Formulation Development, Characterization and In Vitro Antibacterial Activity Evaluation of Cefazolin Loaded Mesoporous Silica Nanoparticles

: The main aims of this manuscript are to: i) investigate the high drug loading of cefazolin and its characterization, ii), demonstrate the bioactivity of the cefazolin particles in vitro on Staphylococcus aureus. From our results, it is observed that the cefazolin loading into MCM-41 particles is 34 wt %. Furthermore, particles showed the burst release of cefazolin at pH 6.8. At higher concentration, MCM-41 particles are comparatively more cytotoxic as compared to lower concentration. Finally, cefazolin loaded particles showed higher in vitro antibacterial activity against Staphylococcus aureus as compared to cefazolin only.


Synthesis of MCM-41
MCM-41 particles were prepared using a previously published protocol with slight modification [11]. Briefly, CTAB (1 g) was dissolved in 480 mL of deionised water to which 3.5 mL of NaOH (2 M) was added, and the temperature raised to 80℃. TEOS (6.7 mL) was slowly added to this mixture and stirred for 2 h at 80℃. The sample suspension was then vacuum filtered and washed with deionised water. The filtrate was dried overnight at room temperature followed by calcination at 550℃ for 5 h in a muffle furnace at a temperature ramp rate of 5 ℃/min for up-ramp and 10℃/min for down-ramp.

Loading of cefazolin into MCM-41 using immersion method
To obtain 30 % mass loading, CEF (35 mg) and pristine or functionalized MCM-41 (65 mg) were added in 5 mL of water for the preparation of nanoparticles. The solution was magnetically stirred at 4 ℃ for 24 h. Drug loaded particles were freeze-dried overnight and stored at 4 ℃.

Characterization of nanoparticles
TEM images of MSNs were acquired by Hitachi 7700 (Hitachi, Japan) electron microscope at acceleration voltage of 100 kV. Nitrogen adsorption -Brunauer-Emmett-Teller (N2-BET) (Tristar, Micromeritics-II, Norcross, GA, USA) was operated to measure the pore size, volume, and surface area of MSNs. The particle size and surface charge were calculated in water (solvent) by dynamic light scattering (DLS) and zeta potential measurements (Malvern, Nano-ZS, ATA Scientific, Taren Point, Australia). Thermogravimetric analysis (TGA) (Mettler Toledo, TGA/DSC 2, columbus, OH, USA) was performed with a heating rate of 10 ℃/min in an airy ambiance. The HPLC analytical (Waters 2695 Separations Module, Waters Corporation, Milford, MA, USA) method for CEF was developed based on with slight modifications. Briefly, the system consisted of 0.05 M KH2PO4: acetonitrile (90:10, v/v) pH 5.0 as the mobile phase at an isocratic flow rate of 1.5 mL/min, and detection wavelength 254 nm [12]. Chromatographic separations were performed on Phenomenex ® C8 RP-HPLC column (250 mm × 4.6 mm, 5 μ). Standard curve was plotted with n = 3.

In vitro release study
The CEF-MCM with 500 µg equivalent cefazoline were dispersed in 5 mL of the united states pharmacopoeia (USP) specified simulated gastric fluid test solution without enzymes (pH 1.2) and simulated intestinal fluid test solution without enzymes USP (pH 6.8) and stirred at 150 rpm and 37. Aliquots (200 µL) were taken at 0.08, 0.5, 1, 2, 4, 6, 8, and 24 h and centrifuged at 12000 rpm for 3 min. Equal volume of fresh buffers (pH 1.2 and 6.8) were added to rinse the pellet and put back the suspension to the total volume of release buffer. Samples were analyzed using the established HPLC method explained in the previous section. All experiments were performed in triplicate.

Cell viability assay
MCM-41particls were tested for their potential cytotoxic effect on RAW 264.7 macrophages. RAW 264.7 macrophages was cultured in high glucose DMEM medium, containing 100 μg/mL streptomycin sulfate and 100 units/mL penicillin, 10% (v/v) FBS, and 4 mM L-glutamine. The cells were grown at 37 °C in a humidified incubator with 5% CO2. RAW 264.7 macrophages (2 × 10 4 cells/well) were seeded into 96-well plates and grown for 24 h. The weighed mass of MCM-41 (25, 50, 100, 250, 500, and 1000 μg/mL) were incubated for 24 h. Cells with medium only were used as controls for each plate. After incubation, 25 μL/well of MTT reagent was added for a further 4 h at 37 °C. The cell culture medium was then aspirated, and the formazan crystals were dissolved by adding 100 μL/well DMSO. The absorbance signal of formazan was measured at 595 nm using a microplate reader.

Characterization of the nano-systems
The synthesised particles were first characterised using transmission electron microscopy (TEM) to assess the success of the synthesis. As shown in Figure 1, the MCM-41 particles were mostly spherical in shape with a slightly rough outer surface, and hexagonal pore arrangement. Furthermore, Figure 2 and Figure 3 show dynamic light scattering data in water including particle size and surface potential of these particles before and after CEF loading, respectively. The hydrodynamic size of MCM-41 is 263 nm, which increased to 345 nm after CFE loading (CEF-MCM-41).. The MCM-41 and CEFMCM-41 particles had negative zeta potentials of −17.2 and −17.7 mV, respectively. We next performed Nitrogen (N2) sorption isotherm analysis, pore size distribution analysis, and BET surface area plots on MCM-41 and their drug loaded particles. N2 adsorption-desorption analysis demonstrated that all particles displayed typical IUPAC type-IV isotherms, indicating the mesoporous nature of the silica samples (Figure 4). The particles also displayed a steep capillary condensation step at a relative pressure (P/Po) range of 0.2-0.4, characteristic of MCM-41 type mesoporous materials (Figure 4). BET surface area plots demonstrated that the pristine MCM-41 had a surface area of 970 m 2 /g which was reduced upon drug loading with CEF to 342 m 2 /g (Figure 3a). Similarly, the pore size of MCM-41 (2.2 nm) was reduced to 1.7 nm (CEF-MCM). Finally, pore volume of MCM-41 (0.90 cm 3 /g) was reduced to 0.78 cm 3 /g for CEF-MCM. These changes in physical properties induced by drug loading of the nanoparticles are consistent with previous reports [10,15]. Drug loading was also analysed using Fourier transform infrared (FTIR) spectroscopy. As shown in Figure 5, CEF shows a sharp intense peak at 1757 cm −1 (which clearly shows presence of carbonyl group), which confirms the carboxylic group in CEF. Some of the other notable peaks seen in cefazolin containing samples include that at 1647 cm −1 and 1593 cm −1 indicating C=N stretching and the presence of amide [16]. For MCM-41, a peak at 3430 cm −1 is related to the stretching vibration of -OH groups (Si-OH), while other characteristic peaks at 1068 cm −1 and 802 cm −1 are assigned to Si-O and Si-O-Si vibrations of silanol groups [17]. However in case of CEF-MCM-41, the characteristic peak of CEF at 1768 cm −1 can be seen with lower intensity , which confirm the loading of CEF in MCM-41. TGA was performed to determine the % drug loading. As shown in Figure 6, 34 % CEF was loaded into MCM-41 nanoparticles.

In Vitro Release Study
As seen from Figure 7, CEF-MCM released over 70% (burst release) of CEF in pH 6.8 in the first 30 min. After that, there was no significant increase in the drug release and total amount of drug release in 8 h was 76%.

In vitro antibacterial activity of CEF-MCM
The minimum inhibitory concentration (MIC90) of CEF-loaded nanoparticles was investigated using broth micro-dilution followed by EUCAST guidelines. We used gram-positive (S. aureus) bacteria (both ATCC and clinical strain) to determine the antibacterial effect of MPN formulations. As summarized in Table 1, MIC value for CEF-MCM particles is half as compared to free drug (CEF). Therefore, our nanoparticles showed the potential to significantly decrease the MIC value of the Pure CEF.

CONCLUSION
In summary, we demonstrated that encapsulation of the cefazolin within MSNs represents an effective strategy for improving its physicochemical properties. MCM-41 nanoparticles showed good drug loading (34 %). Furthermore, drug loaded silica showed improved anti-bacterial activity compared to free drug against both ATCC and clinical strains of S. aureus. Our results demonstrate that mesoporous silica-based carriers have the potential to improve the antibacterial activity of cefazolin.