Diosmin Nanocrystal–Loaded Wafers for Treatment of Diabetic Ulcer: In Vitro and In Vivo Evaluation

Journal of Pharmaceutical Sciences 108 (2019) 1857-1871

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Pharmaceutical Nanotechnology

Diosmin NanocrystaleLoaded Wafers for Treatment of Diabetic Ulcer: In Vitro and In Vivo Evaluation Nouran M. Atia 1, Heba A. Hazzah 1, *, Passent M.E. Gaafar 2, Ossama Y. Abdallah 3 1

Department of Pharmaceutics, Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria, Alexandria, Egypt Department of Pharmaceutical Sciences, College of Pharmacy, Arab Academy for Science, Technology and Maritime Transport, Alexandria, Egypt 3 Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt 2

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 October 2018 Revised 23 November 2018 Accepted 20 December 2018 Available online 30 December 2018

This work aimed at loading of diosmin nanocrystals into alginate-based wafers for treatment of highly exuding diabetic ulcer in rats using topical route of administration. For this purpose, different formulation variables and preparation techniques to enhance the flexibility and adhesion properties of the prepared sodium alginate (SA) wafers were carried out. The prepared wafers were characterized regarding hydration capacity, bioadhesion, scanning electron microscope, and Fourier-transform infrared spectroscopy. Efficacy of treating diabetic ulcer was studied using diabetic-induced rat model using streptozotocin. Results obtained showed that using SA:gelatin with 1.5%/1.5% w/w gave acceptable wafers with a sustained release of diosmin over 8 h. A complete re-epithelialization, well-organized dermal layers, well-formed granulation tissue, and mature collagen bundles were observed in treated rats. It was concluded that combination of gelatin with SA provided an excellent wafer as a promising medicated wound dressing holding diosmin nanocrystals while maintaining its stability. © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Keywords: diosmin nanocrystal wafers sodium alginate gelatin diabetic ulcer wound dressings

Introduction Diabetes is a complex metabolic disorder involving different organs, and it leads to the development of various vascular complications, such as cardiomyopathy, neuropathy, nephropathy, retinopathy, and foot ulcer.1,2 Wound healing is a well-organized and highly controlled multifactorial process characterized by 4 overlapping phases. During the wound-healing process, coordination of blood cells, fibroblasts, keratinocytes, endothelial cells, growth factors, and cytokines plays a vital role in the rate of wound repair.3 Once a diabetic foot ulcer has developed, there is an increased risk of wound progression that may lead to amputation where more than 85% of foot amputations in patients are caused by diabetic foot ulcer.4 The medical treatment of diabetic ulcers is a major clinical problem as it is associated with some pathologic conditions such as hemodynamic abnormalities, hypoperfusion, abnormal angiogenesis, and neuronal ischemia, or extrinsic factors due to infection and continued trauma that delays wound healing.5

Conflict of interest: Authors declare there is no conflict of interest. * Correspondence to: Heba A. Hazzah (Telephone: þ201005038347). E-mail address: [email protected] (H.A. Hazzah).

Treatment of diabetic ulcer can be approached by removing dead, damaged, or infected tissue to improve the healing potential as well as using antibiotics, tissue grafts, proteolytic enzymes, and corticosteroids. However, these therapies have side effects that limit their use; in addition, they provide relief only to a fraction of patients.6 This potentiates the need of finding and using other therapeutic alternatives with minimal side effects.7 Flavonoidal compounds, as diosmin, possess strong antiinflammatory, antioxidant, antiulcer, anticancer, antiatherogenic, hepatoprotective, and neuroprotective activities, in addition to their potential metal-chelating and free radicalescavenging properties.8 Furthermore, flavonoids are highly capable of inhibiting cyclooxygenases and protein kinases enzymes involved in cell proliferation and apoptosis. However, to our knowledge, only 2 studies have reported the use of diosmin topically for treatment of wound,9 and hemorrhoids.10 The lack of data and work on diosmin in this regard despite its high potential is attributed to its poor solubility. An ideal wound dressing should present adequate properties to create a favorable environment for the healing process, such as flexibility, durability, permeability to water vapor, adequate mechanical properties, and adherence to the tissue.11,12 Wafersdas dressingdoffer several advantages such as maintaining moist environment and absorption of large amount of wound exudates due to their high porosity. After application to the

https://doi.org/10.1016/j.xphs.2018.12.019 0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

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wound surface, lyophilized wafers revert to a highly viscous fluid or resilient gel upon absorbing exudate, thus ensuring longer in situ residence time providing extended drug release.13 The incorporation of therapeutic agents into wound dressings is an attractive approach to control the inflammatory reaction, prevent infections, and promote tissue regeneration.14 With the increase in pharmaceutical industrial and public awareness of health hazards of using synthetic active compounds and polymers, our interest was raised regarding the combination of natural polymers and active ingredients. To our knowledge, by far none has reported the use of diosmin nanocrystals topically for treatment of diabetic wound, nor reported its loading into a sodium alginate (SA) wound dressing. That could be attributed to the difficult physical nature and properties of diosmin. Thus, this work aimed at loading of diosmin nanocrystals in alginate-based wafers for treatment of highly exuding diabetic ulcer in rats. In addition, the study aimed at investigating formulation variables and preparation techniques to enhance the flexibility and adhesion properties of the prepared SA wafers. This work is the first to report the preparation and in vivo evaluation of wafer loaded with diosmin nanocrystals for treatment of wound in diabeticinduced rats.

orthophosphate, sodium hydroxide, sodium lauryl sulfate, and boric acid (El Nasr Pharmaceutical Co., Cairo, Egypt), SA of molecular weight 3-5  106 g/mol (BDH Chemical Ltd., Poole, UK), gelatin (GE) type B (Gelita Limited Company, Eberbach, Germany), streptozotocin (STZ; Sigma-Aldrich), citric acid monohydrate and sodium citrate dihydrate (Fisher Scientific, Co., Ltd.). Formulation of Placebo Wafers Placebo gels (PL) were prepared containing different concentrations of SA and GE (Table 1). The gels were prepared by dispersing the polymers in heated distilled water (40 C) with continuous stirring until they completely dissolved yielding a homogenous gel. The concentration of total polymers was kept at 3% w/w. Glycerol (GL) as a plasticizer was added at a concentration of 5% w/w (based on the solid content). In addition, mannitol was added as a cryoprotectant at a concentration of 0.25% w/w (based on the total weight). Twenty grams of each gel was poured in 8-well plastic molds (2.5 g/wafer) (diameter 1.8 mm and thickness 8 mm) and frozen at 25 C before the lyophilization step using lyophilizer at vacuum 40 mTorr, for 10 h. Preparation of Diosmin Nanosuspension

Materials and Methods Diosmin (Jianshi Yuantong Bioengineering Co., Ltd., Hubei, China), hydroxypropyl methyl cellulose (HPMC E15) and methyl cellulose (MC) (Zhengzhou Tianying Chemicals Co., Ltd., Yixing, China), poloxamer 407 (Kolliphore 407) (kind gift from BASF, Ludwigshafen, Germany), dimethyl sulfoxide (DMSO) (Oxford Chemicals, Mumbai, India), mannitol, potassium dihydrogen

Diosmin nanosuspension was prepared using antisolvent precipitation technique; 150 mg diosmin was dissolved in 5 mL of DMSO (solvent phase). The organic solution was then added into aqueous solution of distilled water (50 mL; antisolvent phase) containing HPMC E15 or MC as a stabilizer at a concentration of diosmin-stabilizer 1:1 weight ratio with continuous mixing over 30 min at 500 rpm.

Table 1 Composition of Different Placebo and Diosmin-Loaded Wafer Formulations Formulation Code

SA (% w/w)

GE (% w/w)

Glycerol (% w/w)

Mannitol (% w/w)

Freeze-Dried Powder

PL SA3GL0M0 PL SA3GL0M0.25 PL SA3GL5M0 PL SA3GL5M0.25 PL GE3GL0M0 PL GE3GL0M0.25 PL GE3GL5M0 PL GE3GL5M0.25 PL SA/GE 2.25/0.75 GL0M0 PL SA/GE 2.25/0.75 GL0M0.25 PL SA/GE 2.25/0.75 GL5M0 PL SA/GE2.25/0.75GL5M0.25 PL SA/GE 1.5/1.5 GL0M0 PL SA/GE 1.5/1.5 GL0M0.25 PL SA/GE 1.5/1.5 GL5M0 PL SA/GE 1.5/1.5 GL5M0.25 HPMC SA/GE 2.25/0.75 GL5M1 HPMC SA/GE 2.25/0.75 GL10M1 MC SA/GE 2.25/0.75 GL5M1 MC SA/GE 2.25/0.75 GL10M1 HPMC SA/GE 1.5/1.5 GL5M1 HPMC SA/GE 1.5/1.5 GL10M1 MC SA/GE 1.5/1.5 GL5M1 MC SA/GE 1.5/1.5 GL10M1 HPMC SA/GE 1.5/1.5GL10M0.03 HPMC SA/GE 1.5/1.5 GL10M2 MC SA/GE 1.5/1.5 GL10M0.03 MC SA/GE 1.5/1.5 GL10M2

3.00 3.00 3.00 3.00 d d d d 2.25 2.25 2.25 2.25 1.50 1.50 1.50 1.50 2.25 2.25 2.25 2.25 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50

d d d d 3.00 3.00 3.00 3.00 0.75 0.75 0.75 0.75 1.50 1.50 1.50 1.50 0.75 0.75 0.75 0.75 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50

d d 5.00 5.00 d d 5.00 5.00 d d 5.00 5.00 d d 5.00 5.00 5.00 10.00 5.00 10.00 5.00 10.00 5.00 10.00 10.00 10.00 10.00 10.00

d 0.25 d 0.25 d 0.25 d 0.25 d 0.25 d 0.25 d 0.25 d 0.25 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

d d d d d d d d d d d d d d d d F1.DM1 F1.DM1 F2.DM1 F2.DM1 F1.DM1 F1.DM1 F1.DM1 F2.DM1 F1.DM0.03 F1.DM2 F2.DM0.03 F2.DM2

Codes represented. Nanocrystal stabilizer either hydroxylpropyl cellulose (HPMC) or methyl cellulose (MC). The mixture of polymers used: sodium alginate (SA) and gelatin (GE) in weight ratios (2.2.5/0.75 or 1.5/1.5). Mannitol in the freeze-dried powder (calculated based on the total weight). Glycerol (calculated based on the total solid content). PL, placebo; SA, sodium alginate; GE, gelatin; GL, glycerol; M, mannitol.

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Lyophilization of Diosmin Nanosuspension Formulations

Preparation of Wafers Loaded With Diosmin Nanocrystals

Diosmin nanosuspension formulations F1 and F2 were subjected to lyophilization using mannitol as a cryoprotectant at a concentration of 1% w/w (F1.DM1 and F2.DM1). The nanosuspensions were frozen at 25 C before lyophilization step using lyophilizer at vacuum 40 mTorr (Labconco, Fort Scott, KS). Two control lyophilized formulations were prepared without cryoprotectant for comparison (F1.DM0 and F2.DM0).

Preparation of Wafers Loaded With Diosmin Nanocrystals (Thickening Method) Diosmin nanocrystals were prepared using antisolvent method; the selected diosmin nanosuspension formulations (F1 and F2) were then thickened using SA/GE mixture in either 2.25/0.75 or 1.5/ 1.5 weight ratio. GL was added at a concentration of 5% w/w. Mannitol was added at a concentration of 1% w/w. The formed gels were molded and freeze-dried as previously described.

Characterization of Prepared Diosmin Nanosuspension/Lyophilized Powder Particle Size Analysis The average size of diosmin nanosuspension/lyophilized powder was measured by Malvern Zetasizer at 25 C. All measurements were performed in triplicates. Fourier-Transform Infrared Spectroscopy The Fourier-transform infrared (FT-IR) spectra for diosmin, HPMC E15, MC, F1.DM1, F2.DM1, and physical mixture for these formulations were recorded using an FT-IR spectrometer (PerkinElmer Life and Analytical Sciences, Shelton, CT). The FT-IR spectra were recorded at spectral resolution of 2 cm1 with an average of 20 scans. X-Ray Diffraction The crystallinity of diosmin in the selected freeze-dried diosmin formulations was assessed by powder X-ray diffraction (XRD) (X’Pert PRO diffractometer; PANalytical, Almelo, Netherlands). A copper radiation source was used as the anode material. The diffraction pattern was performed in a step scan model with a voltage of 40 KV and a current of 40 mA in the range of 10 < 2q < 40 . Samples investigated were crude diosmin, F1.DM1, and F2.DM1. In Vitro Drug-Release Study Diosmin Release Under Nonsink Condition Using Dissolution US Pharmacopeia Type II Apparatus (Borate Buffer pH 10). An amount equivalent to 3 mg diosmin was transferred to a dissolution vessel containing 500 mL borate buffer pH 10. The paddle speed was set at 100 rpm, and temperature was maintained at 37 C ± 0.5 C. Then, 5 mL of sample was withdrawn at predetermined time intervals (5, 10, 15, 30, 45, 60, 90, 120, and 180 min), which was replaced by an equal volume of fresh dissolution media. The solution was then filtered through 0.22-mm millipore filter and analyzed spectrophotometrically at l max 266 nm.15 In Vitro Release of Diosmin From Nanosuspension Formulations Using Dialysis Bag Technique. Dialysis of an amount equivalent to 1 mg diosmin using F1 or F2 formulation was carried out through a cellophane bag 4 cm long with a 28-kDa cutoff (VISKING dialysis tubing; SERVA Electrophoresis, Heidelberg, Germany). The bags were transferred to amber glass bottles containing 20 mL of borate buffer pH 10, in a horizontal shaking water bath at 100 rpm, and the temperature was adjusted to 37 C ± 0.5 C. Samples were withdrawn at different time intervals (0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, and 24 h) and replaced with an equivalent volume of a previously warmed fresh medium and measured spectrophotometrically at l max 266 nm. Transmission Electron Microscopy Morphology of diosmin nanosuspension formulations (F1 and F2) was examined using a transmission electron microscope. One drop of diluted dispersion was placed on a copper-coated grid leaving a thin film, followed by air drying. The dried films were then viewed on a transmission electron microscope and photographed.

Preparation of Wafers Loaded With Lyophilized Diosmin Nanocrystals (Dried Nanocrystal Method) Four formulations were developed (HPMC SA/GE 2.25/0.75 GL10M1, HPMC SA/GE 1.5/1.5 GL10M1, MC SA/GE 2.25/0.75 GL10M1, and MC SA/GE 1.5/1.5 GL10M1) (Table 1). These 4 formulations were prepared using the lyophilized formulations (F1.DM1 and F2.DM1). The freeze-dried powders were dispersed in gel matrix containing SA/GE mixture at a concentration of 2.25/0.75 or 1.5/1.5. Diosmin content was fixed at a concentration of 6.8 mg/wafer. GL was added at different concentrations. Mannitol was added in the gel formulation at a concentration of 1%. Then, the formed gels were molded and subjected to lyophilization as previously described (Table 1). Characterization of Placebo and Loaded Wafers Physical Appearance The prepared wafers were physically evaluated regarding brittleness, separation or stickiness to the mold walls, and integrity. Moisture Content Samples of placebo wafers were carefully weighed (Wi) and heated to 100 C till constant weight (Wf), and the moisture content was calculated as a % weight loss using infrared moisture determination balance according to the following equation:

.
Wi  100 %Weight loss ¼ Wi  Wf Porosity The porosity for placebo wafers was calculated mathematically in terms of total pore volume. The true (theoretical volume) volume of the total ingredients composing 1 wafer before lyophilization process was calculated according to the following equation16:

Theoretical volume ¼ ðm1 =r1 Þ þ ðm2 =r2 Þ þ … where m and r are the mass and density of each ingredient/wafer. The practical (bulk) volume of wafer was calculated by the wafer dimension according to the following equation:

Practical volume ¼ area  height The total pore volume was calculated as follows according to the following equation:

%porosity ¼

ðpractical volume  theoretical volumeÞ  100 theoretical volume

Hydration and Erosion Capacity The hydration and erosion capacity of placebo and selected loaded wafers were carried out as previously described by Boateng et al.17 The percentage of water uptake was calculated as follows17:

Water uptakeð%Þ ¼ ðWs  Wi Þ=Wi  100 where Ws is the weight of the hydrated wafer, and Wi is the initial weight of wafer.

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FT-IR Spectroscopy The FT-IR spectra for SA, GE, namely (SA/GE 1.5/1.5 GL0M0), and physical mixture for this formulation were recorded using an FT-IR spectrometer (similar conditions as in section Fourier-Transform Infrared Spectroscopy). Scanning Electron Microscopy The external surface and cross section of selected placebo wafers (PL SA/GE 2.25/0.75 GL5M0.25 and PL SA/GE 1.5/1.5 GL5M0.25), in addition to selected formulations (MC SA/GE 1.5/1.5 GL10M1) and wafer containing diosmin powder, were examined by scanning electron microscopy (SEM). The samples were placed on double-sided adhesive carbon tape on labeled stainless steel stubs. The samples were placed on the exposed side of the carbon adhesive taking care not to damage the surface topography of the wafer. The wafers were then sputter coated with gold placed in the chamber of a SEM, and images acquired using i-scan 2000 software. Particle Size Analysis The average particle size of nanocrystals loaded in wafer formulations was measured by Malvern Zetasizer (Malvern, Instruments Ltd., Malvern, UK) at 25 C. All measurements were performed in triplicates. Drug Content Drug content assay was carried out by randomly taking weight from different wafer formulations (weight equivalent to 1.5 mg diosmin) and dissolved in 100 mL 1 N sodium hydroxide (1.5 mg %). The absorbance at l max 266 nm was measured using UV-visible spectrophotometer. All measurements were performed in triplicates. In Vitro Release Testing (Cup Method) A fragment of wafer equivalent to 3 mg diosmin was weighed and placed in a stainless steel cup (10-mm diameter and 3-mm depth) and covered with polyester gauze acting as a mechanical barrier to prevent gel escape without interfering with drug release. The gauze was fixed to the cup through a specially designed stainless steel ring. In the cup was placed 500 mL borate buffer pH 10, paddle speed was set at 100 rpm, and temperature was maintained at 37 C ± 0.5 C. Then, 5 mL of sample was withdrawn at predetermined time intervals after 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, and 480 min, which was replaced by an equal volume of fresh dissolution media. The solution was then filtered through a 0.22-mm syringe filter and analyzed spectrophotometrically at l max 266 nm.18 Release Kinetics The mechanism of drug release for selected formulations (HPMC SA/GE 1.5/1.5 GL10M1 and MC SA/GE 1.5/1.5 GL10M1) was investigated using the following mathematical models. Zero-order kinetics, first order, Higuchi, and Korsmeyer-Peppas.19 In Vitro Mucoadhesion Assessment In vitro wound adhesion studies were performed on wafers using texture analyzer. The wafer was attached to the upper arm of a 25-mmdiameter probe using double-sided adhesive tape. A 75-mm-diameter petri dish containing 20 g of GE solution (6.67% w/w) was equilibrated with 0.5 mL phosphate buffer pH 7.5 to represent the wound surface.20 The experiment was performed by lowering the probe until the wafer came in contact with the set GE gel surface for 60 s to provide optimal contact. The adhesion force was determined as the force required to break the bond between the wafer and the simulated wound surface.

The cohesiveness (stickiness) was estimated by the total distance (in millimeters) traveled by the probe before complete detachment of the wafer from the GE gel surface.21 Effect of Aging Effect of aging was conducted on the selected formulation (MC SA/GE 1.5/1.5 GL10M1) which was kept in a desiccator at room temperature for 6 months. It was assessed for any change in average particle size and polydispersity index by Malvern Zetasizer at 0, 1, 2, 3, and 6 months of storage. Sample was also assessed for any change in the in vitro drug-release profile by monitoring dissolution rate in borate buffer pH 10 over the same period of time. In Vivo Wound Healing Animals All studies were approved by the ethics committee of the Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria. The animals were kept under standard laboratory conditions and veterinary supervision with no restriction on water and food. Sixteen male Sprague Dawley rats weighing 210-250 g, each of them kept in a separate cage, were used. Food and water were maintained in a 12-h light/12-h dark cycle in a controlled room temperature of 20 C-25 C. Induction of Diabetes Diabetes mellitus was induced in 16 male Sprague Dawley rats weighing 210-250 g using STZ. Rats were fasted for 12 h before injection of STZ (55 mg/kg body weight) dissolved in 1 mL 0.1 M citrate buffer (pH 4.5). STZ was administered via intraperitoneal route (i.p.) in a single dose. For blood glucose measurements, blood sample was withdrawn from the tail vein and measured using glucometer. Blood glucose level was measured before STZ injection and 3 days after injection. Rats having blood sugar level above 250 mg/dL were selected for this study. Rats with blood glucose level less than 250 mg/dL were excluded from the experiment22 (4 rats were excluded). Incision of Ulcer Just before ulcer formation, the 12 selected diabetic rats were anesthetized using ketamine (75 mg/kg, i.p.) and xylazine (10 mg/ kg, i.p.).23 The back of each rat was shaved using a shaving machine and the shaved area was sterilized with alcohol and povidoneiodine. Three full-thickness, half-circle-shaped ulcers (with a diameter of z15 mm) were created using sterile scissors and forceps.24 The rats were divided randomly into 2 groups of 6 according to the treatment applied on the formed ulcers (n ¼ 6). Each rat had 3 ulcers and was kept in a separate cage. Group I was used for examination of ulcers treated with MC SA/GE 1.5/1.5 GL10M1 wafer (selected formulation), diosmin powder wafer, and placebo wafer. Group II ulcers were treated with MC SA/GE 1.5/1.5 gel, diosmin powder gel, and control untreated ulcer. Diosmin powder wafer was prepared by dispersing diosmin powder in gel matrix SA/GE1.5/1.5 containing the same percentage of mannitol and GL and then subjected to freeze-drying. Placebo wafer was prepared with the same technique containing the same percent of mannitol and GL but only lacking diosmin nanocrystal. MC SA/GE 1.5/1.5 gel was also prepared. Simple diosmin gel containing only diosmin powder was prepared by dispersing diosmin powder in gel matrix SA/GE 1.5/1.5.

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Dose Application of Wafer and Gel The daily dose applied to each ulcer was equivalent to 3.4 mg diosmin (1.25 g gel or half wafer by weight). Only during application of gel, rats were anesthetized with xylazine (10 mg/kg, i.p.). After dose application, ulcers were covered with adhesive tape. Before application of the subsequent dose in the following day, the ulcer was washed with sterile saline. Evaluation of Diabetes and Ulcer Healing Blood Glucose Level and Body Weight Changes in Diabetic Rats. Rats were evaluated for blood glucose level and weighted periodically before diabetes induction and every 3 days during the study to show the effect of hyperglycemia on blood glucose level and body weight. Morphologic Examination of Ulcers. Ulcers were monitored visually and digitally photographed with maintaining constant optical zoom for monitoring scar formation, redness, and hyperpigmentation. Measurement of Ulcer Area and Calculation of Ulcer Closure %. The progressive changes in ulcer area were measured periodically every 2 days. Vernier caliper was only used to determine the diameter of the half circle; this value was then analyzed using Image analysis software (Image J; National Institutes of Health) that set a scale for the image and then can determine the area of the irregular ulcer automatically. Furthermore, the wound closure % was calculated according to the following equation23:


%wound closure ¼

initial wound area  wound area at Nth day



initial wound area

 100 Histopathologic Examination. Pancreas biopsy from diabetic rats was taken at the end of the study and was compared histologically to the nondiabetic ones to show the effect of diabetes on B-cell structure. In addition, full-thickness skin biopsies with surrounding tissues were collected at day 10 for different wounds receiving the different therapies in addition to the control one. Specimen was fixed in 10% formaldehyde, then embedded in paraffin, cut into 5mm slices, and stained with hematoxylin and eosin. Fields were visualized under a light microscope at 100 magnification. Fields were examined regarding the degree of epithelialization, presence or absence of inflammatory cells, and the nature of collagen and fibrous tissue. Statistical Analysis

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ratios (2.25/0.75 and 1.5/1.5) also showed physically accepted wafers without any shrinking. Wafers prepared containing only GL (5% w/w) (based on the solid content) showed good flexibility and were easily separated from the mold. Addition of mannitol (0.25% w/w) (based on the total weight) as a cryoprotectant was also beneficial during freezedrying step. From this finding, it was concluded that presence of GL and mannitol as a plasticizer and cryoprotectant, respectively, is essential for production of elastic wafers. Therefore, GL and mannitol were kept at a constant minimum concentration of each. This result was in agreement with that reported by Hazzah et al.16 Characterization of Placebo Wafers Moisture Content The remaining moisture content in the lyophilized wafers ranged from 5% to 8%, which was beneficial in maintaining wafer elasticity. Higher amount of residual water was observed in formulations plasticized with GL, which can be attributed to the hygroscopic nature of GL. This result was also in agreement with that observations reported by Hazzah et al.16 Hydration and Erosion Capacity for Placebo Wafers Hydration capacity is an important parameter for biological application and wound healing. It presents the capacity of wafer to adsorb wound exudates, making it suitable for moderate to high exuding wounds.26 Alginate is a weak polyacid, with pKa values of 4.0 and 3.5 for 1,4linked (beta)-D-mannuronic acid units and 1,4-linked (alpha)-L-glucuronic acid units, respectively. GE type B is a polymeric ampholyte with carboxyl (COOH) and amido (NH) groups with an isoelectric point of pH 4.9. Under pH 7.5 which simulates wound fluid exudate, both SA and GE exist as polyanions owing to the ionization of the carboxyl groups whereas the amido of GE remains unionized. This ionization improves the formation of hydrogen bond with water and enhances the hydration capacity.27 The hydration and swelling capacity was observed to be higher in case of using GE alone as it reached to 1190.7% after 15 min. On the other hand, when SA was used alone, the swelling capacity was only 255.4% after 15 min. Moreover, in case of SA and GE mixtures, when the amount of GE was increased in the mixture from 0.75 to 1.5, there was an increase in the swelling capacity from 497.7% to 717.6% after 15 min. Therefore, increasing the amount of GE in the mixture (SA/GE 1.5/1.5) offered higher hydration capacity when compared to SA/GE 2.25/0.75 (Fig. 1). These findings were in agreement with those of Abruzzo et al.,28 who reported that the presence of a greater amount of GE provided higher water uptake ability. This behavior could be attributed to the abundancy of ionized amino acids in GE structure and consequently

Data analysis was carried out using Microsoft Excel 2010. Results were expressed using mean and standard deviation. Statistically significant differences were determined using 2-tailed and Student t-test. p < 0.05 was described as the level of significance. Results and Discussion Screening of Different Polymers for Wafer Formulation SA and GE were used in preparation of wafers. GE solely failed to produce mechanically stable wafer, which is in a good agreement with the study by Kuijpers et al.25 who attributed that to poor mechanical properties and low thermal stability of GE. These problems can be improved by cross-linking and combining with other polymers. Moreover, using a mixture of SA and GE in different

Figure 1. Hydration and erosion capacity of placebo wafers in phosphate buffer pH 7.5 at 25 C.

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Figure 2. SEM micrographs of placebo wafers. (a) Cross section of PL SA/GE GL5M0.25, (d) surface view of PL SA/GE 1.5/1.5 GL5M0.25 (magnified 150).

2.25/0.75

GL5M0.25, (b) cross section of PL SA/GE

to the presence of free charges favoring water uptake. The opposite was reported by Choi et al.,29 which might be attributed to using other grades of SA and GE. The formulated wafers were characterized by a delayed erosion capacity. This is beneficial in decreasing frequent dose administration. SA wafers were completely degraded and eroded in 120 min. On the other hand, GE wafers were degraded in 360 min. Mixtures of SA and GE with different ratios were shown to have intermediate hydration, and erosion rates at 1.5/1.5 and 2.25/0.75 showed complete degradation after 240 and 165 min, respectively. Porosity During the freeze-drying stage, porous structure wafers were formed. SA wafers showed 1487.12% porosity whereas GE wafers showed more porous structure of 3051.01%. Furthermore, the mixtures of SA and GE at a concentration of 2.25/0.75 and 1.5/1.5 showed porosity percentages of 1712.51% and 2012.81%, respectively. Increasing the amount of GE resulted in a more porous wafer structure. A similar observation was reported by Nguyen et al.30 who reported that addition of higher percentages of GE resulted in a highly porous scaffold. Consequently, these findings were considered as a further confirmation for the results obtained by hydration test. Thus, mixtures of SA and GE (2.25/0.75 and 1.5/1.5) were selected to be loaded with diosmin nanocrystals. Morphologic Examination Using SEM As shown in Figure 2, SA and GE mixture in different weight ratios (2.25/0.75 and 1.5/1.5) showed a highly porous structure in both cross sections and surface views. More porous structure was observed in PL SA/GE 1.5/1.5 GL5M0.25, as concluded from hydration and porosity results. FT-IR Spectroscopy Figure 3 shows the FT-IR spectra of SA, GE, physical mix (1:1), and PL SA/GE 1.5/1.5 GL0M0. As shown from the IR spectrum, it was confirmed that there was no interaction between GE type B and SA.

1.5/1.5

GL5M0.25, (c) surface view of PL SA/GE

2.25/0.75

Thus, using mixture of both would lead to formulation of successful wafers, making GE type B an optimum choice. Preparation and Evaluation of Diosmin Nanocrystals Preliminary investigation of most suitable stabilizer regarding particle size and stability was carried out. HPMC E15, MC, and poloxamer 407 were screened at different concentrations (diosmin-stabilizer weight ratio, 1:0.5, 1:1, and 1:2) (data not shown). From the preliminary results obtained, diosmin nanocrystals prepared using antisolvent technique and using HPMC E15 or MC at a ratio of 1:1 as stabilizer were selected for loading into wafers, where both F1 and F2 showed particle sizes of 276.9 nm and 295.8 nm with PDI of 0.43 and 0.44, respectively. In Vitro Drug-Release Testing The poor solubility of diosmin makes its in vitro release testing a very challenging step. The presence of a suitable amount of sodium hydroxide in the release medium is a must to achieve sink condition. Sodium orthophosphate buffer of pH 12 was the only buffer system that was reported for diosmin dissolution medium due to its solubilization capacity for diosmin.31 The use of sink condition in release testing for nanosuspension would not be discriminative between different formulations which were also investigated (data not shown). Therefore, nonsink condition was adopted for the dissolution testing for better discrimination between different formulations.32 In Vitro Drug-Release Study in Nonsink Condition pH 10 In vitro release experiment using nondiffusion system was conducted in borate buffer at pH 10. The saturation solubility in borate buffer at pH 10 was about 4-fold higher than at pH 9. Thus, it was selected as a discriminating medium for the present study. Diosmin nanosuspension formulations were compared with the raw diosmin powder. A significant difference in dissolution rate between nanosuspension formulations and the raw diosmin

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Figure 3. IR spectrum of (a) sodium alginate, (b) gelatin, (c) physical mixture (1:1), and (d) PL SA/GE

powder (control formulation) was observed (p ¼ 0.00164, <0.05; Fig. 4a). The dissolution rate in control formulation did not exceed more than 15% over 3 h. Based on the previous results, F1 and F2 were the most stable formulations with higher dissolution rate, making them suitable candidates for further experiments and to be converted into lyophilized form. In vitro release experiment in borate buffer at pH 10 using dialysis method was also conducted. This was done to avoid any possible misleading absorbance results obtained by nanoparticles of size less than 220 nm that could pass through millipore filter. As shown in Figure 4b, the percent released from nanosuspension formulations F1 and F2 was about 5-fold more than the control formulation. Furthermore, this experiment showed a similar release as that observed in case of using dissolution method. Therefore, this confirmed the ability of using dissolution method for evaluation of nanocrystal formulations. Transmission Electron Microscopy Spherical diosmin nanoparticles (158-249 nm) were formed when HPMC E15 was used as a stabilizer (Fig. 5). In addition, spherical-less aggregated nanoparticles (159-185 nm) was

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1.5/1.5

GL0M0 wafer.

observed when MC was used as a stabilizer. Particle size results were lower than the mean data obtained from particle size analysis. Both HPMC E15 and MC accumulated on the surface of nanoparticles and provide steric hindrance (Fig. 5). Lyophilization of Nanosuspension The ultimate challenge for a pharmaceutical nanosuspension to be commercially available is the stability issue. Therefore, drying using lyophilization technique is considered a must. Before lyophilization process, a cryoprotectant is often added to the nanosuspension for maintaining the structure of the lyophilized powder. Characterization of Lyophilized Powder Particle Size Analysis. The average particle size of lyophilized diosmin nanosuspension formulations was 308.2 and 304.0 nm for F1.DM1 and F2.DM1, respectively, when mannitol was used at a concentration of 1%. In Vitro Drug Release The amount released from nanosuspension (F1) after 45 min was decreased from 68.3% to 29.8% when converted to freeze-dried

Figure 4. (a) In vitro release profile of diosmin nanosuspension formulations (F1 and F2) and freeze-dried formulations by dissolution method (dissolution medium 500 mL borate buffer pH 10, 100 rpm, 37 C). (b) In vitro release profile of different diosmin nanosuspensions by dialysis bag method (dissolution medium 20 mL borate buffer pH 10, 100 rpm, 37 C).

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Figure 5. TEM micrograph of diosmin nanosuspension. (a) F1 (diosmin-HPMC E15 1:1), (b) F2 (diosmin-MC 1:1).

powder having no mannitol (F2.DM0). The underlying reason for this dissolution behavior is agglomeration of the nanoparticles during freeze-drying process, as dissolution rate is clearly particle size dependent. Therefore, addition of a matrix-former to these nanosuspensions before freeze-drying was a must, in order to preserve the dissolution advantage offered by nanosizing. The amount released from F2.DM1 after 45 min was increased to 63% as mannitol was added at a concentration of 1% w/w. Similar observations were found for F2. XRD Analysis The XRD pattern of diosmin powder revealed high sharp intensity peaks, suggesting the crystalline nature of the drug. The diffraction peaks of F1.DM1 and F2.DM1 showed that both formulations were partially amorphous as there was a decrease in the intensity of diosmin characteristic peaks (Fig. 6).

Preparation of Wafers Loaded With Diosmin Nanosuspension (Thickening Method) For loading diosmin nanosuspension into wafers, both F1(diosmin-HPMC E15 1:1) and F2 (diosmin-MC 1:1) were thickened using either SA/GE 2.25/0.75 or SA/GE 1.5/1.5, and using GL as plasticizer at a concentration of 5% w/w (based on solid content). Moreover, mannitol was used at a concentration of 1% w/w (based on total weight). It was observed that all the 4 formulations were physically unacceptable as the formed wafers shrunk and no porous structure was formed. The structure formed was similar to film structure in appearance. This could be attributed to the mechanical properties of GE that are determined by the triple-helix content in GE solution.33 The addition of water-miscible organic solvents as DMSO might have destructed the structure of GE network (conversion of triple helix

Figure 6. XRD patterns of (a) F2.DM1, (b) F1.DM1, and (c) coarse diosmin powder.

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Table 2 Average Particle Size (nm) and PDI of Diosmin-loaded Wafers (With Different Percentage of Mannitol) After Dispersion in Water Formulation Code

Average Particle Size (nm) ± SD

F1 (Diosmin nanosuspension before lyophilization) HPMC SA/GE 2.25/0.75 GL10M1 HPMC SA/GE 1.5/1.5 GL10M1 HPMC SA/GE 1.5/1.5 GL10M0.03 F2 (Diosmin nanosuspension before lyophilization) MC SA/GE 2.25/0.75 GL10M1 MC SA/GE 1.5/1.5 GL10M1 MC SA/GE 1.5/1.5 GL10M0.03

276.9 349.0 321.1 490.9 295.8 358.4 313.3 497.0

± ± ± ± ± ± ± ±

16.49 19.66 26.25 22.62 9.77 31.97 37.52 18.46

PDI ± SD 0.43 0.50 0.46 0.55 0.44 0.46 0.40 0.60

± ± ± ± ± ± ± ±

0.02 0.01 0.06 0.04 0.04 0.03 0.07 0.02

PDI, polydispersity index; SD, standard deviation.

to random coiled structure). Thus, the addition of DMSO perturbed the structural stability of SA/GE system.34 Based on this, for the present study, it was planned to lyophilize the nanosuspension before dispersing into the gel matrix.

Preparation of Wafers Loaded With Lyophilized Diosmin Nanocrystal (Dried Nanocrystal Method) Wafers were prepared by dispersing the freeze-dried powders into gel matrix SA/GE at a concentration of either 2.25/0.75 or 1.5/1.5. Twelve formulations were prepared for testing 2 variables: GL (plasticizer) content and the overall mannitol content per wafer (Table 1). All the developed formulations were physically acceptable with a wafer-like structure. Wafers with 5% w/w GL were more brittle and tough. On the other hand, using 10% w/w GL yielded more flexible and nonbrittle wafers. Thus, GL at a concentration of 10% w/ w was selected for further trials (13.8 mg/wafer). Additional 4 formulations were prepared using different mannitol concentrations to evaluate its effect. The first 2 formulations (HPMC SA/GE 1.5/1.5 GL10M0.03 and MC SA/GE 1.5/1.5 GL10M0.03) were prepared by dispersing F1.DM0.03 or F2.DM0.03 in gel matrix 1.5/1.5. The freeze-dried powder was sticky and difficult to be dispersed in the gel matrix (Table 1). The second 2 formulations were prepared by dispersing F1.DM2 or F2.DM2 in gel matrix 1.5/1.5. The freeze-dried powder was easily dispersed in gel matrix. However, the formulated wafers were hard,

brittle, and nonflexible as the mannitol content was very high per each wafer, so they were physically unacceptable. Based on the previous results, 6 successful wafer formulations were selected for further investigation: HPMC SA/GE 2.25/0.75 GL10M1, HPMC SA/GE 1.5/1.5 GL10M1, MC SA/GE 2.25/0.75 GL10M1, MC SA/GE 1.5/1.5 GL10M1, HPMC SA/GE 1.5/1.5 GL10M0.03, and MC SA/GE 1.5/ 1.5 GL10M0.03. Characterization of Diosmin-Loaded Wafers Particle Size Analysis of Diosmin Loaded Into Wafers. As shown in Table 2, incorporation of diosmin nanocrystals stabilized by HPMC E15 in either gel matrix SA/GE 2.25/0.75 or 1.5/1.5 using mannitol at a concentration of 1% w/w had a particle size of 349.0 or 321.1 nm, respectively, compared to F1 nanosuspension before lyophilization (276.9 nm). This indicates that the process does not affect the particle size. However, using mannitol with lower concentration (0.03% w/w) increased the average particle size to 490.9 nm. Similar finding was observed for nanocrystals stabilized by MC where only slight increase in particle size was detected (358.4 or 313.3 nm when incorporated in both gel matrices 2.25/0.75 or 1.5/ 1.5, respectively. This could be attributed to the presence of mannitol that minimized aggregation of the freeze-dried nanocrystals. In Vitro Drug Release (Cup Method). In vitro release testing using cup method for HPMC SA/GE 2.25/0.75 GL10M1, HPMC SA/GE 1.5/1.5 GL10M1,

Figure 7. In vitro release profile comparing release rate of nanosuspension (F1 and F2), freeze-dried powder (F1.DM1 and F2.DM1), and wafers loaded with diosmin nanocrystals (HPMC SA/GE 2.25/0.75 GL10M1, HPMC SA/GE 1.5/1.5 GL10M1, MC SA/GE 2.25/0.75 GL10M1, and MC SA/GE 1.5/1.5 GL10M1; dissolution medium 500 mL borate buffer pH 10, 100 rpm, 37 C).

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Figure 8. Hydration and erosion capacity of diosmin-loaded wafers in phosphate buffer pH 7.5 at 25 C.

MC SA/GE 2.25/0.75 GL10M1, and MC SA/GE 1.5/1.5 GL10M1 in comparison with control (diosmin powder loaded into wafer) was conducted. The results showed that incorporation of diosmin nanocrystals into a wafer resulted in a sustained-release pattern over 480 min when compared with nanosuspension and freeze-dried powder formulations (Fig. 7). This could be due to the presence of the polymer which controls the release with one or more physical processes including polymer hydration and swelling.35 Upon contact of a dry polymeric wafer with a moist wound surface, wound exudate penetrates into the polymer matrix. This causes hydration and subsequent swelling of the dressing to form a gel over the wound surface. The swelling behavior of the polymer to form a gel acts as a barrier to drug diffusion. The thick gel layer formed on the swollen wafer surface is capable of preventing matrix disintegration and controlling additional water penetration and retarding drug release. Therefore, the rate of drug release is determined by the rate of diffusion of dissolution medium (exudates) into the polymer matrix.35,36 It is worth mentioning that less than 20% of diosmin was released from control formulation, which contained only raw diosmin, indicating that the nanosystem we developed helped increasing the release rate of the diosmin.

MC SA/GE 1.5/1.5 GL10M1 was selected for the in vivo study via topical route of administration. Unexpected release behavior might occur during diosmin release at skin temperature (32 C). Therefore, further confirmation for the release study was done at 32 C for the selected formulation and other formulations that will be used during in vivo study (MC SA/GE 1.5/1.5 gel, diosmin powder wafer, and diosmin powder gel). To explain the release kinetics of diosmin from the selected formulations (HPMC SA/GE 1.5/1.5 GL10M1 and MC SA/GE 1.5/1.5 GL10M1), the data of in vitro release experiment were fitted into different models: zero-order, first-order, Higuchi model, and Korsmeyer-Peppas model. The in vitro release profiles of diosmin from both formulations could be expressed best by KorsmeyerPeppas model, as they showed high linearity (high r2 values). In addition, values of n were 0.5 in both formulations, confirming Fickian diffusion. Hydration and Erosion Capacity for Diosmin-Loaded Wafers As shown in Figure 8, diosmin nanocrystals stabilized by either HPMC or MC dispersed in gel matrix SA/GE 1.5/1.5 showed a delay in the erosion rate. The wafers were completely degraded and eroded after 300 and 390 min for HPMC E15 and MC, respectively.

Figure 9. In vitro mucoadhesion profile of placebo and loaded wafers using Brookfield texture analyzer.

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Figure 10. Histopathologic examination of islets of Langerhans in pancreas of (a) normal rat and (b) diabetic rat (magnified 100).

Furthermore, when the same nanocrystals were incorporated in the other gel matrix SA/GE 2.25/0.75, faster erosion rate was observed. The wafers were completely degraded in 210 and 225 min in case of HPMC E15 and MC, respectively (Fig. 8). This might be due to the more hydrophilic nature of HPMC E15 compared to MC.37 Furthermore, during the experiment, wafers loaded with MC maintained their structure, whereas HPMC E15-loaded wafers were converted into to a gel-like mass within 1 h. This might hinder their application on skin wounds and burns. Based on the previous results, MC-based wafers provided promising results. Thus, they were selected for mucoadhesion testing. In Vitro Mucoadhesion Testing Adhesion plays an important role in determining an ideal wound dressing as it improves the bioavailability of the drug.38 As shown in Figure 9, placebo wafers with higher GE content (SA/GE 1.5/1.5) showed higher adhesive force than SA/GE 2.25/0.75 due to the higher hydration capacity of GE (Fig. 1). Diosmin-loaded wafers showed higher cohesiveness (stickiness) when compared with placebo wafers as it contains higher percent of GL. Similar observation was reported by Boateng et al.,17 where the increase in GL concentration offered higher increase in the stickiness, as the OH groups in GL structure form hydrogen bond with the wounded skin model (GE equilibrated with phosphate buffer pH 7.5). Thus, the formed wafers would have higher capacity to adhere to the wound surface and protect the wound from external environment.

Based on the previous studies, MC SA/GE 1.5/1.5 GL10M1 wafer offered the highest degrees in hydration and mucoadhesion capacity in addition to a sustained diosmin release over 8 h. Thus, this formulation was selected for further tests and for in vivo study. Effect of Aging It is worth mentioning that the average particle size of diosmin nanocrystals was increased from 295.8 to 825.0 nm after 6 months of storage. The increase in particle by time was an indication for the presence of agglomerations and crystal growth formation. However, a slight increase in particle size from 313.3 ± 37.52 to 395.4 ± 36.22 was observed upon storage for 6 months. This indicates that wafers as a dosage form were able to maintain nanocrystal stability regarding the particle size. Nonsignificant decrease in release pattern was observed after 6 months of storage (p ¼ 0.1256, >0.05). The amount released after 90 min was decreased from 50.2% to 46% after 6 months of storage. Results indicated that incorporation of diosmin nanocrystals in wafer matrix maintained nanocrystal stability. In Vivo Evaluation Induction of Diabetes STZ is an antimicrobial agent (2-deoxy-2-(3-methyl-3nitrosoureido)-D-glucopyranose) synthesized by Streptomyces achromogenes. It has been also used as a chemotherapeutic

Figure 11. Application of wafer on the induced ulcer in diabetic rats. (a) Application of half wafer daily, and (b) application of intact wafer day after day.

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Table 3 Stages of Ulcer Healing of Different Therapies Formulation Code MC SA/GE

1.5/1.5

Day 0

Day 2

Day 4

Day 6

Day 8

Day 10

GL10M1 wafer

Diosmin powder wafer

Placebo wafer

MC SA/GE

1.5/1.5

gel

Diosmin powder gel

Control (untreated)

alkylating agent. In 1963, it was reported that STZ is diabetogenic.39 STZ is a hydrophilic compound that is not able to freely pass the cellular membrane without additional action of specific protein transporters. So, it enters B cells via GLUT2 transporters and this mechanism is tightly connected to its glucose-like structure. Therefore, considering a similar structure of STZ and glucose, and also the fact that it enters beta cells through GLUT2 transporters, glucose can competitively limit STZ uptake by B cells. This means that glucose is able to protect STZ-induced toxicity in B cells. As glucose protection against the toxicity of STZ is concentration dependent, a long fasting is performed before STZ administration.40 The beginning of disease is considered to be the state of permanent hyperglycemia which occurs 48 h after STZ injection. Therefore, blood glucose level was measured before STZ injection and for 3 days after injection. Rats having blood sugar level above 250 mg/dL were selected for study.

Monitoring of Body Weight. While studying the hyperglycemia in rats, loss in body weight was monitored. The body weight decreased from 225.0 ± 17.90 g to 187 ± 15.55 g and from 220.0 ± 13.36 g to 181.4 ± 12.40 g in group I and group II, respectively. The loss in body weight could be explained by the insufficient insulin that prevented the body from getting glucose from blood into the body cells to use as energy. The body started burning fats and muscles to get energy that led to overall body weight reduction. The same observation was reported by Coskun et al.41 where a decrease in body weight from 228 ± 8 g to 201 ± 7 g was observed in diabetic rats that did not receive treatment. Histopathologic Examination. At the end of the study period, pancreatic biopsies of normal and diabetic rats were examined in order to confirm that diabetes was not reverted. As shown in Figure 10b, destructive, degenerated, necrotic changes and shrinking in the islets of Langerhans were observed compared to those for normal rats (Fig. 10a).

Evaluation of Maintenance of Diabetes Monitoring of Blood Glucose Level. During the experiment, blood glucose level was monitored periodically every 3 days to confirm that diabetes was not reverted.

Dose Application of Wafer and Gel As the diabetic ulcers were highly exuding, gel suffered from short residence time on the ulcer site. The applied gel rapidly absorbed fluid

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from ulcer and lost its rheological characteristics and detached easily form skin, decreasing the efficacy of gel. To overcome such limitation, rats were anesthetized and the gel was added dropwise while making sure that it was well absorbed before the addition of the following drop, which was continued till the desired dose was reached. It was observed that the wafer showed faster healing rate compared to the gel form of the same formulation, which was attributed to longer retention time of wafers on the ulcer, with no loss in diosmin dose. The daily dose applied for each ulcer was equivalent to 3.4 mg diosmin (1.25 g gel or half wafer by weight). Each wafer contained 6.8 mg diosmin. Therefore, the amount applied for each ulcer was half wafer daily. In the pilot experiment, when comparing the healing effect of a half wafer daily or intact one day after day, a similar effect was observed (Fig. 11). During the last days in the study and after formation of crusts on ulcers, the application of gel was very tedious as the gel penetration into the ulcer was hindered by the crust formed. However, the strong mucoadhesive properties of the wafers prevented its detachment from the ulcer. It is worth mentioning that, to prevent infection of ulcer, adhesive tape was applied around the ulcer after applying either gel or wafers. Evaluation of Ulcer Healing Ulcers were monitored visually and digitally photographed every 2 days for 10 days (Table 3). The progressive changes in ulcer area were measured periodically every second day. Vernier caliper was used to determine the diameter of the half circle which was fitted to Image analysis software (Image J, National Institute of Health). This software is able to use the given data to set a scale for the image and then determine the area of the irregular ulcer automatically (Fig. 12a). In addition, the wound closure % was calculated (Fig. 12b). Shrinkage of ulcer is a necessary feature for the healing process; therefore, the wound area was measured every 2 days (Fig. 12a). In addition, percent of wound closure was calculated to estimate the healing efficiency of different formulations (Fig. 12b). Diosmin nanocrystals loaded in either wafer or gel significantly enhanced wound closure % compared to diosmin raw coarse powder dispersed in gel (p ¼ 0.00208, <0.05; Fig. 12b). At day 6, the percent of wound closure in wafers loaded with diosmin nanocrystals or coarse particles was 70.7% and 28.13%, respectively. Furthermore, gels loaded with diosmin nanocrystals or coarse particles showed 59.66% and 28.00% wound closure, respectively, at day 6 (Fig. 12c). Therefore, particle size reduction in diosmin enhanced its healing and anti-inflammatory effect. It was found that the percentage of wound closure in wafertreated groups was higher than that for the gel-treated groups (Fig. 12b). This might be attributed to the porous structure of wafers which was beneficial for presenting the effective support and adherence to skin tissue for optimum wound-healing process in addition to the higher accuracy in delivering the required dose in case of wafers as previously mentioned. Diosmin-free wafers (placebo wafers) showed slow rate of healing compared to diosmin-loaded wafers in the form of either nanocrystals or coarse powder (Table 3). This is a further confirmation that the healing effect was attributed to the diosmin nanocrystals. At day 8, homeostasis was clear in case of diosmin-treated groups in the form of either nanocrystals or coarse powder compared to the bloody wound observed in non-diosmin-treated groups (Table 3), resulting in deep skin scar in case of ulcers treated with placebo wafer and control group. Wafers loaded with diosmin nanocrystal (MC SA/GE 1.5/1.5 GL10M1 wafer) showed the highest healing efficiency (Table 3 and Fig. 12b), as wafers possess high porosity and strong mucoadhesive

Figure 12. (a) Ulcer area of different groups of rats receiving different treatments for 10 days. (b) Percentage of ulcer closure of different groups of rats receiving different treatments for 10 days. (c) Percentage of wound closure at day 6 for different groups of rats receiving different treatments.

activity. Consequently, wafers as a dosage form might be considered superior over gels. Histopathologic Examination. For further confirmation of the quality and maturity of the healed tissues, full-thickness sections of normal healthy skin, treated ulcers, and control untreated ones were collected and stained with hematoxylin and eosin for microscopic examination of skin layers. The in vivo study revealed acceleration of wound closure rate in ulcers treated with either MC SA/GE 1.5/1.5 GL10M1 wafer or MC SA/ GE 1.5/1.5 gel compared to other treatment groups including control (untreated) one. This indicates the efficiency of diosmin nanocrystals in the healing process by reducing neutrophils and

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Figure 13. Histopathologic examination of (a) normal skin, (b) treated with MC SA/GE 1.5/1.5 GL10M1 wafer, (c) treated with MC SA/GE 1.5/1.5 gel, (d) treated with diosmin powder wafer, (e) treated with diosmin powder gel, (f) treated with placebo wafer, (g) control untreated group, at day 10 post-ulcer induction (magnified 100).

subsequent over-inflammation responses. As a result of the decrease in neutrophil number, macrophages invaded and refilled the wound site. Re-epithelialization starts with the migration of epithelial cells from the surrounding epidermis at the wound edge. The consequent rapid re-epithelialization prevents water loss and thus reducing the time of exposure to potential infections. Figure 13 shows the histopathologic images for the 2 study groups receiving different therapies after 10 days of ulcer incision. The results showed that MC SA/GE 1.5/1.5 GL10M1 wafers (Fig. 13b) had a great impact on decreasing time of wound healing and fast tissue regeneration. A complete re-epithelialization, well-organized dermal layers, well-formed granulation tissue and mature collagen bundles were also observed. Despite the acceleration of wound healing by diosmin powder in the form of either wafer or gel (Figs. 13d and 13e) compared to placebo wafer and control ones (Figs. 13f and 13g), there was still incomplete re-epithelialization associated with inflammatory cell infiltration and a very low organized granulation tissue. Furthermore, as shown in Figure 13g, the presence of edema and perivascular hemorrhage and a marked reduction in capillary ramification were observed in the control group. It also exhibited thicker epithelial layer, inflammatory infiltrates, and disorganized fibroblasts with the absence of collagen fiber deposition. The fast wound closure rate and dense epidermis were noticed in MC SA/GE 1.5/1.5 GL10M1 wafer-treated group, indicating that the application of these wafers triggered healing by decreasing water loss and bacterial infection during wound-healing process.

Furthermore, these diosmin nanocrystaleloaded wafers potentially enhanced the healing process by hindering the infiltration of neutrophils and stimulating re-epithelialization. Therefore, diosmin nanocrystal wafers were found to be superior in promoting the formation of the collagen and making re-epithelization in lesser time compared to using diosmin nanocrystals in a gel form or using diosmin powder in either a gel or a wafer. Despite the great improvements in skin wound care, chronic wounds as diabetic ulcers still remain challenging. Selection of proper treatment is also a challenging step. Skin wound healing is impaired in STZ-hyperglycemic rats, making this a suitable model for impaired wound healing in diabetic patients. Diabetes-impaired healing process is complex and includes vascular, neuropathic, immune function, and biochemical abnormalities.42 The massive expansion in medical and pharmaceutical wound care has led to considerable attention being placed on the development of wound dressing materials. An ideal dressing material should ensure that the wound remains free of infection and moist with exudates but without any maceration. Moreover, it should permit the exchange of gases and maintain an impermeable layer to microorganisms in order to prevent secondary infection.43,44 The use of alginate in dressing can be attributed to its ability to form gels in contact with moisture. Its high moisture absorption occurs via the formation of a strong hydrophilic gel, which limits wound secretions and minimizes bacterial contamination and offers a high level of exudation absorbency. Alginate dressings maintain a physiologically moist environment that promotes

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healing and the formation of granulation tissue. Furthermore, alginate fibers trapped in a wound are readily biodegraded.45,46 GE is another natural polymer that has been used in a wide variety of wound dressings owing to its high water absorption capacity and hemostatic properties in bleeding wounds. It is a collagen-derived connective tissue protein with unique gelation properties attributed to a physical cross-linking of the triple-helix conformation of native collagen.43 Conclusion In conclusion, the developed diosmin nanocrystaleloaded wafer using a mixture of SA and GE showed to be of great value owing to its characteristics: high porosity, high swelling index, prolonged degradation time, and high mucoadhesion properties. In addition, the prepared wafers had good strength and the ability to enhance tissue regeneration through providing the necessary balance of moisture and controlling inflammation. Based on their proven ability in treatment of highly exuding diabetic ulcers in our study, we expect that the formulated wafers could add significantly to the pharmaceutical wound-healing dressing market. Not only the prepared wound dressing would add to the pharmaceutical benefits regarding efficacy but also the use of natural products and a biodegradable polymer provided a more patient- and environment-friendly system. References 1. Khuwaja AK, Khowaja LA, Cosgrove P. The economic costs of diabetes in developing countries: some concerns and recommendations. Diabetologia. 2010;53(2):389-390. 2. Kandhare AD, Raygude KS, Kumar VS, et al. Ameliorative effects quercetin against impaired motor nerve function, inflammatory mediators and apoptosis in neonatal streptozotocin-induced diabetic neuropathy in rats. Biomed Aging Pathol. 2012;2(4):173-186. 3. Shukla A, Rasik AM, Patnaik GK. Depletion of reduced glutathione, ascorbic acid, vitamin E and antioxidant defence enzymes in a healing cutaneous wound. Free Radic Res. 1997;26(2):93-101. 4. Snyder BJ, Waldman BJ. Venous thromboembolism prophylaxis and wound healing in patients undergoing major orthopedic surgery. Adv Skin Wound Care. 2009;22(7):311-315. 5. Hinchliffe R, Valk G, Apelqvist J, et al. A systematic review of the effectiveness of interventions to enhance the healing of chronic ulcers of the foot in diabetes. Diabetes Metab Res Rev. 2008;24(S1):S119-S144. 6. Vivas A, Escandon J, Lebrun E, Choudhary S, Tang J, Kirsner RS. New therapies for treatment of diabetic foot ulcers: a review of current clinical trials. Surg Technol Int. 2010;20:83-96. 7. Blakytny R, Jude EB. Altered molecular mechanisms of diabetic foot ulcers. Int J Low Extrem Wounds. 2009;8(2):95-104. 8. Benavente-García O, Castillo J. Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity. J Agric Food Chem. 2008;56(15):6185-6205. 9. Acar T, Aydin R, Aid E, Hospital T. Efficacy of micronized flavonoid fraction on healing in thermally injured rats. Int J Angiol. 2009;15(2002):13-16. 10. Tajana A, Bocassanta P, Micheletto G, Orio A. Results of the use of topical diosmin (venosmine) in the treatment of acute hemorrhoid pathology. Minerva Med. 1988;79(5):387-390. 11. Sikareepaisan P, Ruktanonchai U, Supaphol P. Preparation and characterization of asiaticoside-loaded alginate films and their potential for use as effectual wound dressings. Carbohydr Polym. 2011;83(4):1457-1469. 12. Naseri-Nosar M, Ziora ZM. Wound dressings from naturally-occurring polymers: a review on homopolysaccharide-based composites. Carbohydr Polym. 2018;189:379-398. 13. Boateng JS, Auffret AD, Matthews KH, Humphrey MJ, Stevens HNE, Eccleston GM. Characterisation of freeze-dried wafers and solvent evaporated films as potential drug delivery systems to mucosal surfaces. Int J Pharm. 2010;389(1e2):24-31. 14. Elsner JJ, Egozi D, Ullmann Y, Berdicevsky I, Shefy-Peleg A, Zilberman M. Novel biodegradable composite wound dressings with controlled release of antibiotics: results in a Guinea pig burn model. Burn. 2011;37(5):896-904. 15. Freag MS, Elnaggar YSR, Abdallah OY. Lyophilized phytosomal nanocarriers as platforms for enhanced diosmin delivery: optimization and ex vivo permeation. Int J Nanomedicine. 2013;8:2385-2397. 16. Hazzah HA, Farid RM, Nasra MMA, El-Massik MA, Abdallah OY. Lyophilized sponges loaded with curcumin solid lipid nanoparticles for buccal delivery: development and characterization. Int J Pharm. 2015;492(1e2):248-257.

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