Journal of Pharmaceutical Sciences xxx (2016) 1-9

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Journal of Pharmaceutical Sciences

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Pharmaceutical Nanotechnology

Glibenclamide Nanocrystals in a Biodegradable Chitosan Patch for

Transdermal Delivery: Engineering, Formulation, and Evaluation
Hany S.M. Ali 1, 2, *, Ahmed F. Hanafy 1, 3
1
Department of Pharmaceutics and Pharmaceutical Technology, College of Pharmacy, Taibah University, Al-Madinah Al-Munawwarah, Saudi Arabia
2
Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut, Egypt
3
Research and Development Department, European Egyptian Pharmaceutical Industries, Alexandria, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Glibenclamide (GBD) nanocrystals (D50 ¼ 429 nm) were engineered by applying combined precipitation
Received 29 July 2016 and homogenization procedures. GBD crystallinity was maintained during the nanonization process as
Revised 28 September 2016 revealed by differential scanning calorimetry and X-ray analyses. Nanonized and micronized GBD were
Accepted 13 October 2016
incorporated into chitosan solutions to fabricate transdermal delivery systems (TDDSs), nano- and micro-
GBD, respectively. The fabricated TDDSs displayed satisfactory physicochemical characteristics without
substantial aggregation of GBD nanocrystals during the casting and drying procedures. Within 24 hours,
Keywords:
nanoparticles about 85 ± 3.1% of the GBD content was released from nano-GBD, compared to 61 ± 3.9% from micro-
transdermal delivery systems GBD. Cumulative permeation of GBD from nano-GBD after 24 hours was 498 ± 33.35 compared to
chitosan 362 ± 25.25 mg/cm2 from micro-GBD. The calculated flux across rat skin for nano-GBD was 23.14
pharmacodynamics
compared to 13.64 mg/cm2/h for micro-GBD, with an enhancement factor of 1.7. In vivo assessment clearly
permeability
revealed the enhanced efficacy of nano-GBD to reduce blood glucose levels and counteract the induced
hyperglycemia in tested animals compared to micro-GBD (p < 0.5). Simultaneously, the nano-GBD was
able to maintain higher drug concentration for longer time (24 hours, p < 0.5) and minimize intense
action and hypoglycemia associated with GBD oral therapy (p < 0.5).
© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Introduction hypoglycemic agent given to manage noneinsulin-dependent

(type II) diabetes mellitus.4 As the Biopharmaceutics Classification
Particle size diminution to the nanostate is a well-established System, GBD is a class II drug (i.e., poorly soluble with highly
approach to enhance drug absorption, particularly when bioavail- permeability). Accordingly, size diminution could be beneficial to
ability is dissolution rate limited.1 Basically, nanosized drug parti- improve bioavailability of the drug.5
cles can be generated by top-down or bottom-up procedures. The Transdermal drug delivery is applied to topical administration of
top-down nanonization comprises breakup of drug macro- pharmaceutically active ingredients to the healthy skin mostly for
particles as in wet milling and high-pressure homogenization.2 systemic therapy. Transdermal drug delivery systems (TDDSs) have
Alternatively, nanonization via bottom-up techniques involves gained attention of pharmaceutical researchers for decades when
building up of nanoparticles from molecules in drug solution transdermal patches were popular in systemic drug delivery.6
through precipitation by admixing with miscible nonsolvents. A TDDSs represent a noninvasive mean to avoid exposure to the
distinctive drawback of the bottom-up nanonization procedures is first-pass metabolism and can sustain plasma levels within the
the application of organic solvents. Residues of organic solvents in therapeutic window over extended periods.7 Transdermal patches
the generated nanosuspensions represent a major difficulty espe- are usually well accepted and easy to apply with the possibility of
cially in large-scale manufacturing. Despite the high energy an ease and immediate cessation of drug administration by patch
consumption and costs, top-down processes are generally removal. TDDSs also offer an appreciated alternative when oral
preferred in industry up till now.3 Glibenclamide (GBD) is an oral administration is not feasible or may result in erratic bioavail-
ability.8 Achieving these aims would lower variability in drug
responses and considerably improve patient compliance.9,10 How-
* Correspondence to: Hany S.M. Ali (Telephone: þ966-502864018; Fax: þ966-4-
ever, transdermal rout remains a challenge for drug delivery
8475027).
E-mail addresses: [email protected], [email protected] (H.S.M. Ali), through the impermeable epithelium of the skin. Chitosan, a pol-
[email protected], [email protected] (A.F. Hanafy). ycationic polysaccharide, has been extensively used in transdermal

http://dx.doi.org/10.1016/j.xphs.2016.10.010
0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
2 H.S.M. Ali, A.F. Hanafy / Journal of Pharmaceutical Sciences xxx (2016) 1-9

formulations because of its favorable properties as biodegrad- dispersion media by centrifugation, washing with distilled water
ability, bioadhesion, permeability-enhancing ability, and inter- and recentrifugation. The residues were gathered and dried gently
esting physicochemical characters.11,12 using a heated vacuum desiccator at 35 C and 25 inch Hg.13 DSC
Engineering of nanosized GBD through precipitation- thermograms of unprocessed, precipitated, and homogenized GBD
homogenization processes was proposed in the present study. were recorded. For each measurement, a sample of 5-mg weight
Aim was extended to preserve the size characteristics of the was introduced in a sealed aluminum pan and scanned in the
nanocrystals through fabrication of chitosan transdermal systems. temperature range 30 C-300 C. A heating rate of 10 C/min was
In vitro and in vivo characteristics of the developed formulation used, and the thermal analysis was performed under dynamic ni-
were evaluated compared to the micronized drug formulation. trogen atmosphere.

Methodology Powder X-Ray Diffractometry

X-ray powder diffraction patterns of unprocessed, precipitated,
Materials and homogenized GBD were generated using a Shimadzu XRD
6000 diffractometer (Shimadzu Corporation, Kyoto, Japan).
GBD was kindly obtained from the European Egyptian Diffraction patterns were recorded over an angular range of 10 -60
Pharmaceuticals Company (Pharco Corporation; Alexandria, Egypt). 2q using a graphite monochromator and a copper radiation source
Poloxamer 188 was obtained from Spectrum Chemicals (New (l ¼ 1.5418 Å) with a scanning speed of 0.04 /min.
Brunswick, NJ). Chitosan medium molecular weight was obtained
from Sigma-Aldrich (Darmstadt, Germany). Lactic acid and dimethyl Formulation of GBD-Loaded TDDSs
sulfoxide (DMSO) were obtained from Fisher Scientific (Tokyo, Japan).
Other materials were of pharmaceutical grades and used as received. Chitosan solutions, 2% w/w, were prepared in dilute lactic acid
solution (2% w/w). Viscosity of chitosan solution was measured by
GBD Nanonization the Cannon LV 2020 Rotary Viscometers (Cannon Instrument
Company, State College, PA). Calculated volumes (25 mL) of the
GBD nanosized suspension was generated through combined freshly formed GBD nanosuspension (5 mg/mL) and the unpro-
bottom-up and top-down procedures. First, the organic phase was cessed micronized GBD suspension (5 mg/mL) were added to 50 mL
prepared by dissolving 250 mg of GBD in 1 mL of DMSO, whereas of chitosan solution and stirred by propeller stirrer (IKA, Staufen,
the aqueous phase was 49 mL of deionized water containing Germany) for 10 minutes to disperse the drug. Mixtures were
poloxamer 188 (0.5%, w/v) as a stabilizer. The GBD solution was poured into Petri dishes and dried at 50 C (BINDER oven, Tut-
then slowly infused at a rate of 0.5 mL/min using a syringe needle tlingen, Germany). Aluminum foil was used as a backing film. The
(0.5-mm diameter) into the aqueous phase under sonication at developed TDDSs were coded as nano- and micro-GBD for formu-
25 C using energy output of 75 W (Ultrasons-HD; JP Selecta, lations containing nanonized and micronized GBD, respectively.
Barcelona, Spain). The obtained GBD dispersion (5 mg/mL) was
centrifuged at 4180 g for 30 minutes (Centrifuge Z 206 A; Hermle Characterization of TDDSs
Labortechnik GmbH, Wehingen, Germany). Particles were carefully
gathered, washed with deionized water, and recentrifuged simi- Thickness
larly to remove residues of the organic solvent. The collected Thickness of the prepared patches was determined by a digital
particles (about 80% of the initially added drug amount) were Vernier caliper (Mitutoyo, Kawasaki, Japan) at 4 different locations.
redispersed in an appropriate volume of aqueous solution of The average values and standard deviations were determined.14
poloxamer 188 (1% w/v) to prepare a drug dispersion of 5 mg/mL.
The formed dispersion was processed using an Ultra-Turrax T25 Weight Variation
digital homogenizer (IKA-Werke, Staufen im Breisgau, Germany) For weight variation test, 5 films (1 cm2) were weighed sepa-
at 10,000 rpm for 2 minutes. The resultant dispersion was then rately by an analytical balance (ADAM Equipment, Milton Keynes,
homogenized using Avestin C-5 homogenizer (Avestin Inc., UK), and the average weight was calculated.
Ottawa, Canada) with initial homogenization cycles performed at
100, 500, and 1000 bar. Finally, the suspension was homogenized Drug Content
for several cycles at 1500 bar until obtaining a drug nano- Known weights of the films (50 mg) were dissolved in a phos-
suspension of a constant particle size. For comparison, a slurry of phate buffer (pH 7.4, containing 2%, v/v lactic acid). The solutions
the unprocessed GBD was homogenized similarly without the were then sonicated for 15 minutes and filtered and properly
initial precipitation step. diluted (dilution factor ¼ 1000). GBD was determined spectro-
photometrically at lmax of 237 nm against a blank prepared simi-
Size Characterization larly without GBD. Formulations were tested in triplicate to
calculate the mean and standard deviation.
Particle size of the prepared GBD NS was determined by
dynamic light scattering using Microtrac S3500 (Microtrac Inc., Folding Endurance
Montgomeryville, PA). Diluted samples (dilution factor ¼ 2) of The folding endurance is used to evaluate the mechanical
the prepared nanosuspension were measured 3 times for strength and flexibility of the films that is needed for handling. Film
120 seconds at 25 C. strips (2 cm  1 cm) were evenly cut and folded repetitively at the
same place till they broke. The number of times the film could be
Assessment of Drug Crystallinity folded without breaking was recorded.15

Differential Scanning Calorimetry Moisture Content

Thermal analysis by differential scanning calorimetry (DSC) was Moisture content of the prepared films was determined gravi-
carried out using Netsch thermal analyzer (Netsch F3 Maia, metrically at 105 C using a moisture analyzer (HB43-S Halogen
Münster, Germany). GBD solid samples were collected from the Moisture Analyzer; Mettler Toledo, Greifensee, Switzerland).
 H.S.M. Ali, A.F. Hanafy / Journal of Pharmaceutical Sciences xxx (2016) 1-9 3

Figure 1. Particle size distribution of unprocessed glibenclamide (a) and nanonized glibenclamide (b).

Samples were tested in triplicates, and the mean values were introduced in individual baskets where drug matrix side in contact
recorded.16 with the medium of release. Phosphate buffer (500 mL, pH 7.4) kept
at 32 ± 0.5 C and stirring rate of 50 rpm was used as the release
Moisture Uptake medium. At predetermined intervals, 5 mL was withdrawn, filtered
Films of known weights were placed in a desiccator containing by 0.45-mm membrane (Millipore, Cork, Ireland), and analyzed
saturated solution of potassium chloride at room temperature. spectrophotometrically for GBD content at lmax of 237 nm after
Three days later, the films were removed and reweighed. Difference appropriate dilution. Withdrawn samples were replenished with
between the final and initial weights with respect to initial weight equal volumes from a fresh medium.
was used to calculate the percentage of moisture uptake.15
Ex Vivo Skin Permeation Studies
Redispersibility of Drug Particles From TDDS
Skin Preparation
Films were introduced into certain volumes of deionized water Animal studies were carried out in the Medical Research
for film disintegration. Particle size was determine as described Institute, Alexandria University, according to the guidelines and
previously after 10 minutes of magnetic stirring.17 regulations for the appropriate use of animals in biomedical
studies provided by the Egyptian ministry of higher education.
Drug Release Permeation studies were performed on skin obtained from albino
rats (150-200 g). Rats were sacrificed using anesthetic ether. The
Release of GBD from TDDSs was characterized using USP hair of the tested animals was removed, and skin was isolated
dissolution tester (type II).18 Tested transdermal formulations were from the abdominal region as previously reported.19
4 H.S.M. Ali, A.F. Hanafy / Journal of Pharmaceutical Sciences xxx (2016) 1-9

Figure 2. DSC thermograms of the unprocessed (GBD-RAW), precipitated (GBD-PPT),

and homogenized glibenclamide (GBD-PPT-HPH).

Permeation Experiments
Figure 3. X-ray diffractograms of the unprocessed (GBD-RAW), precipitated (GBD-
The permeation studies were carried out in a modified Franz’s PPT), and homogenized glibenclamide (GBD-PPT-HPH).
diffusion cell having a receptor compartment volume of 50 mL and
cross-sectional area of 2.64 cm2. Skin sections were mounted on
diffusion cells between the 2 compartments of the diffusion cell with intraperitoneally. After 24 hours, the degree of hyperglycemia was
stratum corneum facing the donor compartment and the dermal assessed, and rats having blood glucose levels >250 mg/dL were
side of the skin toward the receiver compartment. The tested TDDS chosen to complete the study. Similar procedures applied for
was placed over the skin in a way that the release surface of the TDDS normal rats were followed to assess the hypoglycemic actions of
is kept in direct contact with the stratum corneum side of the skin the tested formulations (nano-GBD, micro-GBD, and control) in
and the backing membrane upward. The receptor fluid was phos- diabetic rats.
phate buffer pH 7.4 containing ethanol, 30% (v/v) to guarantee the
sink condition. The receptor medium was constantly stirred with a Glucose Tolerance Test
magnetic stirrer at 300 rpm and kept at a temperature of 32 ± 0.5 C. Rats were fasted overnight and divided into 4 groups (n ¼ 5).
Samples were withdrawn at specified time intervals and replen- Group 1 served as control and administered with a plain TDDS.
ished with equivalent volumes of fresh medium to maintain sink Groups 2, 3, and 4 were treated with oral GBD (5 mg/kg), micro-
conditions. Withdrawn samples were assayed spectrophotometri- GBD (3 mg), and nano-GBD TDDS (3 mg), respectively. After
cally for GBD content after filtration and appropriate dilution. Under 2 hours, oral glucose (3.5 g/kg) was given to the tested groups.
similar experimental conditions, another set was run using a drug- Blood glucose was monitored just before (0 hour), 0.5, 1, and 2
free transdermal patch to serve as a blank. hours after glucose feeding. Changes in blood glucose levels were
determined relative to the control group.
Permeation Data Analysis
Cumulative amounts of GBD (mg/cm2) permeated within Statistical Analysis
24 hours were plotted versus time (h). Skin permeation rate or flux,
J (mg/cm2/h) was computed from the slope of the regression line All values were expressed as means ± SD. Statistical analysis was
fitted to the linear part of the curve. The enhancement factor (EF) performed using SPSS software (SPSS Inc., Chicago, IL). Results were
for the developed formulation was obtained by dividing nano- processed using the Student t test. Difference was considered sig-
GBDflux with micro-GBDflux.20 nificant when the associated p value was <0.05.

In Vivo Studies Results and discussion

Studies in Normoglycemic Rats Nanonization Process and Particle Size Analysis

Male Wistar rats (n ¼ 5) were fasted overnight while water was
available ad libitum. Tested TDDSs (nano- and micro-GBD) con- Nanosuspensions are thermodynamically unstable systems due
taining 3 mg of GBD, in addition to a control drugefree TDDS, were to the extra surface areas and interfaces created during the nano-
applied to the shaved dorsal skin regions of the tested rats. Animals nization process. If they are not effectively stabilized, nano-
were housed in individual cages under controlled temperature and suspension will have a high tendency to reduce their total energy
light conditions. Blood was withdrawn periodically from the orbital by particles aggregation.21 Based on a number of preliminary
sinus at prespecified intervals, and blood glucose levels were experiments, poloxamer 188 at a concentration of 0.5% (w/v) was
determined using Accutrend Alpha Glucometer (Roche Diagnostics, the stabilizer of choice. During the precipitation phase, poloxamer
Mannheim, Germany). Oral GBD was given (5 mg/kg) to another was added to decrease the free energy, preventing aggregation and
group (oral-GBD), and blood glucose levels were estimated simi- Ostwald ripening of the generated drug particles.22 Subsequently,
larly. The hypoglycemic effect was determined from the difference in the homogenization process, poloxamer helps reduce drug
in glucose levels at the 0 hour and subsequent times for each rat. particles to a value of 415 nm within 5 homogenization cycles.
Efficiency of particle fragmentation during the homogenization
Studies in Hyperglycemic Diabetic Rats process depends on the state of particles dispersion. As the parti-
Male Wistar rats (n ¼ 5) were fasted for 24 hours before study. cles are less aggregated, they are more prone to fragmentation
Diabetes was induced by injecting streptozotocin 55 mg/kg during their passage through the narrow gap of the homogenizer.
 H.S.M. Ali, A.F. Hanafy / Journal of Pharmaceutical Sciences xxx (2016) 1-9 5

Figure 4. DSC thermograms of chitosan (CH), chitosan lactate (CHL), chitosan lactate loaded with glibenclamide (GBD-CHL), glibenclamide after recovery from patches (GBD-REC),
and processed glibenclamide (GBD). The inset shows a magnification of the highlighted peak in GBD-CHL.

Surfactants, such as poloxamer, offer more effective wetting of the unprocessed GBD could be a consequence of particle size diminu-
drug particles than polymers resulting in better particle dispersion tion.26 Smaller particles have a greater fraction of molecules at
and a greater extent of particle size reduction.23 Adsorption of surface with lower fraction of the nearest neighbor molecules
poloxamer on drug particles also provides an additional steric compared to larger particles. Consequently, very small particles
hindrance resulting in more effective prevention of particle (such as nanosized particles) are more weakly bound with less
aggregation and crystal growth. Particle size studies (Fig. 1) showed constrained thermal motion relative to molecules within larger
the starting particle size, D50, of the unprocessed micronized GBD crystal structures.26 The enthalpy change results revealed that
was 4.84 mm (Fig. 1a). Direct homogenization of these particles crystallinity of GBD nanocrystals was 80% of the unprocessed GBD.
reduced the particle size down to a D50 of 1.32mm. However, for the Further evaluation by X-ray analysis was performed to confirm
formerly precipitated GBD, the homogenization process yielded a state of drug crystallinity. As shown in Figure 3 (bottom), the PXRD
particle size D50 of 0.429 mm (Fig. 1b) in shorter time (4-5 cycles). In pattern of unprocessed GBD showed sharp and intense peaks at 2q
no doubt, time shortening is advantageous with regard to energy values of 12.28, 19.58, 21.62, and 23.76 indicating its crystalline
consumption and economic and ecologic issues. The enhanced GBD nature. According to Figure 3, the precipitation and homogeniza-
nanosizing efficiency clearly is a consequence of the precipitation tion processes did not interfere with GBD crystalline state as the
process. The precipitation at first step reduced the particle size of diffraction patterns for GBD. All peaks of the unprocessed GBD were
initial drug crystals from D50 of 4.84-2.90 mm. In addition, the fast observed with reduced intensity and slight peak broadening. Such
precipitation process induces particle friability, drug crystal defects, features could be attributed to the “particle size broadening” phe-
and dendritic morphology facilitating the particle size reduction of nomenon that was associated with PXRD profiles of small-sized
the homogenization stage.24,25 crystalline materials.27,28 Crystallinity is beneficial for physico-
chemical stability of the drug where particle mobility is insignifi-
Assessment of Drug Crystallinity cant for crystalline phase relative to amorphous one.
DSC was also used to study the crystallinity and GBD
DSC thermogram of the unprocessed micronized GBD displayed compatibility within chitosan formulations (Fig. 4). Chitosan
a sharp endothermic melting peak at 177 C (Fig. 2), whereas DSC shows an endothermic peak at 70 C that could be assigned to
thermograms of the precipitated and homogenized GBD showed evaporation of water associated with the hydrophilic groups of
minor changes in the melting peak of GBD (171 C and 174 C for the chitosan. The second exothermic peak at 295 C is connected to
precipitated and homogenized GBD, respectively). Such slight the decomposition of amine units.29,30 Thermal profile of chito-
depression of melting peaks compared to the peak of the san lactate is characterized by broad endothermic peaks in the

Table 1
Physical Characteristics of Glibenclamide Patches

Formulation Code Thickness (mm) Weight (mg/cm2) Drug Content (%) Moisture Content (%) Moisture Uptake (%) Folding Endurance

Micro-GBD 0.12 ± 0.03 45 ± 1.70 92 ± 0.70 4.32 ± 0.23 5.52 ± 0.2 >85
Nano-GBD 0.10 ± 0.04 52 ± 0.60 96 ± 0.63 4.85 ± 0.63 6.97 ± 0.18 >100
6 H.S.M. Ali, A.F. Hanafy / Journal of Pharmaceutical Sciences xxx (2016) 1-9

Figure 5. Particle size distribution of glibenclamide nanosized particles after film redispersion.

temperature range 70 C-120 C and a second broad endothermic and biodegradability, chitosan possesses good film-forming ability
peak at round 150 C-200 C, which is attributed to a loss of water and bioadhesive and absorption enhancing characteristics.34 In the
of crystallization and the melting point of the mixtures.31 present study, chitosan transdermal patches were prepared in the
Although the second endothermic peak obscured the GBD peak, absence of plasticizers to evaluate the impact of particle nanosizing
a combined forked peak resulting from an overlap of GBD and exclusively on characteristics of the fabricated patches and to
chitosan lactate second peak is noticed in thermogram of simplify interpretation of the results. Viscosity of chitosan solution
GBD-loaded patches (the highlighted part and inset in Fig. 4). was found to be 2550 cP. Findings of physical characterization of
Confirmation was performed on GBD residues recovered from TDDSs are displayed in Table 1. Generally, the fabricated chitosan
drug-loaded patches after dissolution and centrifugation. In the films were thin, visually acceptable with an average thickness of
latter case, GBD showed a clear peak at 167 C. 0.10 ± 0.04 and 0.12 ± 0.03 for the nano- and micro-GBD films,
respectively. The low values of SD reflected homogeneity of the
films. No signs of aggregated particles or sedimentation
Formulation and Characterization of GBD-Loaded TDDS were noticed on the lower film surface. The GBD contents of the
films, estimated at different parts of the films, were 96 ± 0.63% and
The rationale behind selecting chitosan to fabricate GBD TDDS is 92 ± 0.70% for the micro- and nano-GBD. Content uniformity sig-
to preserve the size characteristics of the obtained nanosized drug nifies the suitability of chitosan rheologic and mechanical proper-
crystals. During the common methods of drying, nanoparticles ties to permit homogeneous distribution of the drug during the
could aggregate into larger particles with the loss of the enhanced casting and drying procedures.20 Folding endurance was found to
surface area properties.32 These aggregates can be slowly redis- be >100 and >85 for nano- and micro-GBD, respectively, ensuring
persed into nanoparticles in water.33 In addition to biocompatibility satisfactory strength, elasticity, and maintenance of integrity with
general skin folding after application. The percent moisture loss in
the patches ranged between to 4.32 ± 0.23 and 4.85 ± 0.63 for the
micro- and nano-GBD, respectively. The percent moisture uptake of
the patches was 5.52 ± 0.2 for micro-GBD and 6.97 ± 0.18 for nano-
GBD, with a slight increase in case of nano-GBD. Low values of

Figure 6. Release of glibenclamide from (nano-GBD) and (micro-GBD) transdermal Figure 7. Transdermal permeation profiles obtained after application of nano-GBD and
formulations. micro-GBD transdermal formulations.
 H.S.M. Ali, A.F. Hanafy / Journal of Pharmaceutical Sciences xxx (2016) 1-9 7

Figure 8. A diagram explains skin permeation of glibenclamide nanocrystal and microcrystal formulations.

moisture content keep patches stable, flexible, and free from brit- from nano-GBD was 498 ± 33.35 compared to 362 ± 25.25 mg
tleness. Moreover, small values of moisture uptake are beneficial to from micro-GBD. The flux across rat skin calculated for nano-
suppress microbial growth and minimize bulkiness of the GBD was 23.14 compared to 13.64 mg/cm2/h for micro-GBD,
formulations.18 with an EF of about 1.7. As seen, results revealed an improve-
ment in skin permeation of GBD via nanosizing. Upon contact
Redispersion of Drug Particles From TDDS with the hydrated skin, rapid dissolution for the nanosized drug
crystals in nano-GBD results in buildup of considerable levels of
Recovery of drug nanoparticles from the dried formulations GBD on the skin. This leads to an increased concentration
represents a major concern either in in vitro testing or after gradient between dermal formulation and skin (Cs  Cskin), thus
in vivo administration.32 For assessment of the recovery and increasing the GBD flux into the skin. In addition, size diminu-
redispersibilty of the nanosized drug particles, a comparison of tion generates higher fractions of molecules at the surface
the particle size distribution of the initially used nanosuspension rather than within the interior of substances. As a result, the
with that generated from redispersion of the dried nano- surface area of the unit mass of nanosized crystals is consider-
suspension. Figure 5 shows the particle size distribution after ably greater than macrosized crystals. The larger surface area of
film redispersion. The D50 values were 415 and 475 nm for the drug nanocrystals also increases the number of effective contact
particles before and after redispersion, suggesting that no sub- points between drug crystals and the skin. Furthermore, the
stantial aggregation of drug nanocrystals occurred during the individual drug nanocrystal is bordered by what is called a
drying process. “diffusional corona” of the dissolved drug molecules.38 Drugs
demonstrate a greater saturation solubility with decreasing
Drug Release Studies radius of particles, as per Kelvin equation. Although it is not
significant for larger particles, this enhancing effect is more
Figure 6 displays release profiles of GBD from micro- and nano- prominent in particles <1 mm.39,40 Accordingly, the skin surface
GBD. In comparison with their micron-sized counterparts, GBD is efficiently covered by overlay of diffusional coronas of the
nanocrystals significantly enhanced (p < 0.5) the percent drug nanocrystals (Fig. 8).38
released after 0.5 and 1 hour (20.5 ± 0.43% and 33.5 ± 0.6% for
nano-GBD compared to 11 ± 0.5% and 19 ± 0.6% for micro-GBD,
respectively). Within 24 hours, about 85 ± 3.1% of the GBD con- In Vivo Assessment
tent was released from nano-GBD compared to 61 ± 3.9% from
micro-GBD. DSC and X-ray analyses revealed that crystallinity of Monitoring of plasma glucose level was considered to evaluate
the drug was greatly maintained after nanonization. Therefore, the in vivo performance of the hypoglycemic agent, GBD. Results of
amorphization of particles was not expected to considerably impact
the drug release process. The enhancement of drug release is
plausibly a result of the improved dissolution of the nanosized
particles. Particle size diminution generates larger effective surface
area accessible for drug dissolution. Furthermore, an improvement
of thermodynamic solubility of the small particle is predicted
because of the high curvature at the particle interface according to
the OstwaldeFreundlich theory and the Kelvin equation.35,36 The
Prandtl equation also proposes a decrease in diffusional distance
with increase in the rate of drug dissolution by increasing particle
curvature.37

Permeation Studies

Findings of the ex vivo skin permeation of GBD from TDDSs

are displayed in Figure 7. After 0.5 and 1 hour of testing, drug
permeation through rat skin from nano-GBD was 148 ± 5.43 and
177 ± 10.6 mg/cm2, respectively. These values are significantly
higher (p < 0.5) than corresponding values of 107 ± 4.3 and 119
± 8.6 mg/cm2 for the micro-GBD. After 24 hours, the cumulative Figure 9. Changes of blood glucose levels after administration of nano-GBD and
amount of GBD permeated through skin unit area (cm2) micro-GBD compared to oral GBD in normal rats.
8 H.S.M. Ali, A.F. Hanafy / Journal of Pharmaceutical Sciences xxx (2016) 1-9

Figure 10. Changes of blood glucose levels after administration of nano-GBD and
micro-GBD formulations in comparison with oral GBD in diabetic rats.
Figure 11. Glucose tolerance curve after administration of oral and transdermal gli-
benclamide formulations.
in vivo activity of transdermal formulations (nano- and micro-GBD)
relative to oral GBD in normal and diabetic rats are displayed in
In general, results of in vivo study clearly demonstrated that the
Figures 9 and 10. Reduction in glucose levels was clear in normal rats
nanosized GBD formulation was more effective to reduce blood
for all tested formulations group (control normal; p < 0.05; Fig. 9).
glucose levels and counteract the induced hyperglycemia in tested
When given orally, GBD induced a marked degree of hypoglycemia
rats than the microsized drug formulation owing to higher drug
(transdermal patches, p < 0.05), reaching a maximum reduction
release and skin permeation of the drug nanocrystals. At the same
percentage of 48.71 ± 3.4% at 4 hours. The hypoglycemic action
time, the transdermal formulation of nanosized GBD was able to
declined quickly after 4 hours, which could be attributed to the short
maintain an effective drug concentration for longer time and
biologic half-life of GBD,41 and only a reduction level of 8.8 ± 2.1%
minimize the intensity of GBD action and hypoglycemia associated
was recorded after 24 hours. On the other hand, the hypoglycemic
with the drug oral therapy.
action increased gradually to levels of 19.6 ± 1.84% for nano-GBD and
10.4 ± 1.9 for the micro-GBD after 2 hours. Maximum reduction in
glucose levels was observed after 6 hours (33 ± 2.3% for the nano- Conclusion
GBD and 24 ± 2.5% for the micro-GBD) and thereafter decreased
calmly to corresponding values of 28.6 ± 2.1% and 20.4 ± 20% at Nanocrystals of GBD were successfully engineered using a
24 hours. The control group did not demonstrate remarkable combined bottom-up and top-down techniques. Chitosan was used
changes in blood glucose level during the study. effectively to preserve size characteristics of the generated nano-
For diabetic rats, all treated groups showed significant hypo- crystals and fabricate a transdermal delivery formulation. Findings
glycemic effects compared to diabetic control group (Fig. 10). The of this study revealed enhancement in the release profile and skin
recorded hypoglycemic effects after 2 hours of treatment were permeation of GBD when formulated as nanocrystals. Transdermal
34.7 ± 2.9%, 18.7 ± 1.8%, and 10.2 ± 1.3% for the oral, nano-GBD, delivery of nano-GBD from biodegradable chitosan patch provided
and micro-GBD, respectively. As noticed, significant hypoglyce- better maintenance of glucose levels of drug in blood for a pro-
mia induced by oral GBD (transdermal patches p < 0.05) can be longed period with less incidences of hypoglycemia associated with
overcome by transdermal systems. The maximum recorded hy- oral therapy. Therefore, it could serve as a potential alternative to
poglycemic effects were 52.53% at 4 hours for oral GBD, 35.32% at peroral GBD for enhanced bioavailability and better patient
6 hours for nano-GBD, and 26.20% at 6 hours for micro-GBD. compliance through simple application and immediate cessation of
Besides, after 24 hours, the hypoglycemic effect declined to drug administration by patch removal.
levels of 7.61 ± 1.2% for oral GBD, 18.41 ± 1.1% for micro-GBD, and
26.8 ± 1.2 for nano-GBD. For nano-GBD, the reduction value at 24
hours was significantly higher than micro-GBD (p < 0.05) and oral Acknowledgments
GBD (p < 0.05).
Findings of glucose tolerance test (GTT) are displayed in The authors acknowledge the Deanship of Scientific Research at
Figure 11. The untreated control group displayed considerable rises Taibah University for providing support and facilities to carry out
in glucose levels (p < 0.05) after glucose feeding (þ66 ± 4.9%, þ 53 the study. Hany S. M. Ali wishes to express most sincere gratitude to
± 3.41%, þ31 ± 3.5%, and þ13 ± 1.9% at 0.5, 1.0, 1.5 and 2.0 hours, his former PhD supervisor, Professor Peter York, for invaluable help
respectively). These elevated values of glucose levels in the GTT and professional support. Acknowledgment is extended to the
curve were markedly suppressed or even reversed in experimental European Egyptian Pharmaceuticals Company (Pharco Corpora-
animals that received GBD therapy. For the groups treated tion) for the kind supply of glibenclamide.
with TDDSs, the blood glucose levels declined to levels
of þ10 ± 2.8%, þ8 ± 4.1%, 8 ± 2.5%, 21 ± 3.4% for nano-GBD References
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