Preparation and in vitro, in vivo evaluations of norfloxacin-loaded solid lipid nanopartices for oral delivery

Abstract

This work aims to develop norfloxacin-solid lipid nanoparticles (NFX-SLN) as an oral delivery formulation. Hot homogenization and ultrasonic technique was employed to prepare NFX-SLN using stearic acid as lipid matrix and polyvinyl alcohol as surfactant. The physicochemical characteristics of SLN were investigated by optical microscope scanning electron microscopy and photon correlation spectroscopy. Antibacterial experiments of NFX-SLN were carried out by broth dilution technique. Pharmacokinetics was studied after oral administration in male Sprague-Dawley rats. The results showed that NFX-SLN was spherical and the SLN of the optimized formulation had diameters 301 ± 16.64 nm, polydispersity index 0.15 ± 0.04, zeta potential -30.8 ± 0.69 mv, loading capacity 8.58 ± 0.21% and encapsulation efficiency 92.35 ± 2.24% with good stability at 4 °C. The NFX-SLN had sustained release effect and sustained bactericidal activity. Cytotoxicity studies in cell culture demonstrated that the nanoparticles were not toxic. NFX-SLN resulted in significantly higher plasma drug concentration than native NFX. The SLN increased the relative bioavailability of NFX by 12 folds, prolonged the plasma drug level above the average minimum inhibition concentration from 14 to 168 h. These studies demonstrate that NFX-SLN could be a promising oral formulation for enhanced bioavailability and pharmacological activities.

Keywords::

• Norfloxacin
• solid lipid nanoparticles
• antibacterial activity
• sustained release
• bioavailability

Introduction

Norfloxacin (NFX) is a widely used third generation quinolone synthetic antibiotic which has the characteristics of broad bacterium contradicting, small side-effects and cross-resistance with other drugs (Han et al., 2005). NFX is used against both gram-positive and gram-negative bacteria as well as trimethoprim/sulfonamide resistant microbes (Preheim et al., 1987). It is also active against Mycoplasma (Brown et al., 1996). This antibiotic shows promise as an antimicrobial agent for a variety of bacterial diseases (Wolfson & Hooper 1988; Carratalá et al., 1995). NFX kills bacteria on a concentration-dependent basis (Ferrero et al., 1995). The minimum inhibitory concentration for 90% of organisms (MIC₉₀) was below 0.12 μg/ml for E.coli, Salmonella spp, Klebsiella pneumonia, Klebsiellaoxytoca, Proteus vulgaris, Haemophilus influenzae (Lavy et al., 1995; Andersson & MacGowan 2003). NFX has been used extensively as both oral and injectable formulations for human and many domestic animal species (Hans & Li 1991). Oral administration is a fundamental route for therapy and prevention such as the standard of therapy in the prophylaxis of bacterial infections in cirrhotic patients with gastrointestinal hemorrhage; oral treatment regimen for uncomplicated gonococcal infection or oral prophylaxis suppressing infection caused by aerobic gram-negative bacilli during anti-leukemic therapy (Fernández et al., 2006; Kaplowitz et al., 1987; Verhoef & Rozenberg 1989).

However, there are drawbacks to limit the usage of NFX. NFX is scarcely dissolved in pure water, ethanol, methanol or aether while freely soluble in hydrochloric acid and natrium hydroxydatum with a low bioavailability mainly attributed to its low aqueous solubility and low permeability (Guyot et al., 1995; Breda et al., 2009). Human pharmacokinetic data for NFX show fraction absorption indicative of a poor permeability (Hooper & Wolfson 1985). The absolute bioavailability of NFX in humans and in laboratory animals is reported 40% post oral administration (Gips & Soback 1996). Examination of the individual serum concentration/time curves of the volunteers indicated that there may be a “first-pass” effect after oral administration and the site of metabolism is the liver, first-pass hepatic metabolism may be in part responsible for their lesser bioavailability (Hooper & Wolfson 1985). Poor bioavailability may provide lower antimicrobial activity and give rise to the development of resistance by the microorganisms (Aleem et al., 2008). Furthermore, repeated administrations are required to attain a steady-state level of drug. The common usage of NFX is twice a day for 7 to 10 days or even longer for uncomplicated lower urinary tract infections leading to a frequent dosage (Arredondo et al., 2004). Prophylactic NFX to patients with cancer who were neutropenic or likely to develop cytotoxic therapy–induced neutropenia or gastrointestinal hemorrhage are given orally 400 mg twice daily lasting more than 7 days (Fernández et al., 2006; Carratala et al., 1998). Repeated dosage may result in higher incidence of adverse effect. Additionally, treatment of some diseases requires high dosage, such as urinary tract infection required high dose NFX for months (Boerema & Saene 1986). Long-term high dose therapy has caused lenticular opacities, decreased spermatogenesis and testicular atrophy in experimental animals nausea and headache in patients(Smith 1987; Pittman et al., 1993).

Solid lipid nanoparticles (SLN) are valuable as an oral delivery carrier to increase the absorption of a poor water soluble drug and enhance the permeability and retention effect (Li et al., 2009; Maeda 2001; Zhang et al., 2008). It can improve the bioavailability of poorly hydrophilic and lipophilic drugs when these drugs are encapsulated in SLN (Luo et al., 2006). The mechanisms of bioavailability enhancement by SLN is that they possess adhesive properties that make them adhere to the gut wall and release the drug exactly where it should be absorbed (Müller & Keck 2004). In addition, the lipids are known to have properties that promote the oral absorption of lipophilic drugs and drugs in general (Porter & Charman 2001; Charman 2000). SLN can protect labile macromolecules from stomach acid and from the “first-pass” metabolism in the gastrointestinal tract (Manjunath & Venkateswarlu 2005). Better absorption and bioavailability enhancement enables dosage reduction and minimizes side effects (Munoz et al., 1994; Kalaria et al., 2008). The use of biodegradable materials for nanoparticles preparation allows for sustained drug release within the target site over a period of days or even weeks, lowering the frequency of administration (Panyam & Labhasetwar 2003; Vasir et al., 2003).

Stearic acid is a saturated 18-carbon chain fatty acid and usually synthesized from acetyl-CoA, metabolized by β-oxidation and expected to have good biocompatibility and low toxicity in body. The fatty acid is solid at room or body temperature, thus it is chosen for commonly pharmaceutical use as lipid matrix to prepare SLN (Zhang et al., 2000). Polyvinyl alcohol (PVA) is the most commonly used surfactant in the formulation of nanoparticles due to its excellent mechanical strength, biocompatibility, and non-toxicity and has been approved by the US Food and Drug Administration for medical and food applications (Davda & Labhasetwar 2002). In this work, NFX-SLN was prepared using stearic acid as lipid matrix and PVA as surfactant. The performances and the pharmacological activities of NFX-SLN were studied in vitro and in vivo.

Materials and methods

Materials

Stearic acid was obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). PVA and MTT (3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) were purchased from Sigma (St. Louis, MO, USA). NFX was bought from Shandong Lukang Pharmaceutical Co., Ltd. (Jining, China). NFX reference standard and Staphylococcus aureus (CCVCC2248) were purchased from China Institute of Veterinary Drug Control (Beijing, China). The solvents used for HPLC were of liquid chromatography (LC) grade. Methanol and acetonitrile were purchased from Fisher Scientific (Fair Lawn, NJ, USA). The water for HPLC was prepared with a Milli-Q system (Millipore, Bedford, MA). The citric acid was purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and the ammonium acetate was obtained from Beijing Chemical Reagents Company (Beijing, China). Other chemicals and reagents not specified in the text were of analytical grade or equivalent. Baby hamster kidney cell line (BHK-21) was from American Type Culture Collection (ATCC) (Manassas, VA, USA). Sprague-Dawley (SD) rats were from Academy of Military Medical Sciences (Beijing, China). They were acclimatized for 1 week before use with free access to food and water. All animal procedures were conducted with the approval of the China Agricultural University Institutional Animal Care and Use Committee. Humane care of all animals was stringently followed throughout all animal-involved studies.

Preparation of norfloxacin-loaded nanoparticles

Norfloxacin-solid lipid nanoparticles were formulated by hot homogenization and ultrasonic technique (Xie et al., 2010). Briefly, NFX was mixed with 3 g stearic acid in a 50 ml tube and heated in a boiling water bath. After the lipid was melted and the drug was dissolved in the melted lipid, 30 ml PVA solution preheated on a boiling water bath was poured into the lipid phase under magnetic stirring to form an o/w emulsion. The emulsion was sonicated for 5 min (VC X 750 Vibra-CellTM, Sonics & Materials, Inc., Newtown, CT, USA, using the 13 mm microprobe with amplitude 35%) to form a nanoemulsion. The nanoemulsion was quickly poured into 200 ml cold water to obtain a nanoparticle suspension. The nanoparticles were collected by centrifugation at 9000 rpm (Centrifuge 5810 R; Eppendorf, Germany) for 30 min and washed 3 times with distilled water. The SLN were re-suspended in 100 ml distilled water and lyophilized for 72 h (LGJ-12 Freeze Dryer; Beijing Songyuanhuaxing Science Technology Development Co., Ltd., China). The control SLN was prepared similarly without adding NFX.

Determination of mean diameter (MD), polydispersity index (PDI) and zeta potential (ZP)

The mean diameter, PDI and zeta potential of the SLN were determined by photon correlation spectroscopy (PCS) using Zetasizer Nano ZS90 (Malvern Instruments, UK). The samples of SLN were suspended in distilled water at a concentration of 2.7 mg/ml for particle size and PDI analysis, and a concentration of 0.3 mg/ml for zeta potential determination.

Determination of loading capacity (LC) and encapsulation efficiency (EE)

The wavelength scan using a UV spectrophotometer (U-1800, Hitachi Tech Co., Japan.) showed that NFX had a specific absorption at 272.4 nm. To determine the drug content in SLN, a weighed amount of freeze-dried SLN was suspended in 1% NaOH solution in a 15 ml tube and heated on a boiling water bath for 5 min. After cooling down, the solution was centrifuged at 9000 rpm (Centrifuge 5810 R; Eppendorf, Germany) at 4 °C for 15 min. The supernatant was analyzed at 272.4 nm. The control nanoparticles without NFX were treated similarly as blanks for measurements. Loading capacity (LC) was calculated directly. NFX in total SLN was calculated by LC multiply the recovered freeze-dried SLN. Loading capacity (LC) and encapsulation efficiency (EE) are defined as follows:

Microscopic analysis

One milligram of NFX-SLN prepared with 3g strearic acid, 300mg NFX and 30ml 1% PVA was suspended in 1 ml distilled water. Two microliters of the suspension were placed on a microscope slide and the nanoparticles were dried at room temperature for 5 min. Photomicrographs of the SLN were taken using an inverted optical microscope (Olympus 1X71, Olympus, Japan).

Morphology analysis

The morphology of NFX-SLN prepared with 3g strearic acid, 300mg NFX and 30ml 1% PVA was studied by SEM (SE S-3400N; Hitachi, Japan). Briefly, 1 mg samples were suspended in 1 ml distilled water and 2 µl of the suspension were placed on a cover glass and dried at room temperature over 30 min. The samples were coated with gold and examined at an accelerating voltage of 20 kV.

In vitro release study

Norfloxacin-solid lipid nanoparticles (3 mg) prepared with 3g strearic acid, 300mg NFX and 30ml 1% PVA were suspended in 2 ml 0.1 mol/ml hydrochloric acid (HCl) to mimic the in vivo stomach decomposition process (Luo et al., 2006) in a dialysis bag (MW: 8000–14400) and dialyzed against 38 ml 0.1 mol/ml HCl in a 50 ml tube at 37 °C under magnetic stirring at 60 rpm. Since NFX was freely soluble in hydrochloric acid, the volume of receiver solution was large enough to completely dissolve the released drug. Native NFX suspension with the same drug concentration was used as the control. To determinate the NFX amount diffused through the dialysis bag, at fixed time intervals samples (2 ml) were withdrawn from the receiver solution and the same amount of 0.1 mol/ml HCl was added to maintain a constant volume. NFX in the release medium was measured spectrophotometrically at 272.4 nm (U-1800, Hitachi Tech Co., Japan). The control nanoparticles without NFX were treated similarly and used as blanks for measurement. To determine the drug remained in SLN within dialysis bags after release, the SLN was collected by centrifugation of the suspension and the measurement was carried out by the method in determination of LC. All the experiments were carried out in triplicate.

Stability studies

Stability studies of SLN were performed after the samples were stored at 4 °C for 6 months. The values of MD, PDI, ZP and LC were measured for the evaluation of the physical stability of the nanoparticles. In vitro release was also carried out and compared with that of newly prepared SLN.

Antibacterial activity studies

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth dilution technique of National Committee for Clinical Laboratory Standards (NCCLS; now renamed as Clinical and Laboratory Standards Institute, CLSI, 2000). Briefly, 11 mg NFX-SLN (containing 944 μg NFX) was suspended in 4 ml distilled water and serial dilutions were made with distilled water (NFX concentration: 1.2, 0.9, 0.6, 0.45 0.3 and 0.225 µg/ml). The dilutions (2.7 ml) were mixed with 10 µl Staphylococcus aureus (CCVCC2248) containing 0.5 McFarland (1.5 × 10⁸ CFU/ml) to a final bacterial concentration of 5.5 × 10⁵ CFU/ml. The mixture was incubated at 37 °C for 12 h and 24 h in an incubator (Jiangsu Taicang Experimental Equipment Company, China) with shaking at 130 rpm. The MIC of NFX was defined as the lowest concentration inhibiting visible growth of bacteria. The MBC is measured by subculturing the broths used for MIC determination onto fresh agar plates. The MBC is the lowest concentration of the drug that results in killing 99.9% of the bacteria being tested. The native NFX and the released NFX were also made serial dilutions to the final concentrations as above and the MIC and MBC were measured by the same way.

Sustained antibacterial studies were conducted by broth dilution technique. Ten microliters bacterial culture containing 0.5 McFarland (1.5 × 10⁸ CFU/ml) of organisms were added to 2 ml Mueller-Hinton broth medium in a 4 ml tube containing NFX or NFX-SLN with a drug concentration of 0.34 µg/ml. Control nanoparticles (CNP) and Mueller-Hinton broth (MHB) were used as controls. The mixtures were incubated at 37 °C in an incubator with shaking (130 rpm). At fixed time points (6, 12, 24, 36, 48, 72 h), and ten serial dilutions of the mixtures were cultured on Luria broth agar plates at 37 °C. The colonies were counted when they could be observed by naked eyes. Growth graphs were plotted by calculating the number of colonies in 1 ml of the mixture versus the incubating time (hour). All experiments were carried out in triplicates.

Cytotoxicity evaluation

Cytotoxicity evaluation of NFX-SLN was conducted by MTT assay in BHK-21 cell line. The cells were seeded into a 96-well microplate at a density of 1 × 10⁴ cells per well in 0.1 ml DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics and cultured in a humidified 5% CO₂ incubator at 37 °C. After 24 h, the medium was replaced by 100 µl DMEM complete medium containing 58.3 µg NFX-SLN (drug concentration: 50 µg/ml). Native NFX or control nanoparticles (CNP) at same concentrations were also used. After 24 h co-incubation, cell viability was assessed by MTT assay. Typically, 5 mg/ml of MTT in PBS were added to each well reaching a final MTT concentration of 0.5 mg/ml and incubated for 4 h. Then the supernatants were removed and the formazan crystals were dissolved in 150 µl DMSO. Aliquots were drawn from each well and the absorbance at 490 nm was determined by a microplate reader (Bio-Rad 680). Cells without adding SLN or NFX were taken as control and set to 100% viability. The cell viability (%) compared to control cells were calculated by (OD_490sample/OD_490control) × 100.

Pharmacokinetics

A group of 20 male SD rats weighing on an average of 360 ± 10 g were used for the pharmacokinetic studies. They were randomly divided into 4 groups with 5 animals in each group. NFX was administrated to them orally (20 mg/kg of body weight) in the form of either NFX or NFX-SLN suspended in 2 ml sterilized double-distilled water. Each treatment contained 2 groups. At different times (0, 0.17, 0.5, 1, 1.5, 2, 3, 4, 8, 14, 24, 36, 48, 72, 96, 120, 144, 168 h) post dosage, blood samples were collected from tail vein and at one time point only one group of each treatment were collected. The blood samples were collected by turns from 2 groups over the observation period. The experimental protocols concerning the handling of rats were in accordance with the requirements of the Institutional Animal Care and Use Committee at China Agricultural University.

HPLC assay

Sample extracts were prepared by mixing 150 µl plasma with 300 µl mixture of methyl alcohol and acetonitrile (1/1 v/v) and vortexed for 3 min, and then centrifuged at 14000 rpm (Sigma 1-14; Sartorius, Germany) for 25 min. Two hundred microliters supernatant were taken for high performance liquid chromatography (HPLC) analysis using VP-ODS: 250 mm × 4.6 mm (Shimadiu Coperation, Kyoto, Japan). The HPLC condition was: mobile phase: (0.05 mol/l citric acid and 0.01 mol/l ammonium acetate, pH of the aqueous phase was adjusted by triethylamine to 4.5)/methyl alcohol (65/35, v/v); flow rate: 1 ml/min; UV detector wavelength: 278 nm; the injection Volume: 50 µl. The plasma concentration of NFX was found to be linear over the range 0.125–10 µg/ml. The correlation coefficient was 0.9991. The limit of quantification (LOQ) of UV detection was 0.1µg/ml. The relative standard deviations (R.S.D.) of accuracy and precision for three different plasma concentrations of NFX (0.1, 1 and 4 µg/ml) were 12.33%, 5.57% and 0.78% for intra-day analysis, and 7.10%, 6.58% and 6.03% for inter-day analysis.

Statistical analysis

The values of maximum serum concentration (C_max) and time to peak serum concentration (T_max) were obtained directly from the concentration–time plotting. The area under the concentration–time curve (AUC_0-inf), mean residence time (MRT) and elimination half-life (T_½) was analyzed based on noncompartmental pharmacokinetics statistical moment. The relative bioavailability at infinity was calculated as: Fr = AUC_{NFX-SLN, 0-inf} /AUC_{NFX, 0-inf}. Results were expressed as mean ± SD. Statistical analysis was performed using the one-way ANOVA. The differences were considered significant at the level of P < 0.05. Microsoft Excel (2003) (Microsoft Inc., USA) was used for all statistical analysis.

Results and discussion

Characteristics of NFX-loaded nanoparticles

The characteristics of SLN were listed in table 1. The values of MD, PDI, ZP, LC and EE were significantly affected by the amount of NFX added in the preparation. With the increase of NFX from 150 to 600 mg, the values of MD, PDI, ZP and LC increased significantly. Although SLN with 600 mg NFX had higher LC value, its EE value was lower than those with both 150 and 300mg NFX, because of more drugs diffusing into the continuous phase. SLN with 300 mg NFX had higher drug loading capacity than SLN with 150 mg NFX and lower values of MD and PDI than SLN with 600 mg NFX, thus, 300 mg of NFX was selected for the preparation of SLN for further studies. NFX was usually orally given a single dose of 400 mg for patients (Wella M et al., 1998; Eandi M et al., 1983), which equaled to 4.662 g of the SLN prepared with 300 mg NFX. Considering the improvement of the drug bioavailability and the enhanced therapeutic efficacy, the dose could be reduced (Pandey R & Khuller GK 2005; Pandey R et al., 2005).

Table 1.  Characteristics of SLN (mean ± SD, n = 3).

Formulation		MD (nm)	PDI	ZP (-mv)	EE (%)	LC (%)
PVA (%)	NFX (mg)
1	150	298.67 ± 17.24^d	0.05 ± 0.23^ad	29.43 ± 1.25^d	83.41 ± 1.23^ad	4.20 ± 0.16^ad
1	300	301.13 ± 16.64^d	0.15 ± 0.04^d	30.80 ± 0.69^d	92.35 ± 2.24^d	8.58 ± 0.75^d
1	600	379.33 ± 10.50^abd	0.53 ± 0.10^abd	31.97 ± 1.43^d	68.86 ± 0.54^abd	16.21 ± 0.64^abd
0.5	300	373.33 ± 44.08^ad	0.72 ± 0.14^ad	29.67 ± 1.38^d	85.31 ± 0.76 ^ad	8.05 ± 0.38^d
2	300	304.67 ± 2.52^cd	0.15 ± 0.05^cd	31.23 ± 0.78^d	93.04 ± 0.34^cd	8.35 ± 0.44^d
1	0	160.31 ± 5.29	0.09 ± 0.01	19.77 ± 0.99	0	0

The SLNs were prepared with 3g strearic acid, 0, 150, 300 or 600 mg NFX and 30ml 0.5%, 1%, or 2% PVA.

MD: mean diameter; PDI: polydispersity index; ZP: zeta potential; EE: encapsulation efficiency; LC: loading capacity.

Statistical significances are p< 0.05 compared with: ^aPVA 1%/NFX 300 mg; ^bPVA 1%/NFX 150 mg; ^cPVA 0.5%/NFX 300 mg; ^dPVA 1%/NFX 0 mg.

PVA concentration had significant effects on the values of MD, PDI and EE. When PVA concentration increased from 0.5% to 1%, EE increased significantly and MD and PDI decreased. But further increase of PVA concentration from 1% to 2% resulted in no significant changes of MD, PDI and EE. Therefore, 1% PVA was chosen for preparation of SLN.

Optical and SEM micrographs showed that NFX-SLN were well dispersed with good particle size distributions (Figure 1a), and were spherical with smooth surfaces (Figure 1b). NFX-SLN had higher values of MD, PDI and ZP than that of control (Table 1), indicating that the drug played an important role in these characteristics. Lipid matrix encapsulated drug leading to a larger volume. NFX with negative charge enhanced the total surface charge. A high ZP (>30 mV) could provide an electric repulsion to avoid the aggregation of particles (Levy et al., 1994). The nanoparticle system with a high LC and EE could reduce the quantity of carrier required for the administration of a sufficient amount of pharmacologically active agent to the target site.

Figure 1.  Micrographs of NFX-SLN: (a) optical microscope (400×); (b) scanning electron microscope (15000×)

In vitro release of NFX from SLN

This 0.1 mol/ml HCl was used to mimic stomach acidity (Li et al., 2000). The in vitro release behavior of NFX-SLN and native NFX is summarized as the cumulative percentage release of NFX-SLN, as shown in Figure 2. The release of native NFX was almost complete by 2h. For the NFX-SLN, an initial fast release was observed during the first 4 h, the release reached 47 ± 2.86% by 2h and 59.09 ± 2.02% by 4h. The fast release phase could be attributed to the large surface area of nanoparticles with drug enrichment or precipitation in outer shell of the particles, leading to a relatively short distance of diffusion and hence fast release of the drug (Mühlen et al., 1998; Hu et al., 2002; Üner 2006). From a therapeutic standpoint, a fast release profile could be considered as an advantage as a sufficient amount of a drug and improvement of the penetration of the drug (Jenninga et al., 2000). After the fast release, it was a sustained release and the total release reached 77.21 ± 3.63% by 24 h. Then a prolonged, relatively steady and slow release was observed. The release reached 88.36 ± 1.27% by 72 h. After 72 h releasing, the remaining drug in SLN within dialysis bags was 8.62 ± 1.39%. This release trend is similar to our previous studies on other drug-loaded SLN (Xie et al., 2008; Han et al., 2009; Xie et al., 2009).

Figure 2.  In vitro release studies (mean ± SD, n = 3). NFX-SLN: norfloxacin-nanoparticles; NFX: native norfloxacin

Stability studies

After 6 months of storage at 4°C, ZP and LC displayed no significant changes, but the values of MD and PDI increased significantly (Table 2). In vitro release studies showed that stored SLN had the same release ability as that of newly prepared SLN. At different time points (Figure 3), the cumulative release values of the stored SLN were nearly the same as those of the newly prepared SLN, indicating the SLN maintained their physical characteristics as a controlled release formulation. These results demonstrate that the SLN had good stability at 4°C.

Table 2.  Comparison of characteristics of stored and newly prepared SLN (mean ± SD, n = 3).

SLN	MD (nm)	PDI	ZP (-mv)	LC (%)
Newly prepared	301.13 ± 16.64	0.15 ± 0.04	30.80 ± 0.69	8.58 ± 0.75
Stored	344.71 ± 64.20*	0.27 ± 0.05*	29.20 ± 2.36	8.37 ± 0.20

MD: mean diameter; PDI: polydispersity index; ZP: zeta potential; LC: loading capacity.

* Statistical significances between newly prepared SLN and Stored SLN are p < 0.05

Figure 3.  Comparison of In vitro releases of stored and newly prepared NFX-SLN (mean ± SD, n = 3).

Antibacterial activity

In vitro antibacterial activity studies showed that the released NFX had the same MIC and MBC as that of the native NFX (Table 3), indicating that the bioactivity of NFX was not changed during the preparation and release procedures. This preparation method avoided the using of organic solvent, at the same time, maintained drug stability since the temperature in the preparation did not exceed 100 °C while the melting point of NFX is 228 °C (Gao 1995). Moreover, the MIC and MBC of NFX-SLN were the same as that of native NFX at 12 h, and lower than that of native NFX at 24 h, suggesting that SLN increased the antibacterial activity of NFX. These results are similar to those obtained by Ranjita Misra et al and Esmaeili et al (Misra et al., 2009; Esmaeili et al., 2007). Fusion of the lipid composition with the bacterial membranes might account for the enhanced antibacterial effect of NFX-SLN (Sachetelli et al., 2000). Due to membrane merging, the antibiotic could easily penetrate inside the bacteria allowing the increased bactericidal efficacy observed with the drug, thus circumventing the normal pathway of penetration (Beaulac et al., 1996; Beaulac et al., 1998).

Table 3.  MIC and MBC of NFX (µg/ml, n = 3).

		12 h	24 h
MIC	Native NFX	0.3	0.6
	Released NFX	0.3	0.6
	NFX-SLN	0.3	0.3
MBC	Native NFX	0.6	0.9
	Released NFX	0.6	0.9
	NFX-SLN	0.6	0.6

MIC: minimum inhibitory concentration; MBC: minimum bactericidal concentration

Sustained antibacterial activity studies were carried out based on the growth kinetics of Staphylococcus aureus and a viable colony count method was conducted. At 6 h and 12 h NFX-SLN was less effective (Figure 4a), but at all other time points, NFX-SLN was much more effective than native NFX (Figure 4b). Lower concentration of NFX might count for the less effectiveness of NFX-SLN at the early stages because of an incomplete release of NFX from the nanoparticles. After 12h, NFX-SLN exhibited its advantage over native NFX. Colony number of native NFX sharply increased between 36 h and 48 h and reached almost the same level as that of the controls by 72 h, indicating the exhaustion of the antibiotic. In case of NFX-SLN, the increase of bacterial colonies was much slower (Figure 4b). NFX-SLN remained effective for a longer period of time owing to the sustained release of the drug. The two curves representing control nanoparticles and Muller-Hinton broth were in the same trend, indicating that control nanoparticles had no antibacterial effect.

Figure 4.  Sustained-antibacterial activity: (a) 6 and 12 h; (b) from 6 h to 72 h (mean ± SD, n = 3) NFX-SLN: norfloxacin-nanoparticles; NFX: native norfloxacin; CNP: control nanoparticles; MHB: Muller-Hinton broth Statistical significances are p< 0.05 compared with: *NFX.

Cytotoxicity

As demonstrated in Figure 5, cytotoxicity study on BHK-21 cell line showed no significant differences among the NFX-SLN, native NFX and the control (P > 0.05) on average cell viability, indicating the nanoparticles were not toxic to the cells. Stearic acid and PVA are commonly used pharmaceutical lipid matrix and surfactant with good biocompatibility and low toxicity.

Figure 5.  Cytotoxicity studies on BHK-21 cells (mean ± SD, n = 5) NFX-SLN: norfloxacin-nanoparticles; NFX: native norfloxacin; CNP: control nanoparticles

Pharmacokinetics

Figure 6 shows the Plasma NFX concentration vs. time plotting after oral administration of NFX-SLN or NFX, table 4 shows their pharmacokinetic parameters. At all time points, the plasma concentrations were significantly higher with NFX-SLN than with native NFX. NFX-SLN reached the peak plasma concentration (C_max) of 2.7 ± 0.6 μg/ml at 2 h. The drug concentration decreased between 2 h and 4 h, then sustained above the MIC₉₀ (0.12 μg/ml) in a steady trend. After fourteen hours, the plasma NFX concentration of NFX-SLN was 0.32 ± 0.03 μg/ml and then maintained over 0.139 ± 0.012 μg/ml for a period of 168 h. In contrast, the plasma drug concentration of native NFX increased rapidly to the peak level of 0.54 ± 0.16 μg/ml at 0.5 h, then declined quickly and was undetectable after 14 h. This result is similar to the in vitro release trend between NFX and NFX-SLN. The elimination half-life (t_1/2) and the mean residence time (MRT) of NFX-SLN were significantly longer than those of native NFX (101.84 ± 7.52 h vs 9.23 ± 0.33 h, and 155.98 ± 5.8 h vs 14.74 ± 0.64 h), which also indicated that the NFX-SLN had a sustained release effect. The perceived advantage of SLN was the encapsulation of the drug in the solid matrix for protection and controlled release purposes, the carrier itself may exhibit certain absorption promoting effect (Müller et al., 2000; Garcia et al., 2002). The lipid protects the drug from chemicals as well as enzymatic degradation, thereby delaying the in vivo metabolism and prolonging the systemic circulation time and controls the release of drug in blood (Luo et al., 2006; Bala et al., 2004; Bhardwaj et al., 2005). Sustained drug release effect over a period of days or even weeks could provide a long-circulation effect in vivo (Panyam & Labhasetwar 2003; Vasir et al., 2003). This NFX-SLN should be a promising formulation to improve the therapeutic efficacy and avoid frequent administrations of NFX for treatments needed multiple and frequent dosages, such as cancer induced neutropenia and gastrointestinal hemorrhage, and consequently reduce the adverse effects. The C_max of NFX-SLN indicates that there was a fast release of SLN which was consistent with the in vitro initial fast release in this study. This kind of curves is desirable for sustained-release products where the initial release rate should be sufficiently rapid to ensure the therapeutic drug serum levels.

Table 4.  Pharmacokinetic parameters after oral administration to SD rats (mean ± SD, n = 5).

Formulation	AUC0-inf	MRT (h)	Cmax (μg/ml)	Tmax (h)	T1/2 (h)	Fr
NFX-SLN	57.81 ± 6.89^*	155.98 ± 5.80^*	2.70 ± 0.60^*	2^*	101.84 ± 7.52^*	12^*
NFX	4.8 ± 0.14	14.74 ± 0.64	0.54 ± 0.11	0.5	9.23 ± 0.33	1

AUC_0-inf: the area under the concentration–time curve; MRT: mean residence time; C_max: the values of maximum serum concentration; T_max: time to peak serum concentration; T_1/2: elimination half-life; Fr: the relative bioavailability at infinity

^*Statistical significances between NFX-SLN and native NFX are p < 0.05

Figure 6.  Plasma NFX concentration vs. time plotting after oral administration of NFX-SLN or NFX: (a) within 24 h; (b) from 24 h to 168 h (mean ± SD, n = 5). NFX-SLN: norfloxacin-nanoparticles; NFX: native norfloxacin.

The AUC_0-inf of NFX-SLN and native NFX were 57.81 ± 6.89 and 4.8 ± 0.14 respectively. SLN enhanced the oral bioavailability of NFX as reflected by AUC_0-inf by 12 folds. The mechanisms of oral uptake include diffusion of particles through mucus and accessibility to enterocyte surface, epithelial interaction, cellular trafficking, exocytosis and systemic dissemination (Hou et al., 2009). Nanoparticles are preferentially absorbed via “M-cells” in the Peyer’’s patches by the process of endocytosis (lymphoid uptake), thereby delivering the drug loaded particles directly into systemic circulation through the lymphatics and circumventing the first-pass metabolism (Jani et al., 1992; Desai et al., 1996). Lymphatic uptake is influenced by lipid nature, chain length, and hydrophobicity of nanoparticles (Hussain et al., 2001; Nordskog et al., 2001). The stearic acid has a long chain fatty acid, which increased in lymphatic uptake of NFX. The size of SLN in the range of 20–500 nm allows the efficient uptake in intestine, particularly in the lymphoid sections of this tissue (Yuan et al., 2007). Due to their small particle size, SLN might exhibit bioadhesion to the gastrointestinal tract wall or enter the intervillar spaces thus increasing their residence time in the gastrointestinal tract (Vasir et al., 2003). This increase in adhesion will result in enhanced bioavailability (Luo et al., 2006). Bioavailability enhancement helps to lower dosage levels (Munoz et al., 1994) and thus decreases toxicity or side effects. The enhanced bioavailability of NFX suggests that the NFX-SLN could be a useful formulation for treatment of diseases specifically depended on high dosage such as treatment of urinary tract infections.

Conclusions

Norfloxacin streaic acid-SLN prepared by hot homogenization and ultrasonic technique had good sustained release effect and stability. The SLN effectively enhanced the bioavailability and antibacterial activity of NFX. It could be a promising system for oral delivery of NFX.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.