Solid lipid micro-dispersions (SLMs) based on PEGylated solidified reverse micellar solutions (SRMS): a novel carrier system for gentamicin

Abstract

The purpose of this study was to formulate and evaluate novel PEGylated solidified reverse micellar solutions (SRMS)-based solid lipid microparticles (SLMs) for improved delivery of gentamicin. Lipid matrix (SRMS) [consisting of 15% w/w Phospholipon® 90G (P90G) in 35% w/w dika wax (Irvingia gabonensis) was formulated and characterized by differential scanning calorimetry (DSC). SLMs were formulated by melt-emulsification using the SRMS, PEG 4000 and gentamicin (1.0, 2.0, 3.0% w/w), and their physicochemical as well as pharmacokinetic parameters determined. In vitro permeation of gentamicin from the SLMs through artificial membrane (0.22 μm pore size) was carried out using Franz’s cell and phosphate-buffered saline (PBS, pH 7.4) as acceptor medium, while bioevaluation was performed using clinical isolates of Pseudomonas aeruginosa and Staphylococcus aureus. Stable and irregularly-shaped gentamicin-loaded SLMs of size range 34.49 ± 2.56 to 53.52 ± 3.09 µm were obtained. The SLMs showed sustained drug permeation and exhibited time-dependent and capacity-limited bioactivity. Overall, SLMs containing 2% w/w SRMS, 3% w/w gentamicin and PEG 4000 entrapped the highest amount of drug, gave highest IZD against the test organisms and highest permeation flux (5.239 μg/cm².min) and permeation coefficient (1.781 × 10⁻⁶cm/min) within 420 min, while pure gentamicin gave the least. Preliminary in vivo pharmacokinetic studies also showed an AUC-₂₄ of 1507 µg/h/ml for the optimized formulation, while that of oral drug solution was 678 µg/h/ml. This showed a 2.2-fold increase in the systemic bioavailability of gentamicin from the optimized formulation. PEGylated SRMS-based SLMs prepared with heterolipid from Irvingia gabonensis would likely offer a reliable delivery system for gentamicin.

• Dika wax (Irvingia gabonensis)
• gentamicin
• lipid-based drug delivery system
• PEGylation
• solid lipid microparticles

Introduction

In recent decades, there has been an increased awareness of the need to develop drug delivery systems to improve the properties of therapeutic compounds, increase their effectiveness and reduce their harmful side effects (Kenechukwu et al., 2011). Oral administration of therapeutic agents represents by far the easiest, safest and most convenient route of drug delivery, especially in the case of chronic therapies. Unfortunately, the oral delivery route is beset with problems such as gastrointestinal (GI) destruction of labile molecules and low levels of macromolecular absorption (Umeyor et al., 2012a). The development of oral forms of many drugs, however, remains a challenge either on account of their stability or their absorption from the GI tract (GIT) thus leading to sub-therapeutic bioavailability (Nnamani et al., 2010; Chime et al., 2013).

PEGylation is one of the most promising and extensively studied strategies for improving the pharmacokinetics and pharmacodynamics of drugs (Attama et al., 2009). Various PEGylation techniques have been used to enhance the delivery of both hydrophilic and lipophilic drugs. PEGylation has been shown to increase therapeutic efficacy by enabling increased drug concentration, improved biodistribution and longer dwelling time at the site of action (Attama et al., 2009). More so, the widening availability of lipidic excipients with specific characteristics offers flexibility of application with respect to improving the bioavailabilty of both lipophilic and hydrophilic drugs and manipulating their release profile (Reithmeier et al., 2001; Attama & Nkemnele, 2005; Rawat et al., 2011). The proven safety and efficacy of lipid-based carriers make them potential alternative drug carrier materials to polymers as well as attractive candidates for preparing lipid-based formulations (You et al., 2005; Barakat & Yassin, 2006; Joshi & Shah, 2008). These formulations allow for controlled/sustained drug delivery, among other advantages (Kumar, 2000; Jaspart et al., 2007; Umeyor et al., 2012b). Solidified reverse micellar delivery systems (SRMDS) are lipid-based biodegradable matrix drug delivery systems (Friedrich et al., 2000; Friedrich & Muller-Goymann, 2003; (Friedrich et al., 2005), and have been widely investigated as potential drug delivery systems for drugs which encounter penetration and absorption problems (Schneeweis & Muller-Goymann, 2000; Umeyor et al., 2012c; Momoh et al., 2013a).

Gentamicin, an aminoglycoside antibiotic, used in the treatment of severe Gram-positive and Gram-negative microbial infections, is limited by poor absorption, low bioavailability and high toxicity (Robert & Walters, 1998; Chang et al., 2006; El-Gendy et al., 2009; Momoh & Esimone, 2012). This broad-spectrum hydrophilic bactericidal antibiotic acts by inhibition of protein synthesis after binding to specific 30S-subunit ribosomal proteins. It is unstable in acidic pH of the stomach and its cationic nature affects its penetration into the mucosal walls of the GIT; hence, it is commonly administered topically, intramuscularly, intravenously and subcutaneously (Stephens et al., 2000; Sundin et al., 2001; Singh et al., 2010). Like other aminoglycosides, gentamicin is toxic to the sensory cells of the ear and also causes nephrotoxicity by inhibiting protein synthesis in renal cells. This mechanism specifically causes necrosis of cells in the proximal tubule, resulting in acute tubular necrosis which can lead to acute renal failure (Drusano et al., 2007).

By tactical engineering of lipid-based drug delivery systems (LBDDS) such as PEGylated solidified reverse micellar solution-based solid lipid microparticles (SRMS-based SLMs), the toxicity, poor oral absorption and penetration problems of gentamicin could be surmounted. Solid lipid microparticles (SLMs) combine the advantages of different traditional carriers; for example, they can be produced on a large industrial scale and allow controlled release of incorporated drug (El-Kamel et al., 2007; Kenechukwu et al., 2011; Chime et al., 2012a,b; Umeyor et al., 2012a,c; Nnamani et al., 2013). Homolipids and heterolipids have gained renewed interests as excipients for LBDDS (Attama & Muller-Goymann, 2006, 2007). Homolipids are esters of fatty acids with various alcohols. Dika wax is an edible vegetable fat derived from the kernel of Irvingia gabonensis Var excelcia (Matos et al., 2009). Dika wax has been evaluated as basis for drug delivery (Chukwu et al., 1991; Ofoefule et al., 1997; Okore, 2000). Previous studies on LBDDS using a heterolipid from dika wax (I. gabonensis) and containing some drugs demonstrated positive results (Chime et al., 2012c). Similarly, Phospholipon® 90 G (P90G) has been shown to be a good excipient in the formulation of SRMS-based SLMs (Attama et al., 2009; Nnamani et al., 2010; Momoh et al., 2013a). To the best of our knowledge, there are no reports in the literature on the delivery of gentamicin using PEGylated SLMs. The novelty embodied in this study lies on the fact that for the first time PEGylation of lipid-based drug delivery system (i.e. PEGylation of solidified reverse micellar solution-based solid lipid microparticles) was employed as a new technique to improve the delivery of gentamicin. It is expected that PEG 4000 would facilitate the release of gentamicin from the lipid matrix core and its subsequent transport across the membrane, more than the commercially available gentamicin formulations; this anticipated enhanced delivery of gentamicin would be of immense benefit in the treatment of microbial infections caused by gentamicin-susceptible organisms.

Consequently, the objectives of this study were to formulate SRMS (lipid matrix) consisting of P90G and heterolipids from I. gabonensis, and PEGylated SRMS-based SLMs containing gentamicin using melt-emulsification technique and evaluate the in vitro permeation, pharmacokinetics and bioactivity of gentamicin from such a delivery system.

Experimental

Materials

The following materials were used: gentamicin pure sample (JUHEL Pharmaceutical Limited, Awka, Nigeria), dika wax (a biodegradable heterolipid was obtained from I. gabonensis and purified in our laboratory), Phospholipon® 90G (Phospholipid GmbH, Köln, Nattermann, Germany), poloxamer 188 (Sigma Aldrich, Madrid, Spain), polyethylene glycol 4000 (Acros Organics, Fair Lawn, NJ), monobasic potassium phosphate, sodium hydroxide and concentrated hydrochloric acid (BDH, Hull, England) and distilled water (Lion water, UNN, Nigeria). All other reagents and solvents were of analytical grade and were used as supplied.

Extraction and purification of heterolipids from I. gabonensis

Irvingia gabonensis was purchased from Nsukka market, Enugu State, Nigeria, in the month of July, 2011. The seed material was authenticated by Mr. A.O. Ozioko, a consultant taxonomist with the International Center for Ethnomedicine and Drug Development (InterCEDD) Nsukka and the voucher specimen was deposited in the herbarium of the Department of Pharmacognosy and Environmental Medicines, University of Nigeria, Nsukka.

Dika wax was extracted by Soxhlet extraction using established procedure (Chime et al., 2012c). Briefly, I. gabonensis seed was milled in a hammer mill and extracted in a Soxhlet extractor using n-hexane at 100 °C. The n-hexane was allowed to evaporate at room temperature. Boiled distilled water which was twice the volume of the wax was poured into the molten wax in order to dissolve the hydrophilic gum contained in the wax. The hydrophilic gum was removed using a separating funnel. Ethyl acetate was equally poured into the molten wax in order to remove the hydrophobic gum from the wax. The principle of adsorption was employed in the purification of the extracted wax using admixtures of activated charcoal and bentonite (2:1) as the adsorbent blend. The molten wax (10 g) heated to 100 °C was allowed to run through the column (1 g). The purified wax was stored in a refrigerator until used.

Preparation of lipid matrix (SRMS) and solid lipid microparticles

Lipid matrix consisting of a mixture of 35% w/w dika wax (heterolipid) and 15% w/w Phospholipon® 90G (P90G) (Phospholipid GmbH, Köln, Nattermann, Germany) was prepared by fusion method (Umeyor et al., 2012a). Briefly, the dika wax and P90G were weighed using electronic balance (Mettler H8, Geneva, Switzerland), placed into a crucible, melted together at 75 °C on a thermo-regulated (water bath shaker (Heto, Copenhagen, Denmark) and stirred thoroughly. Thereafter, the mixture was allowed to cool and solidify at room temperature to obtain the lipid matrix (SRMS).

The melt-emulsification technique (Jaspart et al., 2007; Umeyor et al., 2012c; Momoh et al., 2013a) was adopted for the preparation of the SLMs. In each case, the SRMS was melted at 75 °C, and the aqueous phase containing PEG 4000 and poloxamer 188 at the same temperature was added to the SRMS with gentle stirring with a magnetic stirrer (SR 1 UM 52188, Remi Equip., Mumbai, India), and the mixture was further dispersed with a mixer (T 25 digital Ultra-Turrax®; IKA, Staufen, Germany) at 8000 rpm for 5 min. The SLMs suspension was obtained after cooling at room temperature. The above procedure was repeated using PEG 4000 and gentamicin (1.0, 2.0 and 3.0% w/w) and lipid matrix (4.0, 3.0 and 2.0% w/w), to obtain gentamicin-loaded SLMs (batches A₁–A₃, B₁–B₃ and C₁–C₃). The unloaded SLMs (D₁–D₃) were also prepared. The formulation compositions are shown in Table 1.

Table 1. Formulation compositions of the PEGylated SLMs.

Batches	PEG 4000 (g)	Poloxamer 188 (g)	Gentamicin (%w/w)	Lipid base (15%w/w P90G in 35% w/w DF) (g)	Distilled water, q.s. (% w/w)
A1	1.0	2.0	1.00	4.0	100
A2	2.0	2.0	1.00	3.0	100
A3	3.0	2.0	1.00	2.0	100
B1	1.0	2.0	2.00	4.0	100
B2	2.0	2.0	2.00	3.0	100
B3	3.0	2.0	2.00	2.0	100
C1	1.0	2.0	3.00	4.0	100
C2	2.0	2.0	3.00	3.0	100
C3	3.0	2.0	3.00	2.0	100
D1	1.0	2.0	–	4.0	100
D2	2.0	2.0	–	3.0	100
D3	3.0	2.0	–	2.0	100

Batches A₁–A₃, B₁–B₃ and C₁–C₃ are gentamicin-loaded PEGylated SLMs, while batches D₁–D₃ are unloaded (zero-drug) SLMs; P90G = Phospholipon® 90G, DF = dika wax.

Thermal characterization

Melting transition and change in heat capacity of the lipid matrix, dika wax, P90G, PEG 4000, gentamicin and the formulated SLMs were determined using a differential scanning calorimeter (NETZSCH DSC 204 F1, Munich, Germany). Small quantity of each sample was weighed into an aluminum pan, hermetically sealed and the thermal properties determined at a heating rate of 10 °C/min over a temperature range of 10–400 °C, under an inert nitrogen atmosphere with a flow rate of 20 ml/min. Baselines were determined using an empty pan, and all the thermograms were baseline corrected.

Particle size analysis and morphological characterization of SLMs

The particle size and morphology of the SLMs were determined by computerized image analysis on a polarized light microscope (Lieca, Wetzlar, Germany). Briefly, approximately a drop of the SLMs from each batch was placed on a slide (Marinfield, Germany) using a 1 ml dropper. It was then covered with a cover slip and viewed under a polarized light microscope. With the aid of the software in the microscope, the particle morphologies were observed and photomicrographs were taken. The sizes of the particles were measured and the average taken.

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

Approximately 5 ml of the gentamicin-loaded PEGylated SLMs was added into a microconcentrator (5000 MWCO Vivascience, Hanover, Germany), and was centrifuged (TDL-4 B. Bran Scientific and Instru. Co., London, England) at 3000 rpm for 3 h and the supernatant was collected, filtered (using Whatman filter paper no. 1) and properly diluted with distilled water. The dilute solution was adequately analyzed for gentamicin content spectrophotometrically (Unico 2102 PC UV/Vis Spectrophotometer, New York, USA) at 303 nm. The amount of drug encapsulated in the SLMs was calculated with reference to a standard Beer’s plot for gentamicin to obtain the EE% using the formula (Umeyor et al., 2012c): (1) LC expresses the ratio between the entrapped active pharmaceutical ingredient (API) and total weight of the lipids (Kenechukwu et al., 2011). It is determined as follows: (2) where, W_l is the weight of lipid added in the formulation and W_a is the amount of API entrapped by the lipid.

Time-resolved pH-dependent stability studies

The pH of the SLMs from each batch was determined using a pH meter (Suntex TS - 2, Mumbai, India) after one week, one month and three months of storage.

In vitro permeation studies

In vitro permeation studies were performed using a modified Franz diffusion cell (Franz, 1975) in phosphate-buffered saline (PBS, pH 7.4). A definite volume (2 ml) of the formulated SLMs in each case was placed in the donor compartment of the Franz diffusion cell separated from the receptor compartment by an artificial membrane (Spectrum Laboratories, Rancho Dominguez, Canada) (MWCO = 6–8000; pore size 0.22 μm). The receptor compartment was filled with PBS (pH 7.4) and maintained at a temperature of 37 ± 1 °C by means of a thermostatically controlled water bath, with agitation provided by a magnetic stirring bar at 50 rpm. Aliquot (5 ml) was removed and replaced by an equal volume of the receptor phase at different time intervals up to 420 min and the samples collected were analyzed for drug content spectrophotometrically using a spectrophotometrically (Unico 2102 PC UV/Vis Spectrophotometer, New York, USA) at 332 nm. The receptor phase was replenished with equal volume of phosphate buffer at each sample withdrawal. The results were the mean values of three runs. The cumulative percentages of drug permeated per square centimeter of the formulations were plotted against time. Control experiment was performed using pure sample of gentamicin powder (G₂) and a commercially available gentamicin injection (G₁) added directly into the donor compartment of the Franz diffusion apparatus.

Permeation data analysis

The flux (μg cm⁻²min⁻¹) of gentamicin was calculated from the slope of the plot of the cumulative amount of gentamicin permeated per cm² of artificial membrane at steady state against the time using linear regression analysis (Attama & Nkemnele, 2005). The permeation coefficients were obtained from the steady-state flux values making use of the following equation: (3) where, P is the permeation coefficient, CO is the initial drug concentration in the donor compartment; J represents the steady state flux obtained from Equation (4)(4) . (4) where, Q indicates the quantity of substances crossing the artificial membrane, A is the area of the artificial membrane exposed and t is the time of exposure.

Antimicrobial studies

The antimicrobial activity of the SLMs was tested against clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa by agar diffusion technique (Kenechukwu et al., 2011; Momoh & Esimone, 2012) using samples withdrawn during the in vitro drug release studies. Molten nutrient agar was inoculated with 0.1 ml of S. aureus broth culture. It was mixed thoroughly, poured into sterile Petri dishes and rotated for even distribution of the organism. The agar plates were allowed to set and a sterile cork borer was used to bore three cups in the seeded agar medium. Using a sterile syringe, a definite volume withdrawn from the receptor compartment of the diffusion apparatus at pre-determined time intervals was used to fill the holes. The plates were allowed to stand at room temperature before incubating at 37 ± 1 °C for 24 h. The diameter of each inhibition zone was measured and the average was determined (Singh et al., 2010). The procedure above was repeated for P. aeruginosa.

Preliminary in vivo studies

This study was conducted in accordance with Ethical Guidelines of Animal Care and Use Committee (Research Ethics Committee) of University of Nigeria, Nsukka, following the 18th WMA General Assembly Helsinki, June 1964 and updated by the 59th WMA General Assembly, Seoul, October 2008. Ten (10) Wistar albino rats weighing between 140 and 225 g were used. They were grouped into two groups of five each. Group 1 received the optimized LBDDS formulation (batch C₃) while group 2 received extemporaneous oral solution of gentamicin (batch G₂) in distilled water (since there is no available commercial oral formulation) at a dose of 5 mg/kg. All the animals were fasted 12 h before the test and throughout the experiment. One (1 ml) volume of blood was withdrawn from the rats’ orbital sinus using heparinized hematocrit tubes at 0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0 and 24 h. The withdrawn blood samples were centrifuged at 5000 rpm for 5 min to separate the plasma, which were stored at −4 °C till analyzed. For each sample, 0.2 ml of the plasma sample was deproteinated by diluting with equal volume of acetonitrile and centrifuged at 2000  rpm for 5 min. Then 0.1 ml of the supernatant was diluted in distilled water and assayed for drug content using a digital spectrophotometrically (Unico 2102 PC UV/Vis Spectrophotometer, New York, USA) at 303 nm. The plasma from the blood withdrawn at zero hour was similarly diluted and used as blank and for the preparation of calibration curve. Amounts of drug in the plasma were plotted against time to obtain the plasma concentration time curve, which was further evaluated to obtain the pharmacokinetic parameters (Attama et al., 2011; Momoh et al., 2013b).

Statistical analysis

All experiments were performed in replicates for validity of statistical analysis. Results were expressed as mean ± SD. ANOVA and Student’s t-test were performed on the data sets generated using SPSS (Version 17, SPSS Inc., New York, USA). Differences were considered significant for p values, p ≤ 0.05.

Results

Thermal properties

The results of the DSC thermograms of dika wax, P90G, lipid matrix, PEG 4000 and gentamicin are shown in Figures 1–5. The DSC thermograms of dika wax (Figure 1) showed a sharp endothermic peak at 44.3 °C, while the DSC thermograms of Phospholipon® 90 G (P90G) showed an endothermic peak at 131 °C (Figure 2). The thermogram of the lipid matrix formulated with P90G and dika wax (Figure 3) showed endothermic peak at 43.4 °C. The DSC trace of PEG 4000 (Figure 4) showed a sharp endothermic single peak corresponding to melting at 65.7 °C with an enthalpy of −41.44 mW/mg, respectively. The DSC thermogram of gentamicin (Figure 5) showed a major melting peak at 285.8 °C with an enthalpy of −10.41 mW/mg. Table 2 shows the thermal properties of gentamicin-loaded SLM formulations. The results showed that the formulations gave lower melting peaks as well as enthalpies than gentamicin.

Figure 1. DSC thermogram of dika wax.

Figure 2. DSC thermogram of Phospholipon® 90G (P90G).

Figure 3. DSC thermogram of lipid matrix (dika wax:P90G).

Figure 4. DSC thermogram of PEG 4000.

Figure 5. DSC thermogram of gentamicin.

Table 2. Thermal properties of the drug and PEGylated SLMs.

Batches	Melting point (°C)	Enthalpy (mW/mg)
Gentamicin	285.8	−10.41
A1	112.7	−164.0
A2	129.4	−312.8
A3	127.1	−254.0
B1	111.8	−173.1
B2	122.0	−213.1
B3	119.7	−205.4
C1	110.2	−215.7
C2	123.5	− 200.9
C3	125.8	− 195.0

Batches A₁–A₃, B₁–B₃ and C₁–C₃ contain 1.0, 2.0 and 3.0% w/w of gentamicin, respectively.

Particle size and morphology of SLMs

Table 3 shows the particle sizes of the SLMs. The results indicate that gentamicin-loaded SLMs and unloaded SLMs had a mean particle size (n = 30) range of 34.49 ± 2.56 to 53.52 ± 3.06 µm and 31.49 ± 1.02 to 33.50 ± 2.31 µm, respectively. The photomicrographs (Figure 6) showed that the SLMs were discrete, irregular and green in color.

Figure 6. Representative photomicrographs of the SLM formulations. Batches A₁, B₁ and C₁ are gentamicin-loaded SLMs while batch D₁ is the unloaded (zero-drug) SLMs.

Table 3. Some physicochemical properties of PEGylated SLMs.

		pH^a,c
Batches	Particle size (µm)^a,b	One week	One month	Three months	EE (%)^a,c	LC (g API/100 lipid)^c
A1	34.49 ± 2.56	5.20 ± 0.05	5.25 ± 0.30	5.24 ± 0.04	52.40 ± 1.96	20.00
A2	36.56 ± 2.81	5.31 ± 0.12	5.30 ± 0.08	5.29 ± 0.05	59.61 ± 1.80	28.20
A3	37.53 ± 1.97	5.21 ± 0.17	5.27 ± 0.02	5.23 ± 0.09	62.50 ± 2.73	33.60
B1	42.53 ± 3.15	6.49 ± 0.11	6.46 ± 0.07	6.47 ± 0.25	66.43 ± 3.14	38.00
B2	45.70 ± 2.04	6.50 ± 0.02	6.49 ± 0.10	6.48 ± 0.22	71.68 ± 2.22	44.70
B3	49.54 ± 3.12	6.46 ± 0.17	6.48 ± 0.09	6.45 ± 0.03	75.40 ± 3.99	49.50
C1	50.55 ± 1.09	7.10 ± 2.45	7.13 ± 2.45	7.11 ± 2.45	80.66 ± 3.58	52.00
C2	52.50 ± 2.13	7.15 ± 0.82	7.12 ± 0.82	7.13 ± 0.82	82.52 ± 3.65	57.30
C3	53.52 ± 3.06	7.17 ± 1.97	7.20 ± 1.97	7.18 ± 1.97	84.79 ± 3.81	61.80
D1	31.49 ± 1.02	4.27 ± 0.12	4.30 ± 0.05	4.28 ± 0.07	–	–
D2	32.50 ± 1.70	4.28 ± 0.04	4.27 ± 0.12	4.29 ± 0.08	–	–
D3	33.50 ± 2.31	4.31 ± 0.03	4.30 ± 0.02	4.27 ± 0.11	–	–

^aMean ± SD, ^bn = 30, ^cn = 3; Batches A₁–A₃, B₁–B₃ and C₁–C₃ are gentamicin-loaded PEGylated SLMs, while batches D₁–D₃ are unloaded (zero-drug) SLMs; EE = encapsulation efficiency, LC = loading capacity.

Encapsulation efficiency (EE%) and loading capacity (LC)

The EE% of the SLMs was in the range of 52.40 ± 1.96% to 84.79 ± 3.81%. The EE% (Table 3) increased with increase in the concentration of gentamicin for all batches. So, batches C₁–C₃ gave highest EE% while batches A₁–A₃ gave the least. Table 3 also shows that maximum LC of 52.00, 57.30 and 61.80 g of gentamicin per 100 g of lipid were obtained for batches C₁–C₃ respectively, containing 3% w/w gentamicin.

Time-resolved pH-dependent stability studies

Table 3 also shows the time-resolved pH values of the SLMs. The results indicate that, after three months of storage, unloaded SLMs had an average pH range of 4.27 ± 0.11 to 4.31 ± 0.03, while that of drug-loaded SLMs was 5.20 ± 0.05 to 7.20 ± 1.97.

In vitro permeation of gentamicin

The permeation profiles of gentamicin from the SLMs in PBS are depicted in Figure 7(A–C), whereas the permeation data (permeation coefficients and steady-state permeation fluxes) are presented in Table 4. There was a sustained permeation of gentamicin from the SLMs across the artificial membrane without a burst effect in the formulations over time. Generally, drug permeation followed the order: C₁–C₃ > B₁–B₃ > A₁–A₃. The permeability assessment of gentamicin from the SLMs formulations across the artificial membrane (Table 4) showed permeation fluxes of 4.917, 5.161 and 5.239 µg/cm².min for batches A₁–A₃, respectively; 4.126, 4.572, and 4.717 µg/cm²min, respectively for batches B₁–B₃; then 4.761, 4.839 and 4.961 µg/cm²min, respectively for batches C₁–C₃. The permeation fluxes for gentamicin injection and pure sample of gentamicin were 3.839 and 2.354 µg/cm²min, respectively (batches G₁ and G₂). The permeation coefficients were 9.834 × 10⁻⁷, 1.032 × 10⁻⁶ and 1.781 × 10⁻⁶cm/min, respectively, for batches A₁–A₃; 9.048 × 10⁻⁷, 9.395 × 10⁻⁷ and 9.496 × 10⁻⁷cm/min, respectively, for batches B₁–B₃; 9.522 × 10⁻⁷, 9.678 × 10⁻⁷ and 9.857 × 10⁻⁷ cm/min, respectively, for batches C₁–C₃; while the permeation coefficients of gentamicin injection (batch G₁) and pure sample of gentamicin (batch G₂) were 8.329 × 10⁻⁷ and 7.106 × 10⁻⁷cm/min, respectively.

Figure 7. Permeation profile of gentamicin from (a) A₁–A₃ SLMs; (b) B₁–B₃ SLMs; (c) C₁–C₃ SLMs in PBS, pH 7.4 (n = 3). A₁–A₃, B₁–B₃ and C₁–C₃ contain 1.0, 2.0 and 3.0% w/w of gentamicin, respectively, while G₁ and G₂ are commercial gentamicin injection and plain gentamicin, respectively.

Table 4. Permeation parameters of gentamicin-loaded PEGylated SLMs.

Formulation code	Flux (J) (µg/cm^2 min)	Permeation coefficient (P) (cm/min)
A1	4.761	9.522 × 10^−7
A2	4.839	9.678 × 10^−7
A3	4.961	9.857 × 10^−7
B1	4.126	9.048 × 10^−7
B2	4.572	9.395 × 10^−7
B3	4.717	9.496 × 10^−7
C1	4.917	9.834 × 10^−7
C2	5.161	1.032 × 10^−6
C3	5.239	1.781 × 10^−6
G1	3.837	8.329 × 10^−7
G2	2.354	7.106 × 10^−7

A₁–A₃, B₁–B₃ and C₁–C₃ are PEGylated SLMs containing 1.0, 2.0 and 3.0% w/w of gentamicin, respectively; G₁ and G₂ are commercial gentamicin injection and plain gentamicin, respectively.

Antibacterial properties

The results of the bioactivity recorded as inhibition zone diameter (IZD) (Tables 5 and 6) indicate that gentamicin-loaded SLMs produced very significant IZD against Gram-positive organism (S. aureus) and Gram-negative organism (P. aeruginosa). The formulations recorded increasing IZDs against the organisms with time. Moreover, gentamicin-loaded SLMs gave greater IZDs than the plain gentamicin as well as commercial gentamicin injection against the organisms. Overall, sub-batch C₃ containing the highest PEG 4000 and drug gave the greatest IZD against S. aureus (49.40 ± 3.07 µm) and P. aeruginosa (47.49 ± 2.38 µm) compared with other SLMs after 420 min. The results indicate that the bioactivity of gentamicin entrapped in the SLMs was better against Gram-positive than Gram-negative bacteria employed in the study.

Table 5. Susceptibility of Pseudomonas aeruginosa to gentamicin in the PEGylated SLMs.

	IZD (mm)^a,b
	Time (min)
Batches	60	120	180	240	300	360	420
A1	22.70 ± 0.19	23.89 ± 0.12	25.47 ± 0.11	26.78 ± 0.54	28.15 ± 0.96	30.37 ± 1.45	31.72 ± 1.69
A2	22.85 ± 0.43	24.07 ± 0.35	26.55 ± 0.32	27.83 ± 0.09	29.74 ± 0.85	31.91 ± 1.73	33.53 ± 1.04
A3	23.68 ± 0.81	25.97 ± 0.06	27.66 ± 0.94	29.34 ± 0.87	31.52 ± 1.06	33.81 ± 1.75	36.56 ± 1.50
B1	22.92 ± 0.09	24.37 ± 0.08	27.23 ± 0.19	30.19 ± 1.04	33.62 ± 1.53	36.89 ± 1.44	37.32 ± 1.09
B2	24.02 ± 0.50	25.98 ± 0.17	28.67 ± 0.25	31.87 ± 1.23	34.63 ± 1.09	37.95 ± 1.18	38.64 ± 1.33
B3	24.95 ± 0.67	26.62 ± 0.88	29.03 ± 0.72	32.83 ± 0.98	37.95 ± 1.82	42.56 ± 2.07	44.35 ± 1.98
C1	22.99 ± 0.45	24.59 ± 1.67	27.43 ± 2.20	30.96 ± 1.79	33.82 ± 3.10	47.19 ± 2.48	40.22 ± 3.93
C2	24.03 ± 0.78	27.29 ± 1.09	31.80 ± 1.33	35.24 ± 2.05	38.07 ± 2.41	41.26 ± 0.99	43.87 ± 1.56
C3	25.00 ± 0.82	28.57 ± 1.46	32.23 ± 1.00	36.04 ± 1.72	39.68 ± 2.15	43.80 ± 2.75	47.49 ± 2.38
G1	22.13 ± 0.98	22.46 ± 0.74	23.71 ± 0.19	25.64 ± 0.43	27.98 ± 0.95	29.16 ± 0.79	31.87 ± 1.47
G2	19.80 ± 0.62	22.28 ± 0.39	23.59 ± 0.97	24.77 ± 0.56	25.14 ± 0.79	27.81 ± 0.35	29.77 ± 0.72

^aMean ± SD, ^bn = 3, A₁–A₃, B₁–B₃ and C₁–C₃ are PEGylated SLMs containing 1.0, 2.0 and 3.0% w/w of gentamicin, respectively; G₁ and G₂ are commercial gentamicin injection and plain gentamicin, respectively.

Table 6. Susceptibility of Staphylococcus aureus to gentamicin in the PEGylated SLMs.

	IZD (mm)^a,b
	Time (min)
Batches	60	120	180	240	300	360	420
A1	22.98 ± 0.07	24.09 ± 0.02	26.00 ± 0.17	28.13 ± 0.70	30.25 ± 1.96	32.87 ± 1.04	33.72 ± 0.99
A2	23.00 ± 0.21	25.32 ± 0.45	27.19 ± 0.06	29.08 ± 0.14	31.28 ± 1.77	33.64 ± 2.05	35.00 ± 2.87
A3	24.13 ± 0.94	26.83 ± 0.17	28.08 ± 0.74	30.43 ± 1.25	32.93 ± 0.82	34.48 ± 1.90	37.30 ± 1.56
B1	23.63 ± 0.87	25.82 ± 0.59	28.53 ± 0.62	31.95 ± 0.89	34.27 ± 2.08	37.14 ± 3.00	38.63 ± 1.75
B2	24.46 ± 0.90	26.37 ± 0.67	29.19 ± 0.83	32.44 ± 2.00	35.94 ± 3.01	38.86 ± 1.98	39.46 ± 2.46
B3	25.03 ± 0.76	28.16 ± 0.45	30.62 ± 0.78	33.95 ± 0.83	38.32 ± 0.56	43.18 ± 2.14	45.53 ± 1.93
C1	23.03 ± 0.94	26.18 ± 1.02	29.75 ± 2.31	32.46 ± 1.98	36.72 ± 3.06	39.09 ± 2.84	42.87 ± 3.00
C2	24.59 ± 0.23	27.73 ± 0.94	30.10 ± 1.90	33.89 ± 2.09	37.64 ± 2.52	41.47 ± 1.88	44.59 ± 2.22
C3	25.40 ± 0.19	28.28 ± 0.08	31.98 ± 1.04	34.76 ± 1.22	39.99 ± 1.97	44.00 ± 2.55	49.40 ± 3.07
G1	22.18 ± 0.07	23.64 ± 0.71	24.99 ± 0.01	26.05 ± 0.33	28.19 ± 0.15	30.19 ± 1.07	32.47 ± 1.88
G2	22.00 ± 0.16	23.19 ± 0.05	24.07 ± 0.09	25.38 ± 0.66	26.77 ± 0.90	28.15 ± 0.03	30.98 ± 2.07

^aMean ± SD, ^bn = 3, A₁–A₃, B₁–B₃ and C₁–C₃are PEGylated SLMs containing 1.0, 2.0 and 3.0% w/w of gentamicin, respectively; G₁ and G₂ are commercial gentamicin injection and plain gentamicin, respectively.

In vivo bioavailability

The plasma concentration–time profiles of the drug are shown in Figure 8. The mean plasma concentrations after an oral administration of the optimized PEGylated SLMs (sub-batch C₃) increased at broader peaks than the plasma concentrations of the extemporaneous oral gentamicin solution (pure drug). The results indicate that the concentration of gentamicin in the blood increased rapidly and reached peak values within the first few minutes. There was a sharp fall in gentamicin concentration in the extemporaneous oral solution (pure drug) as compared to the optimized gentamicin-loaded SLMs (sub-batch C₃), which exhibited a slow decrease in plasma drug concentration. In other words, the high peak observed in pure sample was not sustained compared with the optimized gentamicin-loaded SLMs formulation. The mean C_max value for the gentamicin-loaded SLMs was 132 µg/ml compared with 119 µg/ml for the pure drug. The mean T_max values for optimized gentamicin-loaded SLMs and pure drug were not significantly different (p ≥ 0.05). The mean AUC value for the prepared PEGylated SLMs was significantly (p ≤ 0.005) greater than that for the oral solution. The AUCs were 678 µg/mL h and 1507 µg/mL h and the T_1/2 values were 4 h and 9 h for pure drug and gentamicin-loaded SLMs, respectively.

Figure 8. Changes of gentamicin concentration in blood over 24-h study period, of rats orally administered with the optimized gentamicin-loaded PEGylated solid lipid microparticles (C₃) and gentamicin pure sample (G₂) at a dose of 5 mgkg⁻¹ (n = 5).

Discussion

The DSC results of the lipid matrix formulated with P90G and dika wax showed that the lipid matrix gave lower enthalpies than the P90G. Expectedly, higher transition temperature would result due to the melting transition of the P90G, but the phospholipid being amphiphilic, was actually solubilized in the molten dika wax with lower melting point. As a result, two transitions should not be expected as the phospholipid was molecularly dispersed in the dika wax (Attama et al., 2006; Chime et al., 2012b). Reduction in enthalpy generally suggests less crystallinity of lipid matrices (Attama & Muller-Goymann, 2006; Umeyor et al., 2012c). Thus, the lipid matrix generated imperfect matrix (due to distortion of crystal arrangement of individual lipids after melting and solidification), which may have created numerous spaces for drug localization (Attama & Muller-Goymann, 2006; El-Kamel et al., 2007; Jaspart et al., 2007; Umeyor et al., 2012b). The varied fatty acid contents of these lipids may have interacted in such a manner, so as to partly disorder the crystal arrangement of the individual lipids. Low crystallinity of lipids is desirable in order to create more spaces for drug localization and enhance the encapsulation efficiency (EE%) of drugs and increase the loading capacity of the lipids. The DSC data of PEG 4000 confirms the purity of PEG 4000, and that its melting is instantaneous at a high heat capacity. The DSC was also used to detect evidence of good PEGylation and molecular bond interaction between lipid matrix and PEG 4000 for possible drug delivery (Momoh et al., 2013b). Results obtained from this study gave an indication that there was a possible bond formation between lipid matrix and PEG 4000 which resulted in the formation of PEGylated SLMs. The DSC result of gentamicin is in agreement with the melting point range of gentamicin (240–290 °C), and also confirms the purity of the drug. The DSC data of the SLMs showed that the melting peaks as well as the enthalpies depend on the PEG and drug contents of the formulations. The physico-chemical compatibility of the drug and the excipients studied by DSC suggested an absence of drug incompatibility, consistent with similar studies (Jaspart et al., 2005; Long et al., 2006; Eradel et al., 2009; Pilaniya et al., 2011). The results revealed the compatibility of gentamicin and the excipients as well as the stability of the drug in the lipid matrix. This is because the formulations gave lower melting peaks as well as enthalpies than gentamicin, implying that gentamicin exists in amorphous state in the formulations and is also properly solubilized in the matrix system (Umeyor et al., 2012c).

The results of the physicochemical tests on the SLMs showed that high drug loading resulted in large particle sizes, which could lead to retarded drug release from the formulations, consistent with earlier reports (Kenechukwu et al., 2011; Umeyor et al., 2012a,c). Time-resolved pH-dependent stability test was undertaken to assess the pH stability of the SLMs when stored at different time intervals. There was little degradation of the drug and/or the excipients employed in the study within this period of time, based on the time-resolved pH-dependent stability study. Furthermore, the lipid contents improved the EE% of gentamicin in the SLMs as could be seen from the EE% results (Table 3). The values of EE% and LC showed improved solubility of gentamicin in the lipid matrix. Incorporation of P90G in SLMs led to the formation of structured lipid matrix, which invariably enhanced gentamicin entrapment in the core of the SLMs. In addition, PEG 4000 being a hydrophilic surfactant improved the solubilization of the drug within the lipid core (Attama & Nkemnele, 2005; Momoh & Esimone, 2012; Momoh et al., 2013b).

The permeation study was undertaken since it is an important tool that predicts in advance how a drug would behave in vivo, especially for poorly permeable drugs such as gentamicin (Nnamani et al., 2013). The permeation data obtained were within the range obtained for some lipophilic and lipophobic drugs (Friedrich et al., 2000; Stephens et al., 2000; Friedrich et al., 2005; Chang et al., 2006; Attama et al., 2009; El-Gendy et al., 2009; Umeyor et al., 2012c; Nnamani et al., 2013). Since drug-loaded SLMs formulations containing highest amount of drug (i.e. 3% w/w gentamicin) especially batch C₃ gave the highest permeation flux and coefficient, it implies that sustained release gentamicin dosage form might be developed with this formulation, consistent with some drug-loaded SLMs formulations (Jaspart et al., 2005; Long et al., 2006; Eradel et al., 2009; Pilaniya et al., 2011). Generally, batches C₁–C₃ gave greater permeation fluxes and coefficients than corresponding batches B₁–B₃ and A₁–A₃ SLMs formulations. Comparing these with the fluxes and coefficients of pure sample of gentamicin (batch G₂) and gentamicin injection (batch G₁), the gentamicin-loaded SLMs formulations demonstrated enhanced permeation attesting further to improvement in the in vitro performance of the formulations. The drug permeation result presented here strongly indicates improved permeation of the drug through the artificial membrane of the Franz diffusion cell. By implication, PEG 4000 facilitated the release of gentamicin from the lipid matrix core and its subsequent transport across the membrane, more than the commercially available gentamicin injection and the unformulated drug (pure sample of gentamicin). This enhanced delivery of gentamicin would be of immense benefit in the treatment of microbial infections caused by gentamicin-susceptible organisms, consistent with earlier reports on gentamicin formulations (Singh et al., 2010; Kenechukwu et al., 2011; Momoh & Esimone, 2012; Umeyor et al., 2012a).

The microbiological test was performed using samples withdrawn from the in vitro studies to show an increasing IZD over time during the permeation study and to establish that gentamicin did not lose activity during formulation. Gentamicin-loaded SLMs produced very significant IZDs against the test bacteria. Gentamicin is active against S. aureus (Singh et al., 2010) and P. aeruginosa (El-Gendy et al., 2009). It was observed that the greater the amount of gentamicin loaded into the SLMs, the greater the IZD produced, in agreement with earlier reports (Kenechukwu et al., 2011; Momoh & Esimone, 2012). The formulations thus exhibited capacity limited bioactivity. Similarly, the antibacterial activity of the formulations was also time-dependent, manifested by an increasing IZD against the organisms with time. High IZDs recorded against the organisms within 60 min of the study especially with batches C₁–C₃ was an indication that these formulations would have exhibited the fastest release of the entrapped drug, hence the fast antibacterial activities; whereas time-dependent increases in IZDs within 420 min implies that the SLMs had potentials for sustained drug release. Moreover, all batches of the gentamicin-loaded SLMs gave greater IZDs than plain gentamicin and commercial gentamicin injection against the organisms. Overall, batch C₃ gave the greatest IZD against the organisms. This formulation would be a useful alternative for enhanced delivery of gentamicin in the treatment of infections caused by gentamicin-susceptible micro-organisms, thus encouraging further development of this formulation.

Results of the pharmacokinetic analysis (shown in Figure 8) indicate that the concentration of gentamicin in the blood increased rapidly and reached peak values in rats dosed with the formulations. The systemic bioavailability of gentamicin from the optimized formulation was 2.22-fold higher than that of oral solution of the drug. The AUC is an important parameter for measuring bioavailability of drug from dosage forms since it represents the total integrated area under the blood concentration time profile, which represents the amount of drug reaching the systemic circulation (Attama et al., 2011; Momoh et al., 2013b). The AUC of the PEGylated gentamicin SLMs was significantly (p ≤ 0.05) higher than those of the gentamicin oral solution. The C_max was also higher in the PEGylated SLMs than the oral solution, an indication that the PEGylated system could better be used to deliver the drug orally. The peak plasma concentration was reached 30 min after oral administration of the PEGylated gentamicin SLMs and declined slowly unlike in pure sample that showed fast decline in plasma drug concentration. The increased AUC and C_max for gentamicin administered as PEGylated SLMs compared with gentamicin oral solution may be as a result of lowering of systemic drug concentration by gastrointestinal degradation for gentamicin administered as a solution. Lipid particle formulations have been known to be absorbed through the lymphatics, which can avoid the gastrointestinal degradation and consequently, improve bioavailability consistent with reported works (Attama et al., 2011; Momoh et al., 2013b). Since gentamicin-loaded SLMs (sub-batch C₃) showed a slow decrease in plasma concentration when compared to pure drug (G₂), this is an indication that the carrier inhibited the clearance of gentamicin from systemic circulation. Furthermore, the visual behavioral assessment of the rats after dosing of the bland formulation and drug-loaded formulation (sub-batch C₃) to ascertain possible gastrointestinal side effect or other side effects did not reveal any difference between the two groups. This experiment was carried out after washout period of 10 days.

Conclusions

This study investigates gentamicin-loaded PEGylated SRMS-based SLMs prepared by melt-emulsification using PEG 4000, P90G and a heterolipid from I. gabonensis. Compared with commercial gentamicin injection, the bioactivity, pharmacokinetics and in vitro permeation studies undertaken with the formulations across artificial membrane using the Franz diffusion cell provided a basis to establish that PEGylated SRMS-based SLMs could better be used to control the release of gentamicin, enhance its permeation and potentially reduce its toxicity.

Acknowledgements

The authors wish to thank Phospholipid GmbH, Köln, Nattermann, Germany and JUHEL Pharmaceuticals Ltd., Awka, Anambra State, Nigeria, for the generous gift of Phospholipon® 90G and gentamicin, respectively.

Declaration of interest

The authors of this manuscript do not have a direct financial relation with the commercial identity mentioned in this manuscript that might lead to a conflict of interest. The authors do not have any conflict of interest in the preparation of this manuscript and they received no funding for this research work.