reagents LTDA; purity > 99.5%) were used alone and as a surfactant-ethanol mixture in different proportions (v/v %). The sample preparation is shown in Table 1.

Figure 2
Schematic illustration of the preparation method.

Table 1
Sample preparation.

Sample	Acronyms	NOR	Tween 80	EtOH
Norfloxacin + Tween 80	100%	100 mg	50.0 mg	-
Norfloxacin + 75% (v/v) Surfactant-ethanol mixture	75%	100 mg	37.5 mg	12.5 mg
Norfloxacin + 50% (v/v) Surfactant-ethanol mixture	50%	100 mg	25.0 mg	25.0 mg
Norfloxacin + 25% (v/v) Surfactant-ethanol mixture	25%	100 mg	12.5 mg	37.5 mg
Norfloxacin + EtOH	0%	100 mg	-	50.0 mg

The samples were milled at a frequency of 30 Hz (maximum equipment capacity) for 30 minutes¹⁶. A MM400 Retsch mill was used with 10 mL stainless steel jars, 2 balls of the same material (Φ = 7 mm) and about the same total sample mass (≈ 150 mg). The material formed is a white powder.

3. Characterization of Samples

3.1. Powder X-ray diffraction (PXRD)

The samples were analyzed through Rigaku diffractometer, model MiniFlex600. The diffractograms were obtained using copper tube, subjected to 40 kV, current of 15 mA, Cu κα, λ = 1.54056 Å, 5° ≤ 2θ ≤ 50°, with a scanning rate of 4°min^-1 in continuous scanning mode.

3.2. Infrared spectroscopy (IR)

The spectra were obtained using the Thermo Scientific spectrometer, model Nicolet 380, through the technique of total reflectance attenuated, in the range of 675 cm^-1 to 4,000 cm^-1, 4 cm^-1 resolution and 64 accumulations per spectrum.

3.3. Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) was performed using JEOL equipment, model JSM-IY500HR operated at an acceleration voltage of 3 kV. The samples were coated with gold and dispersed in isopropanol with the addition of a silicon disk and dried at room temperature.

3.4. Dynamic light scattering (DLS)

The measurements were conducted at 25 ºC with a dispersion angle of 90º, the samples were dispersed in isopropyl alcohol in the ratio and added to the disposable DLS cuvette (for DLS analysis). The analyses were obtained on the Zetasizer Nano ZS90, from Malvern Instruments.

4. Results

4.1. PXRD Analysis

The PXRD of NOR sample shows a mixture of polymorphs A, C, and sesquihydrate form I, as already reported in other studies¹²,¹⁴, and main diffraction peaks are presented in Table S1 and Figure S1 (Supplementary Material). The PXRD of samples (Figure 3), in general, shows a diffraction pattern characteristic of the sesquihydrate form I of Norfloxacin¹⁴, this conversion occurs due to the highly hygroscopic nature of the drug and the hydrate formation potential of NOR¹⁷,¹⁸. However, for the 100%, 75% and 0% samples the conversion to sesquihydrate form I was incomplete, since diffraction peaks of the starting material are still observed in their diffractograms, these peaks are indicated to ‘*’ on Figure 3.

Figure 3
PXRD patterns of Tween 80, NOR, 100%, 75%, 50%, 25%, 0% and NOR sesquihydrate form I. The peaks of starting material are represented by ‘*’.

This hydration process of materials was probably accelerated by the decrease in the average particle size and, consequently, a significant increase in the surface area.

Furthermore, the diffraction patterns show that no interaction occurred between NOR and Tween 80 during the mechanochemical process, as expected, since the diffraction pattern of the samples is the same as the NOR PXRD patterns found in the literature, which rules out the formation of a multicomponent solid or reaction chemical. The preservation of crystalline

4.2. IR Analysis

IR spectrum of Tween 80 (Figure 4) showed its signature bands at around 3,500 cm^-1 (OH stretching), a large band between 2,974 to 2,800 cm^-1 (CH₂ stretching), a band in 1,735 cm^-1 (C=O stretching) and a band in 1,646 cm^-1 (HOH bending). The data was according to literature¹⁹,²⁰. The NOR spectrum shows the main bands at 3,413 cm^-1 (OH stretching), at 1,615 cm^-1 (C=O stretching of ketone) and at 1,727 cm^-1 (C=O stretching of carboxylic acid), a spectral pattern characteristic of the anhydrous NOR and the presence of neutral molecules²¹,²². The milled samples show a significant decrease in the band at 1,727 cm^-1 (C=O stretching of carboxylic acid), the appearance of bands at 1,580 cm^-1 and 1,379 cm^-1 attributed to the asymmetric and symmetric stretching vibrations of the carboxylate group, respectively, and the appearance of the band between 3,000 and 2,730 cm^-1 attributed to NH⁺ stretching vibrations, this occurs due to the zwitterionic behavior of the hydrate form, which is induced by water-mediated proton transfer¹²,¹⁸.

Figure 4
IR spectra of Tween 80, NOR, 100%, 75%, 50%, 25% and 0%.

4.3. SEM and DLS analysis

SEM images of NOR sample (Figure 5a) show micrometer-sized particles, with a mean diameter of 17.21 µm and morphological variety. The sample contains both single particles and agglomerates. The 100% sample (Figure 5b) shows well-defined morphologically uniform particles and the size distribution data (Figure 6) reveals particle sizes ranging from 78.82 to 6,439 nm. The sample exhibits high polydispersity with three intensity peaks, the largest of which is at 531.2 nm. The 75% sample (Figure 5c) displays particle agglomeration along with surfactant threads, which indicates an excess of stabilizer in the samples²³. The size distribution data (Figure 6) reveals particle sizes ranging from 396.1 to 955.4 nm and exhibits lower polydispersity with a single and narrow peak at 615.1 nm. The 50% sample (Figure 5d) exhibits particle agglomeration; however, the particles are larger and do not have surfactant threads. The size distribution data (Figure 6) reveals particle sizes ranging from 91.28 to 2,305 nm and high polydispersity with a large peak at 825 nm. The 25% sample (Figure 5e) displays well-defined morphologically uniform particles and the size distribution data (Figure 6) reveals sizes ranging from 78 to 2,305 nm, with the highest concentration of particles around 825 nm. The 0% sample (Figure 5f) shows micrometer-sized particles (15.05 µm) with morphological heterogeneity and agglomerated particles.

Figure 5
SEM images of (a) NOR, (b) 100%, (c) 75%, (d) 50%, (e) 25% and (f) 0%.

Figure 6
Particle size distribution data for 25%, 50%, 75% and 100%.

The sample prepared without adding surfactant (0%) does not show a significant decrease in particle size compared to the starting drug (NOR); the particle size decreased by an average of 2 μm, not reaching the nanometric scale, which would not result in a pronounced increase in the dissolution rate. The 25% and 50% samples present approximate results, for both samples the particle size distribution data present two broad peaks, which indicates two ranges of polydisperse particle sizes, however, the highest concentrations of particles are in the range of nanometric size, with an average size of 825 nm. However, the 100% sample does not show an excess of surfactant exhibits size reduction but has several particle size ranges, which would result in different dissolution rates and could compromise therapeutic efficiency. Regarding the entire 75% sample, particles are in the nanometer range and exhibit the lowest polydispersity. Thus, this percentage of ethanol and surfactant was assertive to the obtention of Norfloxacin nanocrystals.

5. Conclusion

Through this study, a reduction in the particle size of Norfloxacin can be observed. The SAG technique proved to be efficient in obtaining reduced-sized particles without altering the nature of the drug. Manipulating different percentages of surfactant and ethanol in the sample highlighted the importance of using surfactant to obtain and control the size of nanometer-scale particles. However, in non-ideal surfactant proportions, significant polydispersity of particle sizes was noted. The 75% proportion shows nanometric particle sizes and homogeneity, making it a potential candidate for future dissolution rate studies.

Supplementary material

The following online material is available for this article:

Table S1. Main peaks of NOR polymorphs (2θ).

Figure S1. DRXP of NOR starting material and NOR’s polymorphs: Form A, Form C and sesquihydrate form I.

6. Acknowledgments

This work was supported by the São Paulo Research Foundation (FAPESP) [grant numbers 2023/07343-2 and 2023/06756-1], Brazilian Council for Scientific and Technological Development (CNPq) [grants numbers 422893/2021-8 and 317282/2021-2], and Coordination for the Improvement of Higher Education Personnel (CAPES) [grant number 88887.900882/2023-0].

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Publication Dates

• Publication in this collection
  21 Oct 2024
• Date of issue
  2024

History

• Received
  30 Apr 2024
• Reviewed
  16 July 2024
• Accepted
  22 Aug 2024