The effect of nanonization on poorly water soluble glibenclamide using a liquid anti-solvent precipitation technique: aqueous solubility, in vitro and in vivo study

Abstract

The aim of the present research was to improve the aqueous solubility and oral bioavailability of glibenclamide (GLB), a BCS class-II drug. A GLB nanosuspension (NS) was prepared using a liquid anti-solvent (LAS) precipitation technique and stabilized using HPMC K15M and lactose. Different in-process variables which directly affect the precipitated particle size have been thoroughly studied and optimized. The effect of a cryoprotective agent which could prevent agglomeration during lyophilisation was investigated. The optimal formulations of GD-H0.3d and GD-H0.4f exhibited a size range of 168.6 and 342.2 nm respectively and did not show any interaction when screened for incompatibility using FT-IR and DSC, but exhibited a decrease in crystallinity. The prepared GLB NPs exhibited superior aqueous solubility and dissolution when compared to pure GLB. The oral bioavailability of optimized formulations was found to exhibit 2.59, 1.67, 1.19, 2.50 and 2.40 folds of increment with respect to C_max, K_el (h⁻¹), t_1/2, AUC_{0–24 h} and AUC_0–∞ for GD-0.3d in contrast to pure GLB.

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1. Introduction

Nearly 70% of new potential drug molecules available in today’s market are classified under the BCS as class-II, posing difficulty in formulating oral dosage forms due to their poor aqueous solubility which ultimately affects drug bioavailability.^1,2 Nanonization is one of the techniques which have been deeply exploited over the past few decades with an aim to achieve successful targeting as well as improving the overall drug bioavailability. Despite numerous attempts to overcome this obstacle, the design of oral dosage forms with a desirable oral bioavailability remains a day to day challenge for researchers.³

Glibenclamide (GLB), 5-chloro-N-(4-[N-(cyclohexylcarbamoyl)sulfamoyl]phenethyl)-2-methoxybenzamide, also known as glyburide, is an anti-diabetic drug belonging to a class of sulphonylureas used for the management/control of type-II diabetes. It activates the β-cells of the pancreas and stimulates insulin release. However being a BCS class-II drug, the aqueous solubility is the rate limiting step which consequently hinders its oral absorption and its bioavailability.^4–6

Nanonization aims at increasing the surface area to volume ratio of an individual particle via particle size reduction which subsequently improves the solubility as well as the dissolution of poorly water-soluble molecules.⁷ Strategies to nanonize GLB and to improve its aqueous solubility and bioavailability have been previously reported by Kumar et al., 2014,⁸ Guan et al., 2014 (ref. 9) and Salazar et al., 2013 (ref. 10) using a precipitation technique, supercritical fluid technology and combinative particle size reduction H 42 technologies. However, these technologies involve complicated procedures and inconvenient processes. Alternatively, the Liquid Anti-Solvent (LAS) precipitation technique is a simple bottom up process for control over particle properties such as size and morphology as well as crystallinity.¹¹ Pointing out its advantages, the LAS technique is a more convenient process under ambient conditions without the need for any specialized equipment and is easily scalable.¹²

The underlying principle behind the LAS technique or any bottom-up technique involves crystallization or precipitation or solvent evaporation. The particles subjected to the LAS technique undergo supersaturation, nucleation and growth during the recrystallization process before precipitating out as nano- or micro-particles. However, the strict optimization of process parameters is a prerequisite for preparing optimized nanocrystals and to avoid undesirable agglomeration and uncontrolled crystal growth from the solvent system comprising of the drug.^13,14 Thorat and Dalvi, 2012 (ref. 12) have highlighted the use of the LAS technique for recrystallizing poorly water soluble drugs with a controlled outcome of the particle size, size distribution and the stabilization of ultrafine particles. Shah et al., 2013 (ref. 15) previously prepared optimized GLB NPs using the LAS technique in order to improve their dissolution characteristics, and optimized the various significant parameters that could affect the response variables by using a Plackett–Burman screening design.

The aim of this research was to optimize and study the process parameters of the LAS technique that directly affect the physico-chemical properties of precipitated GLB NPs and further evaluate the aqueous solubility and oral bioavailability behaviour.

2. Material and methods

2.1 Material

GLB was provided as a gift sample from Wockhardt Ltd, Aurangabad, India. Hydroxypropyl methylcellulose K15M (HPMC K15M), mannitol and lactose monohydrate were purchased from Loba Chemie, Mumbai. All chemicals and buffers used were of analytical grade.

2.2 Preformulation studies

2.2.1 Selection of solvent. In order to select an appropriate solvent for the preparation of GLB NPs using the LAS technique, various solvents and buffers were evaluated for their ability to solubilise GLB. An excess amount of GLB was dissolved in 10 ml of the selected organic solvent like acetone, methanol, ethanol and dimethyl sulfoxide (DMSO) as well as a pH 1.2 HCl buffer and pH 6.8 and 7.2 phosphate buffers, under ambient temperature (25–30 °C). The amount of drug solubilised was quantified at 238 nm using the UV spectroscopy method (UV1800, Shimadzu).

2.2.2 Study and selection of the optimal solvent to anti-solvent ratio and drug concentration. In order to select and evaluate the effect of the solvent to anti-solvent ratio (ml/ml) on the particle size of the GLB nanoparticles (NPs), different ratios of solvent containing a 50 mg ml⁻¹ equivalent concentration of GLB were added drop-wise to the anti-solvent (distilled water) under a constant homogenization speed (10 000 rpm) using a Polytron PT 1600E, Switzerland. The solvent to anti-solvent ratio used for the study was 5 : 5 to 1 : 9 ml/ml with an increment of 0.5 ml. The process time was kept constant at 5 min. The particle size of the GLB suspension was determined by photon correlation spectroscopy (PCS) using a Zeta sizer Nano ZS, Malvern Instruments, Malvern, UK. The optimal ratio that precipitated the minimal particle size was fixed and further considered for optimizing the drug concentration for the preparation of GLB NPs.

Similarly, to select and study the effect of drug concentration on the precipitated particle size of GLB, a pre-fixed ratio of solvent to anti-solvent containing different amounts (10 to 60 mg ml⁻¹) of GLB was used. The method of preparation and evaluation of the particle size of the GLB suspension was the same as mentioned above. The entire study was performed under ambient conditions. Details of the solvent : anti-solvent ratio (ml/ml) and drug concentration used for the study are further enclosed in the ESI.†

2.3 Preparation of GLB nanosuspension by LAS technique

GLB nanosuspensions (NSs) were prepared using the LAS precipitation technique. From the previous study, the optimal solvent : anti-solvent ratio and drug concentration that produced the minimal precipitated particle size were kept constant and to further understand the effect of stabilizer concentration on the precipitated particle size, HPMC K15M and lactose, in different concentrations, were selected for the stabilization of the GLB NSs. A prefixed amount of solvent containing GLB was added drop-wise to the anti-solvent containing HPMC K15M/lactose (0.1–0.6% w/v with an increment of 0.1% w/v) and homogenized at 10 000 rpm for 5 min under ambient temperature. The formed NS was centrifuged and re-suspended into fresh distilled water. This process was repeated twice before subjecting the prepared final NS to lyophilisation to obtain the GLB NPs. The GLB NS was re-suspended and evaluated for particle size. The formulation codes of the different prepared NS batches were coded as GD-H0.1to GD-H0.6 and GD-L0.1 to GD-L0.6, representing formulations stabilized using HPMC K15M and lactose respectively (details of the formulation codes are further enclosed in the ESI†).

Batches which reproduced the lowest particle size from each stabilizer used were subjected to lyophilisation studies. Mannitol in different concentrations (0–5% w/v, with an increment of 1% w/v) was used as a cryoprotective agent. The NSs were added to rubber stoppered vials and frozen using a deep freezer (Remi PVD-185 D, India) at −40 °C for 24 h, followed by freeze drying using a freeze dryer (Ilshin Laboratory Co. Ltd, Korea). The percentage yield of the freeze-dried GLB NPs was calculated gravimetrically (the formulation details are presented in the ESI†).

2.4 Re-dispersibility test

Fifty milligrams of prepared freeze-dried GLB NPs was dispersed in 1.5 ml of distilled water and vortexed for 30 s under ambient conditions. The samples were immediately evaluated for the particle size as mentioned in the previous section,^16,17 and also visually examined and categorized under the following three grade systems: (Grade A) rapidly formed NS, having a clear appearance, (Grade B) rapidly formed NS, but slightly turbid and (Grade C) particles tend to aggregate and fail to redisperse immediately.

2.5 Physico-chemical characterization of GLB NPs

The physico-chemical characterization of the prepared freeze-dried GLB NPs and pure GLB was achieved using Fourier Transform Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), X-ray Diffraction (XRD) and the surface morphology was studied.

FT-IR spectral analysis was done by employing the KBr pellet press method and analysing the samples using an FT-IR spectrometer, Shimadzu, Model 8033. DSC was carried out by crimping the samples in aluminium pans and analysing them using a DSC Dupont9900. XRD analysis was performed using a Rigaku diffractometer coupled with copper as the anode material and a graphite monochromator and operated at 15 mA, 30 kV voltage. A scanning electron microscope (SEM) (Joel-LV-5600, USA) was used for surface morphology determination.

2.6 Evaluation of prepared GLB NPs

2.6.1 Drug content. One hundred milligrams of the GLB NPs was dissolved in acetone and diluted appropriately, followed by measurement of the absorbance at 238 nm using the UV spectroscopy method.

2.6.2 Aqueous solubility of prepared GLB NPs. An excess amount of the prepared GLB NPs and pure GLB were taken in rubber capped glass vials containing 5 ml of distilled water and shaken using a mechanical stirrer for 24 h at ambient temperature (25–30 °C).¹⁸ The resulting solution was filtered using Whatmann filter paper grade 1 and analyzed using a UV spectrometer at 238 nm.

2.6.3 In vitro drug release. Drug release studies of 10 mg equivalent weight of the GLB NPs and pure GLB were performed in 900 ml of pH 6.8 phosphate buffer with 1% w/v SLS using a dissolution USP XXIII attached with a paddle (Electrolab, Mumbai, India) at 75 rpm and 37 ± 0.5 °C.¹⁰ Aliquots were withdrawn at pre-determined intervals and quantified using the HPLC method. Results obtained were statistically analyzed to determine their significance using the paired t-test.

2.6.4 In vivo studies. All animals for this study were acquired after protocol approval through the Institutional Animal Ethics committee, JSS University, Mysore, India (Approval no. 106/2012). The animals authorized for carrying out the study were cared for under the supervision of the pharmacology department, JSS College of Pharmacy, Mysore, India in compliance with the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines. Twelve albino Wistar rats of either sex weighing 230–260 g were divided into two groups and monitored for one week under quarantine conditions prior to the study. The animals were fed a standard chow diet ad libitum and had free access to water.

Initially, the rats were anaesthetized through intraperitoneal injection of urethane (1 g kg⁻¹) and the jugular vein was cannulated to facilitate the collection of blood samples. The two pre-divided groups with six in each, were labelled as test and control. Oral administration of the optimized GLB NPs and pure drug (fixed dose of 5 mg kg⁻¹ in water) to the test and control group was facilitated by a stomach sonde needle respectively. 0.3 ml blood samples were withdrawn, pre- and post-administration of the GLB NPs, from the jugular vein at pre-determined time intervals. Samples were collected in heparinised tubes, followed by centrifugation for 5 min at 10 000 rpm at 4 °C. The plasma was separated and stored at −50 °C for further analysis.

Plasma drug concentration was determined using a validated HPLC method. The drug was extracted from the plasma using 2 ml of methanol and vortexed for 5 min at 10 000 rpm. The supernatant was collected and was injected into a C₁₈ column of HPLC using methanol : water in 80 : 20 v/v as the mobile phase. The pH was adjusted to 3.4 with orthophosphoric acid. The samples were injected into the column at a flow rate of 1 ml min⁻¹ and quantified at 238 nm. Pharmacokinetic parameters such as maximum plasma concentration (C_max), time to reach maximum plasma concentration (T_max), half life (t_1/2), absorption rate constant (K_a) and area under the curve (AUC_0–t) were calculated from the plasma-concentration time profile.

3. Results and discussion

3.1 Development of GLB NPs

GLB NPs were developed using the LAS technique with an aim to improve aqueous solubility and oral bioavailability of GLB. The LAS technique is an attractive bottom up approach, for developing NPs/microparticles (MPs), that can be carried out under ambient temperature conditions. The underlying principal for generating NPs/MPs is precipitation. Steps involve the mixing of solution (drug dissolved in a suitable solvent system) and anti-solvent, generation of supersaturation, nucleation and growth by coagulation and condensation, followed by agglomeration in the case of uncontrolled crystal growth.¹² This further highlights the importance of selecting a suitable solvent system for rapid and high supersaturation as it has a direct effect on the outcome such as size and morphology as well as purity. In order to bypass uncontrolled coagulation and non-uniformity of the NPs, suitable polymers/surfactants are incorporated for preparing stabilized NPs. Therefore, optimizing the different process parameters is a prerequisite prior to the final preparation of the NPs.

3.2 Solubility studies

In order to recognise a suitable solvent/solvent system that can effectively solubilise GLB, different organic solvents and buffers were selected. The results obtained are depicted in Table 1. From the results, DMSO and acetone were found to be suitable candidates for solubilising (25 g l⁻¹ and 7.3 g l⁻¹ respectively) GLB.

Table 1 Solubility data of GLB

Solvent system	Solubility^a (mg l^−1)
a n = 3.
pH 1.2 HCl buffer	5.2 ± 0.043
pH 6.8 phosphate buffer	18.4 ± 0.332
pH 7.2 phosphate buffer	23.5 ± 0.765
Acetone	7300.94 ± 0.798
Methanol	389.4 ± 0.231
Ethanol	4800.07 ± 0.11
DMSO	25 100.43 ± 0.49

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Beck et al., 2010 (ref. 19) concluded that a higher polarity solvent results in increased particle size and vice versa. Considering this theory, DMSO exhibits superior solvent polarity when compared to acetone (7.2 against 5.1 respectively), making the latter a preferable candidate as a solvent system for solubilising GLB, and deionised water (DI) was taken as an anti-solvent.

3.3 Study on the influence of various in process parameters on precipitated particle size

3.3.1 Influence of solvent : anti-solvent. Different ratios of solvent : anti-solvent (ml/ml) were screened thoroughly to identify the appropriate ratio and to understand its influence on the precipitated particle size. The results obtained are depicted in Fig. 1. Pure GLB was found to be 14.3 ± 0.8 μm in size. During this study, it was observed that by decreasing the solvent to anti-solvent ratio from 5 : 5 to 3.5 : 6.5 ml/ml, the GLB mean particle size decreased subsequently from 4931.1 nm to 2815.5 nm (with a 1.751 fold decrease in particle size). However beyond 3.5 : 6.5 to 1 : 9 ml/ml, an increase in the drug mean particle size from 2815.5 nm to 5192.3 nm (with a 1.844 fold increase in particle size) was observed. Even though the particle size is in the micrometer range, a 5 fold decrease in the particle size was observed at the 3.5 : 6.5 ml/ml solvent : anti-solvent ratio when compared to the particle size of the pure drug. In contrast to other prepared ratios, the 3.5 : 6.5 ml/ml ratio offered a rapid reduction in drug concentration which led to a rapid drug precipitation upon addition of the ​solvent to the ​anti-solvent that ultimately resulted in the smallest particle size.^20–22

Fig. 1 Effect of solvent : anti-solvent ratio on particle size.

Therefore based on the results, 3.5 : 6.5 ml/ml was selected and fixed as the optimal ratio for further studies. This study highlights the influence of the solvent/anti-solvent ratio on the resulting particle size of the precipitated drug.

3.3.2 Influence of drug concentration. The effect of drug concentration on the precipitated particle size was studied by varying the drug concentrations from 10–60 mg ml⁻¹ with an increment of 10 mg ml⁻¹ and keeping the above 3.5 : 6.5 ml/ml selected solvent : anti-solvent ratio constant. The results are shown in Fig. 2. Initially, a decrease in particle size from 2815.2 nm to 2276.4 nm was observed as the drug concentration increased from 10–40 mg ml⁻¹, beyond which further increase in concentration from 40–60 mg ml⁻¹ consequently increased the drug particle size from 2276.4 nm to 3089.3 nm. The effect of drug concentration has previously been investigated and reported by Park and Yeo, 2010 (ref. 23) for roxithromycin (ROX) and further reviewed by Abhijit and Sanjaykumar, 2013.²⁰ According to the findings, drug concentration and the precipitated drug particle size are inversely proportional to one another. A decrease in the precipitated drug particle size is observed with a subsequent increase in drug concentration. This further relates the dependency of the nucleation rate on drug concentration in the prepared drug solution. Furthermore, the degree of supersaturation alters the nucleation rate, and it directly relates to the concentration of the drug in the solution. By extrapolating the findings on ROX by Park and Yeo, 2010,²³ the increase in the particle size observed beyond 40 mg ml⁻¹ of GLB in the drug solution might be due to the agglomeration of particles together during the process of precipitation, resulting in poor distribution of both size as well as shape. The observed phenomenon can be a consequence of the formation of several nuclei at the interface with respect to the influence on viscosity by drug concentration. Furthermore, the increased nuclei formation hinders diffusion from the solvent to anti-solvent which ultimately leads to particle aggregation.^24–26 The solvent : anti-solvent ratio (3.5 : 6.5 ml/ml) and drug concentration (40 mg ml⁻¹) were considered as optimized parameters and kept constant for the further course of the study.

Fig. 2 Effect of drug concentration on particle size.

3.3.3 Influence of stabilizers. The theory of stabilizing a NS simultaneously with precipitation during the LAS technique has been outlined by Thorat and Dalvi, 2012.¹² According to their report, the adsorption strength of a stabilizer onto the drug surface depends mainly on two factors i.e., (a) the amount of stabilizer adsorbed onto the surface is inversely proportional to its solubility in the liquid phase and (b) the stabilizer–particle interaction strength over stabilizer–solution interaction strength. HPMC K15M and lactose were screened for their ability to stabilize the GLB NS. Neutral polymers stabilize the system via hydrogen-bonding interactions with that of the drug particle surface and by reduction in the solid–liquid interfacial tension.^21,27–31 A thorough literature survey suggests that the selected stabilizers are neutral in nature.³²

The results obtained for the GLB NS prepared by individually employing HPMC K15M and lactose are presented in Fig. 3. From the results, the formulations GD-H0.3 and GD-L0.4 containing 0.3% w/v of HPMC K15M and 0.4% w/v of lactose, exhibited the smallest particle size of 136.1 ± 17.9 nm and 352.9 ± 20.9 nm respectively. At 0.3% w/v of HPMC K15M and 0.4% w/v of lactose, the interaction of the drug–polymer is superior when compared to the polymer–solvent affinity, subsequently resulting in the decreased precipitated particle size. The effect of the particle size in relation to the stabilizer can be explained by understanding the theory of the interaction parameter of the solvent–polymer–drug. As per the theory, the favourable interaction of drug molecules with the surroundings subsequently causes a quicker diffusion of stabilizer molecules towards the growing particle surface, which further inhibits the addition of drug molecules and controls the precipitated particle size.¹²

Fig. 3 Effect of stabilizer concentration on particle size.

From the results, the formulations GD-H0.3 and GD-L0.4 were considered as optimized formulations depending on their particle size and were subjected to lyophilization to develop GLB NPs.

3.4 Lyophilization of GLB NS and redispersibility test of obtained GLB NPs

During the course of lyophilisation, the precipitated particles tend to fuse with each other resulting in particle agglomeration due to the stress of freezing and dehydration encountered during the process. To prevent this, mannitol in different concentrations ranging from 0–5% w/v, with an increment of 1% w/v, was used as a cryoprotective agent to impart protection during freezing and drying stresses. In order to ensure immediate redispersibility of the freeze-dried NPs, 100 mg of the freeze-dried product was manually agitated for about 30 s and evaluated for the particle size. The results obtained are reproduced in Fig. 4. From the findings, it is evident that for GD-H0.3 trials, a minimal concentration of 3–5% w/v of cryoprotective agent is required for ensuring complete redispersion, whereas for GD-L0.4 trials, a minimal concentration of 5% w/v is required to achieve the same. The results of the redispersibility test findings are given in Table 2. Below the above-mentioned concentrations and in the case of cryoprotective absentia, the particles tend to aggregate and fail to redisperse upon gentle agitation. The above-mentioned concentrations of mannitol ensure redispersion through vitrification at a glass transition temperature (T_g) by the immobilization of the NPs within its glass matrix and prevent its aggregation to give protection against the mechanical stress of ice crystals.³³

Fig. 4 Effect of cryoprotective concentration on prepared GD-H0.3 and GD-H0.4 particle size.

Table 2 Redispersibility test results

Formulation code	Grade^a
a n = 3.
GD-H0.3a	Grade C
GD-H0.3b	Grade C
GD-H0.3c	Grade C
GD-H0.3d	Grade A
GD-H0.3e	Grade A
GD-H0.3f	Grade A
GD-L0.4a	Grade C
GD-L0.4b	Grade C
GD-L0.4c	Grade C
GD-L0.4d	Grade B
GD-L0.4e	Grade A
GD-L0.4f	Grade A

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To conclude, GD-H0.3d and GD-L0.4f GLB NPs prepared using the 3.5 : 6.5 (ml/ml) solvent/anti-solvent ratio, comprising 40 mg ml⁻¹ of GLB and 0.3% w/v of HPMC K15M and 0.4% w/v of lactose respectively, followed by lyophilisation using 0.3% w/v and 0.5% w/v of mannitol as a cryoprotectant were considered as optimized freeze-dried products with an average particle size of 136.1 ± 17.9 nm for GD-H0.3d and 342.2 ± 26.9 nm for GD-L0.4f. Even though the formulations GD-H0.3e and GD-H0.3f produced a desirable particle size upon redispersion, the redispersed particle size was larger than GD-H0.3d and therefore they were not used for further study. The percentage yield was found to be 71.8 ± 1.8% and 73.4 ± 2.3% for GD-H0.3d and GD-L0.4f after final gravimetric analysis with a drug content of 95.1 ± 0.8% and 92.6 ± 1.2% respectively.

3.5 Physico-chemical characterization of GLB NPs

3.5.1 FT-IR, DSC and XRD. In order to determine the compatibility, the drug, NSs (GD-H0.3 and GD-L0.4) and the formulations GD-H0.3d and GD-L0.4f were screened for any possible interactions using a FT-IR spectrometer and thermography investigations. The spectral peaks and DSC thermograms are shown and compared in Fig. 5a and b and 6 respectively. GLB displays characteristic peaks at 3119.00 cm⁻¹, 2930.15 cm⁻¹, 2854.56 cm⁻¹, 1525.40 cm⁻¹ and 1157.31 cm⁻¹ corresponding to N–H stretching, aliphatic C–H stretching, O–H stretching, N=O stretching and C–N stretching respectively. Similar observations with or without any modification in the characteristic peaks of GLB were seen in the NSs (GD-H0.3 and GD-L0.4) and the formulations GD-H0.3d and GD-L0.4f. The thermogram of GLB exhibits a sharp endothermic peak at 175.64 °C demonstrating its crystallinity. On the contrary, similar identical sharp peaks of GLB were found in GD-H0.3d and GD-L0.4f, reflecting the existence of a crystalline state. Hence FT-IR and DSC confirm the absence of any possible incompatibility.

Fig. 5 (a) FTIR spectra of (A) pure GLB, (B) GD-H0.3 and (C) GD-L0.4, (b) FTIR spectra of (A) pure GLB, (B) GD-H0.3d and (C) GD-L0.4f.

Fig. 6 DSC thermograms of GLB, GD-H0.3d and GD-L0.4f.

The XRD data of GLB and the formulations are collated in Fig. 7. The characteristic sharp and intense peaks of GLB are found at 2θ angles of 10.84, 11.70, 18.92, 20.96, 23.06 and 27.26 degrees, indicating its crystalline nature. However, the formulations GD-H0.3d and GD-L0.4f exhibit peaks at similar positions to GLB but with a decreased intensity which represents the lower crystallinity/amorphous nature of the GLB NPs. During the preparation of the GLB NPs through the LAS technique, rapid crystallization retards the formation of compact crystalline structures, resulting in the formation of NPs with amorphous or decreased crystallinity.³¹

Fig. 7 XRD diffractograms of: (A) pure GLB, (B) GD-H0.3d and (C) GD-L0.4f.

3.5.2 Scanning electron microscopy. The surface morphology of the GLB NPs (Fig 8) reflect plate and rod shaped crystals with a smooth surface and narrow particle size distribution ranging from 100–200 nm for GD-H0.3d and 300–500 nm for GD-L0.4f, correlating with their particle size as observed using a Malvern zeta-sizer.

Fig. 8 SEM of glibenclamide nanoparticles: (A) formulation GD-H0.3d; (B) formulation GD-L0.4f.

3.6 Evaluation of GLB NPs

3.6.1 Solubility. GLB in their pure form solubilised 24.56 mg l⁻¹ in distilled water whereas GD-H0.3d and GD-L0.4f exhibited superior aqueous solubilities of 157.77 mg l⁻¹ and 139.03 mg l⁻¹, thereby achieving a 6.4 and 5.6 fold increase in aqueous solubility respectively. This increase in solubility can be related to its amorphous or decreased crystallinity.³⁴

3.6.2 Dissolution studies. The dissolution studies of pure GLB and the prepared GLB NPs (GD-H0.3d and GD-L0.4f) were carried out in pH 6.8 phosphate buffer with 1% w/v of SLS, and an in vitro profile for the same study is compared and presented in Fig. 9. Inside the initial 5 min, the prepared GD-H0.3d and GD-L0.4f exhibited a burst release of 68.5 ± 1.82% and 42.4 ± 1.68% of GLB NPs, whereas for pure GLB, the release was about 3.7 ± 0.36%. Within 20 min, GD-H0.3d managed to release 94.2 ± 1.67% of GLB whereas, it took 35 min for GD-L0.4f to release 91.8 ± 2.42% of GLB. Meanwhile, at the end of 120 min, only 32.25 ± 1.19% of drug release was observed from pure GLB. About a 2.96 and 2.95 fold increase in the dissolution rate for GD-H0.3d and GD-L0.4f was distinguished at the end 120 min when compared to pure GLB. The increased dissolution rates for the prepared GLB NP formulations can be attributed to the transformation of the physical state to a highly disordered amorphous nature.^35,36 SLS is added as a surfactant to the dissolution media to facilitate dissolution as well as drug release of the poorly water soluble drugs.¹⁰.

Fig. 9 In vitro % drug release profile of GLB, GD-H0.3d and GD-L0.4f.

In order to understand the influence of the nanonization of pure GLB on in vitro drug release, the release profiles of pure GLB vs. GD-H0.3d, GD-L0.4f and GD-H0.3d vs. GD-L0.4f were compared by processing the obtained data with a paired t-test. The results indicated a statistically significant (P < 0.005) difference between pure GLB and GD-H0.3d and GD-L0.4f. However, not much significant (P > 0.005) difference was observed within the prepared GD-H0.3d and GD-L0.4f formulations. From the above results, GD-H0.3d was considered as a superior formulation compared to its counter-part GD-L0.4f due to its rapid dissolution rate. Hence, formulation GD-L0.4f was not used for the oral pharmacokinetics study.

3.7 Oral pharmacokinetics study

The plasma concentration–time profile and pharmacokinetic parameters for GD-H0.3d and pure GLB are summarized in Fig. 10 and Table 3. The study reflects that GD-H0.3d shows significantly higher C_max, K_el (h⁻¹), t_1/2, AUC_{0–24 h} and AUC_0–∞, without altering T_max, when compared to the pure drug. The results indicate that particle size reduction has improved the rate and extent of GLB absorption from the GI tract. However, size reduction had no profound impact on T_max. A 2.59, 1.67, 1.19, 2.50 and 2.40 fold iincrease with respect to C_max, K_el (h⁻¹), t_1/2, AUC_{0–24 h} and AUC_0–∞ for GD-H0.3d was found when compared to its counter-part. The influence of nano-sizing a drug to improve its oral bioavailability has been previously discussed by Li et al., 2009 (ref. 37) on rats with an amorphous fenofibrate (FF) NS. The findings observed indicated that nano-sizing of FF significantly enhanced oral bioavailability based on the particle size. These findings can be extrapolated to our present research.

Fig. 10 Plasma concentration–time profile for GLB and GD-H0.3d.

Table 3 Pharmacokinetic parameters after oral administration of GLB and GD-H0.3d to rats

Parameters^a	Cmax (ng ml^−1)	Tmax (h)	t1/2 (h)	Kel (h^−1)	AUC0→24 h (ng h ml^−1)	AUC0→∞ (ng h ml^−1)
a AUC0→∞ is the area under the curve from time 0 extrapolated to infinite time.
GLB	9428.42 ± 897.8	4.0 ± 0.0	5.73 ± 0.42	0.1207 ± 0.0061	65 106.41 ± 60.3	71 680.24 ± 63.6
GD-H0.3d	24 451.14 ± 2170.5	4.0 ± 0.0	9.57 ± 0.23	0.1438 ± 0.0042	162 945.12 ± 241.5	172 383.64 ± 237.2

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4. Conclusion

In the present study, efforts were made to optimize the process parameters of the LAS technique for developing GLB NPs, in order to enhance the aqueous solubility as well as oral bioavailability. Initially, different process parameters such as the selection of solvent, ratio of solvent to anti-solvent, effect of drug, stabilizer and cryoprotectant concentration, which can directly affect the precipitated drug particle size were optimized and thoroughly discussed. The optimized GLB NPs did not show any incompatibility and were found to be readily dispersible upon gentle agitation. In addition, among the optimized GLB NPs, formulation GD-H0.3d achieved a 6.4 fold increase in aqueous solubility, rapid dissolution rate and 2.59, 1.67, 1.19, 2.50 and 2.40 fold increase with respect to C_max, K_el (h⁻¹), t_1/2, AUC_{0–24 h} and AUC_0–∞ when compared to pure GLB. The results obtained summarize a platform to develop GLB NPs and its class of drugs for enhancing their oral absorption. Additionally, this work provides an in depth understanding of several factors that can influence nanoprecipitation and subsequently its effect on physico-chemical properties, aqueous solubility and oral pharmacokinetics.

Acknowledgements

The authors would like to thank Wockhardt Ltd for providing us with the drug sample. Also we would like to acknowledge the J.S.S. College of Pharmacy, Mysuru and J.S.S. University for providing us with financial assistance to carry out the research.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra12678a

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