Accepted Manuscript Title: Lyophilized Sponges Loaded with Curcumin Solid Lipid Nanoparticles for Buccal Delivery: Development and Characterization. Author: Heba A. Hazzah Ragwa M. Farid Maha M.A. Nasra Magda A. EL-Massik Ossama Y. Abdallah PII: DOI: Reference:

S0378-5173(15)00542-6 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.06.022 IJP 14974

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

9-4-2015 12-6-2015 14-6-2015

Please cite this article as: Hazzah, Heba A., Farid, Ragwa M., Nasra, Maha M.A., EL-Massik, Magda A., Abdallah, Ossama Y., Lyophilized Sponges Loaded with Curcumin Solid Lipid Nanoparticles for Buccal Delivery: Development and Characterization.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.06.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Lyophilized Sponges Loaded with Curcumin Solid Lipid Nanoparticles for Buccal Delivery: Development and Characterization. Heba A. Hazzah1, Ragwa M. Farid1, Maha M. A. Nasra2, Magda A.EL-Massik1, Ossama Y. Abdallah2,

5

Heba A. Hazzah: [email protected] "Corresponding Author" Cell phone:

+2 0100 50 38347

Office: +2 (03) 3877122 Pharmaceutics Department 10

Faculty of Pharmacy and Drug Manufacturing Pharos University in Alexandria, Canal el Mahmoudia St., Smouha, Alexandria, Egypt

Ragwa Mohamed Farid:[email protected] 15

Maha Mohamed Adel Nasra:[email protected] Magda Abdelsamie ElMassik:[email protected] OssamaYousef Abdallah:[email protected]

20

Department of Pharmaceutics, Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria, Alexandria, Egypt. 2 Department of Pharmaceutics, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt. 1

1

Graphical abstrct 25

Abstract This study aimed to prepare and evaluate mucoadhesive sponges as 30

dosage forms for delivering solid lipid nanoparticles. For this purpose curcumin (Cur) was formulated as solid nanoparticles (SLN) using Gelucire 50/13, and Polaxomer 407. The prepared CurSLN dispersion was thickened with different mucoadhesive polymers. Different concentrations of glycerol, and mannitol of range (0.25%to 20%), and

35

(0% to 1%) respectively were also examined. The formed gel was poured into oblong molds and freeze dried to form mucoadhesive sponge to be applied to the buccal mucosa. The prepared sponges were evaluated for their, in-vivo residence time, in-vitro and in-vivo drug release, and hydration capacity. Surface morphology for the different

40

sponges were examined using SEM. TEM was also carried out for sponge

fragments

previously

dispersed

into

water.

Infrared

spectroscopy was conducted to investigate interaction between used ingredients. The results showed that the CurSLN loaded HPMC, and Polycarbophil sponges showed 4, and 15 hours in-vivo residence time 45

respectively, providing a considerable amount of curcumin into saliva. The incorporation of glycerol and mannitol at concentration of 1% provided elegant and flexible sponges. The SEM showed that the deposition of CurSLN differed according to the type of polymer used.

2

TEM confirmed the integrity of liberated CurSLN from sponges. IR 50

spectra showed an interaction between HPMC and poloxamer 407, which affected its behaviour as a gelling agent. The obtained results provide an efficient approach for delivering solid lipid nanoparticles in a solid dosage form keeping the nanoparticle characters and integrity. Keywords—

55

Curcumin,

mucoadhesive,

Sponge,

Solid

Lipid

Nanoparticles, Abbreviations: Cur: Curcumin, SLN: Solid lipid nanoparticles, P; polycarbophil sponge, H; HPMC sponge, Gl: glycerol, M: mannitol, G50/13: Gelucire50/13, PX407: poloxamer 407.

60

1.

Introduction

The promises held within drug delivery systems using solid lipid nanoparticles (SLN), necessitate the investigation of the feasibility of delivering such systems through an administrable dosage forms. Generally, the applicable and most commonly used dosage forms for 65

the treatment of oral mucosal diseases are tablets, films or gels [Bruschi and de Freitas 2005]. Tableting or film casting may jeopardize the integrity of the SLN structure and size due to the stress or heat the SLN will be subjected to. Although gels could be considered suitable , its main limitation is the short in-vivo residence

70

time (25 min) [Hazzah HA, Farid RM et al. 2015]. Therefore in attempt to increase the mucoadhesion time, solid dosage form in the form of sponge, a porous structure, was considered for loading SLN.

3

Sponge could be defined as a dispersion of gas (usually air) into a solid matrix to give a porous solid structure [Lai, Abu'Khalil et al. 75

2003]. It is an alternative to tablets but lacks the compression step. Preparation procedure depends mainly on using polymers, and a freeze drying procedure to eliminate all water incorporated leaving a soft solid structure with acceptable flexibility. Lyophilization is a preferred drying method as it overcomes most limitations associated

80

with the formulation of lipid based formulated system. Lyophilized formulations offer stable products, extend shelf life and allow storage of products at room temperature instead of refrigeration. Sponges have been developed in several studies either as wound dressing [Dai, Zheng et al. 2009; Rossi, Marciello et al. 2012; Sandri,

85

Bonferoni et al. 2013] , ocular application [Refai and Tag 2011], and buccal for trans mucosal delivery [Portero, Teijeiro-Osorio et al. 2007; Kassem, ElMeshad et al. 2014]. A special interest has been shed on the natural gift, the golden pigment from the golden spice, Curcumin (Cur). The pace of curcumin research

90

has grown rapidly, with thousands of citations studying its antioxidant [Sharma

1976],

anti-inflammatory[Gupta,

Kim

et

al.

2010],

antimicrobial[Negi, Jayaprakasha et al. 1999], cancer chemopreventive and potentially chemotherapeutic properties [Prasad, Tyagi et al. 2014]. However, it suffers poor chemical stability, rapid metabolism, 95

and photochemical instability[Tønnesen, Karlsen et al. 1986]. Various strategies have been taken to overcome curcumin limitations and to allow its therapeutic application, including the incorporation in

4

delivery systems [Mazzarino, Travelet et al. 2012; Sun, Bi et al. 2013; Peng, Lee et al. 2014; Prasad, Tyagi et al. 2014]. Nanoscale particles 100

may represent a future where activity is ensured, and the problems associated with using medicinal plants are overcome [Bonifácio, da Silva et al. 2014]. Recently, application of curcumin locally to oral mucosa has been reported to be an efficient approach for treatment of precancerous oral lesions at a low dose 6mg/day for six week course

105

treatment [Hazzah HA, Farid RM et al. 2015]. The study adopted the use of mucoadhesive gel loaded with curcumin solid lipid nanoparticle. The results obtained intensify the importance of delivering curcumin to the oral mucosa with higher in-vivo residence time. Solid lipid nanoparticles loaded sponge aiming to target buccal mucosa

110

has not been studied. In this domain, the work aimed at shedding lights on new approaches to optimally deliver CurSLN (curcumin solid lipid nanoparticles) into single unit sponge, in addition to examining the effect of freeze drying process and formulation variables on SLN integrity and sponge characters.

115

2.

Materials:

Curcumin (Hebei food additive Co.,Ltd China), Poloxamer 407 (Kolliphore 407, a sample gift from BASF, Germany), Polycarbophil (Noveon AA-1 a sample gift Lubrizol, Belgium) , Potassium dihydrogen phosphate, Sodium lauryl sulphate (SLS), Glycerol, and 120

Mannitol (El-Nasr pharmaceutical Co. Egypt), Tri-ethanol amine (TEA) (Nice Chemicals, Pvt.ltd, kerla, India), Gelucire 50/13(Stearoyl macrogol-32 glycerides) (Kind gift from Gattefosse, France), HPMC 5

4000, were kindly provided by Pharo Pharmaceutical Co., Borg ElArab city, Alexandria, Egypt, Carboxy methyl cellusloe sodium (CMC 125

sodium), Polyvinyl alcohol PVA (Mwt30,000, Merck ), Gellan Gum, and Sodium alginate (BDH Chemical Ltd., Poole, England), Mucin was obtained from porcine stomach, Type II, (Sigma, St Louis, USA)

130

3. Methods 3.1.

Formulation of sponges

3.1.1.

Preparation of placebo sponge

Six placebo gels of different polymers (CMC sodium, polyvinyl alcohol, gellan gum, HPMC 4000, Polycarbophil, chitosan) were 135

prepared. A calculated amount of each polymer was added to distilled water (except for chitosan was added to 1% w/w acetic acid) on a magnetic stirrer (Wisestir® (DAIHAN-scientific co., ltd. Seoul, Korea)) until homogenous gel was obtained. The concentration of polymer was kept at 2 % w/w. Glycerol (GL) as a plasticizer and mannitol (M) as a

140

cryoprotectant were kept at a concentration of 0.25% w/w of the gel formula (i.e 10 % of solid content of sponge calculated on dry base). The gels were kept in a refrigerator for 2 hours to remove entrapped air bubbles. Ten gram of each homogenous gel was poured into 10 wells plastic

145

moulds of dimensions (0.5cm x1cm x2cm). The molded gel was frozen at -25oC for 4 hours prior lyophilization step using lyophilizer ( Lyph 6

lock ® 4.5,LABconco. Kansas, USA) at vacuum set at 40 mTorr, for 10 hours.

150

3.1.2. Preparation of curcumin solid lipid nanoparticles The lipid (5% Gelucire 50/13) was melted at 55-60 oC followed by the addition of (0.6%) curcumin. Calculated amount of aqueous phase, (water to 100%) with 8% Poloxamer 407 maintained at 70-80 oC squirted gently into the lipid (oil) phase under magnetic stirring at 600

155

rpm. Next, the mixture underwent high-shear dispersion at 12,000 rpm for five minutes using homogenizer T18 ULTRA-TURRAX® (IKA, Germany). The emulsion obtained was cooled gradually to room temperature forming SLN. 3.1.3 Characterization of curcumin solid lipid nanoparticles

160

3.1.3.1. Measurement of particle size (PS) and polydispersity index (PDI)

The average of three for particle size and polydispersity index of the solid lipid nanoparticles formula was determined using Nano-ZS Zeta-sizer (Malvern Instruments, Malvern, UK). SLN dispersion was diluted 165

20 times with double distilled water filtered through 0.45 μ membrane filters to ensure that the light scattering intensity was within the instrument’s sensitivity range.

3.1.3.2. Entrapment efficiency determination

7

One ml SLN dispersion diluted to 10 folds was centrifuged using 170

cooling centrifuge (Centurion Scientific Ltd, UK) at speed 15, 000 rpm, for one hour at 4oC. The supernatant was carefully separated, appropriately diluted, and filtered through syringe milli-pore filter 0.2 um to remove any suspend particulates. The amount of drug in the filtrate was measured spectrophotometrically at λ max 420 nm.

175

%EE was calculated as follows:

(1)

Where Wi is the amount of initial drug and Wf is the amount of free 180

drug

3.1.3.3.

Zeta potential(ZP) measurement

The ZP of the SLN dispersion was measured at 25°C, under an electrical field of 40 V/cm using the Nano-ZS Zetasizer. The measurements were carried out in triplicates.

185

3.1.3.4.

Transmission electron microscopy (TEM)

A drop of the SLN dispersion was placed on a membrane coated grid surface with a filter paper. Uranyl acetate was used for staining. Any excess fluid was removed from the carbon grid after staining whose

8

surface was air dried at room temperature before being loaded onto the 190

transmission electron microscope (TEM, JOEL, CX, Japan).

3.1.3.5.

Differential scanning calorimetric analysis (DSC)

DSC analysis was carried out using Pyris series DSC6 (PerkinElmer, Germany). A thin layer of the selected samples were spread and sealed in aluminum pans under a nitrogen-air atmosphere at a flow rate of 195

20ml/min and evaluated in 35°C–400°C temperature ranges. Selected formula CurSLN (Gelu50/13-PX407) was evaluated in comparison to the references raw materials (Cur, Gelucire50/13, and Poloxamer 407).

3.1.4. Preparation of Sponges loaded with curcumin solid lipid nanoparticles 200

The CurSLN dispersion was thickened using either HPMC or polycarbophil at a concentration of 2%. Glycerol and mannitol were added at different concentration for studying their effect on sponge formation (Table1). Cur dispersion of 0.6% concentration dispersed in 8 % Poloxamer 407 was thickened using same polymers for

205

comparison. Gels containing 0.6% curcumin was prepared for comparison. The formed gels was moulded same as described in section 3.1.1. 3.2.

Characterization of prepared sponges

3.2.1. Physical appearance

210

The formed sponges were physically evaluated concerning brittleness, separation or stickiness from mould, and integrity.

9

3.2.2. In-vivo mucoadhesion residence time

Six human healthy volunteers (22-45 years old, 5 female, one male) agreed to participate in this study. The mucoadhesive sponges (placebo 215

and curcumin loaded ones) were selected for the in-vivo evaluation. The volunteers were asked to place the sponge on the gingiva in the region of the upper canine. They were then asked to monitor the time necessary for complete disappearance of sponge particulate or residual gels. The work described has been carried out in accordance with the

220

Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans [Association. 2001]. Each subject was adequately informed of the aims, methods, sources of funding, any possible conflicts of interest, institutional affiliations of the researcher, the anticipated benefits and potential risks of the study

225

and the discomfort it may entail. The subjects were informed of the right to refuse to participate in the study or to withdraw consent to participate at any time without reprisal.

230

3.2.3. Scanning electron microscopy (SEM)

The external surface of the sponges was examined by SEM by placing them 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 235

sponge. The sponges were then sputter coated with gold placed in the

10

chamber of a SEM (JSM 5300, JOEL, Japan), and images acquired using i-scan2000 software.

3.2.4. Transmission electron microscopy (TEM)

TEM was used for examining the integrity of CurSLN in lyophilized 240

sponge. Sponges loaded with CurSLN, were dispersed in water, and sonicated for 15 minutes, samples were diluted and a drop of the SLN dispersion was placed on a membrane coated grid surface with a filter paper, the sample was treated similarly as described under section 3.1.3.4.

245

3.2.5. Porosity

The porosity was calculated mathematically in terms of total pore volume [Ho, Kuo et al. 2004]. The bulk volume of the total 250

ingredients composing one sponge before lyophilization process was calculated according to the following equation:

(1) Where m, ρ are the mas s and density of each ingredient/sponge 255

The true volume of sponge was calculated by the sponge dimension

(2) 11

260

The total pore volume was calculated as follow:

(3)

265

3.2.6. Moisture content Samples of sponges were carefully weighed (Wi) and heated to 100 oC till constant weight (Wf) the moisture content was calculated as a % weight loss according to the following formula

270

(4)

3.2.7.

Hydration capacity

The hydration capacity of the CurSLN-loaded sponge (of the selected formulae) was carried out in triplicates. Sponges were held on a 275

stainless steel mesh and immersed in 10 mL of phosphate buffer (pH 6.8) simulating salivary pH [Khairy 2014] . The swelling behavior was observed at predetermined time intervals. Samples were removed with the mesh, blotted off carefully on tissue papers to remove the surface-adhered liquid droplets and reweighed to constant weight. The

280

percentage of water uptake was calculated as follows:

(5)

12

where Ws is the weight of the hydrated sponge , and Wi is the initial weight of sponge.

3.2.8. Fourier- Transformed Infrared spectroscopy (FT-IR)

285

IR spectra of HPMC4000, Poloxamer 407, and (HPMC-Poloxamer 407 co-precipitate), were recorded on a Bruker IFS 66V FT-IR spectrometer. IR spectra were obtained over the range 350–4400 cm−1 by the ATR (attenuated total reflection) technique. 3.2.9. In-vitro mucoadhesion assessment

290

Mucoadhesive capacity of sponge was evaluated by applying the sponge on an agar plate normalized with mucin (pH 6.8). The petri dish was incubated at 37oc at an inclination angle 30, distance covered by sponge over different time intervals were recoded (1, 2, 3, and 24 hours). Any change in sponge integrity observed was also recorded.

295

All measurements were performed in triplicates (mean ±SD)[Farid, Etman et al. 2013].

3.2.10. In-vitro drug release testing

A sponge with definite weight equivalent to 0.6 mg Cur was transferred to a cellophane bag 4 cm long with a 12, 14 kDa cut off 300

(VISKING dialysis tubing, SERVA, electrophoresis, Germany). The bags were transferred to ambered glass bottles containing 10 ml of water: ethanol (1:1), in horizontal shaking water bath at 100 strokes/min, adjusted to 37±0.5 oC. The whole volume was withdrawn at different time intervals (0.5, 1, 2, 3, 4, 5 hr) and replaced with an 13

305

equivalent volume of a previously warmed fresh medium.

The

concentration was detected using UV spectrophotometer at λ max 430 nm. The percent release was calculated as the cumulative drug released at these different time intervals [El-Refaie, Elnaggar et al. 2015] [Khairy 2014]. All measurements were performed in triplicates (mean 310

±SD). Similarity factor (f2) was calculated using Excel 2010 data sheet.

3.2.11. In-vivo release of selected sponges

Formulated sponges (HCurSLNGL1M1) (PCurSLNGL1M1) containing 6 mg curcumin were evaluated for their in-vivo release. Five adult healthy 315

volunteers, four females and one male (age range 30–50 years) were enrolled. No restrictions concerning food intake was put in order to mimic normal day activity except 15 minutes before the sampling interval, no eating or drinking was allowed. The subjects were asked not to touch the sponge with their tongue. Blank saliva samples were

320

taken before sponge installation. At fixed intervals, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 12, 14, and 15 hours saliva samples were collected in a tube containing 1ml acetone (as a solvent) and 5mg SLS ( as a stabilizer) and kept frozen at −25 ◦C until analysis. Samples were centrifuged, at 15,000 rpm 4oC, for 15 min, the supernatant was carefully separated,

325

appropriately diluted, and measured using UV-spectrophotometer.

14

330

3.3.

Statistical analysis

Characterization and in-vitro release data were expressed as percentage or mean (±SD) and analyzed by unpaired Student t-test (Excel 2010 data sheet). 4. Results and Discussion 335

4.1.

Screening of different polymers for sponge formulation

HPMC sponges produced without glycerol and mannitol addition were brittle. HPMC sponges prepared containing only glycerol (0.25%) (i.e 2.5 mg/sponge) showed good flexibility; addition of mannitol (0.25%) (i.e 2.5 mg/sponge) was also beneficial. 340

Incorporation of both,

glycerol and mannitol gave more flexible and more elegant structure. From this finding, it was concluded that presence of glycerol and mannitol is essential for production of elastic sponge. In screening of gelling polymers, glycerol and mannitol were added to avoid brittleness and stickiness to mould and were kept at constant minimum

345

concentration of each, so as to eliminate other variables. Placebo sponges were prepared to select the most appropriate gelling agent to form the sponge regarding to: physical appearance, taste, patient compliance, and mucoadhesion characters. The results were discriminative, where the use of PVA showed a complete failure, while

350

it was successful for the other five used polymers (Chitosan, CMC sodium, gellan gum, HPMC and polycarphophil). However, chitosan taste was not quite acceptable where the residual acetic acid caused acidic taste. In addition, the in-vivo adhesion was only 15 minutes. Chitosan sponges swelled and formed cotton-like mass structure that 15

355

rapidly detached from the mucosa. This could be due to precipitation and coagulation of chitosan in the salivary pH. The other CMC sodium and gellan gum based sponges showed very short in-vivo residence time ranging from 15 to 30 minutes. The polymers over swelled and detached from the gingiva rapidly. On the other hand HPMC, and

360

polycarbophil were promising and showed in-vivo residence times 3 and 6 hours respectively. Based on these findings, Polycarbophil and HPMC were selected for further investigation to guarantee high mucoadhesive performance. 4.2.

365

Characterization of curcumin solid lipid nanoparticles

Curcumin solid lipid nanoparticles showed a particle size of 298 ±49.1 nm with a PDI 0.476 ±0.11. The entrapment efficiency was 88.07 % ±0.5, and Zeta potential was (– 15mV). Although , value for zeta potential obtained was low, stearic stabilization can maintain the system stability even if the system is of low zeta potential, where

370

similar results were described by Shah et al [Shah, Eldridge et al. 2014]. In addition, it was assumed that since the formulation will be thickened with a gelling polymer, and transformed to a sponge will help stabilization of the system and prevent particles aggregation.

Investigating the crystallization behaviour of constituents used and 375

SLN using differential scanning calorimetric analysis was carried out. The thermograms of the bulk lipid Gelucire 50/13, Curcumin, Poloxamer 407, and CurSLN(G50/13-PX407), are shown in Figure 1 . The melting process for Gelurice50/13 and curcumin took place with

16

maximum peaks at 53°C and 183°C, respectively. DSC studies showed 380

that the lipid Gelucire50/13 was completely in crystalline form. The Curcumin peak was lost in the formulation, indicating its solubilization in the nano lipid system.

4.3. 385

Characterization of curcumin solid lipid nanoparticles

loaded sponge

4.3.1. Physical appearance For loading CurSLN into sponge, CurSLN(G50/13-PX407) dispersion was thickened using either HPMC 4000 /or Polycarbophil. Table 1 represents different combinations of plasticizer and cryoprotectant, and 390

they were screened for acceptable lyophilization behaviour and physical elegance of the resulting products. For glycerol, (0.25 % to 20% ) (i.e 2.5 to 200 mg/sponge) were used, while for mannitol it ranged from (0% to 1%)( i.e 0-10mg/sponge). There was increased flexibility with increasing glycerol content as it

395

can lower ice surface allowing the sponge to accommodate the stress generated during lyophilization [Kasper and Friess 2011]. Glycerol 1% (10 mg per sponge) produced adequately flexible, tough and non-brittle sponge. However, using high concentration of glycerol 20% (200 mg/sponge) hindered the freezing and icing process which affected the

400

formation of sponge with definite structure. Sponges formed were of indefinite shape, sort of over spilling during the drying phase occurred, where the gel formed did not adequately freeze as a result it could not

17

withstand vacuum during lyophilization process. Moreover, the mass formed was tremendously porous, and sticky. Similar observation was 405

reported by Ayensu et al [Ayensu, Mitchell et al. 2012], when the authors increased the amount of glycerol , wafers produced were sticky. Glycerol, as a plasticizer acts by interposing itself between polymer chains resulting in reduced interaction and, reduction in the

410

intermolecular cohesive forces between polymer chains [Boateng, Stevens et al. 2009]. In conclusion, the optimum concentration for glycerol was 1% (10 mg/ sponge. On the other hand, the cryoprotectant mannitol at a concentration of 1% (10 mg per sponge) was also found effective in terms of visual appearance and flexibility as compared to

415

sponges with mannitol concentrations (0%, 0.25% and 0.5 %). Glycerol and mannitol, structurally and functionally, act as plasticizers and cryoprotectant respectively [Di Tommaso, Como et al. 2010]. Ayensu et al [Ayensu, Mitchell et al. 2012] have reported that using both compounds at reduced concentrations has a better effect while

420

creating a synergistic plasticizing effect compared to the effect achieved by a single agent at a higher concentration. Regarding physical integrity and prevention of sponge structural damage after freeze drying process, formulae containing equal proportions of both glycerol and mannitol 1 % (10 mg/sponge) showed

425

to be the most promising. The prepared sponges were orange, flexible, porous, and of uniform weight, texture and thickness. They were elegant with no visible cracks. They easily regain their original

18

structure upon gentle squeezing. Such optimized characteristics make these formulae suitable for easy application. The mould used was 430

selected to be of oblong-like shaped to provide a dosage form of relatively large surface so it could be easily applied to the oral lesion, in addition to provide sponge with acceptable thickness that would be acceptable by patients. 4.4.

435

Sponge Morphology : Scanning electron microscopy (SEM)

Studying the morphology of sponge was necessary to identify the sponges' structure and better understand their behaviour. Different factors have been studied, to examine their effect on sponge structure. 4.4.1. Effect of curcumin loaded For better evaluation, and comparison, sponges (using the two selected

440

polymers HPMC, and Polycarbophil) were loaded with: Cur powder directly dispersed in the gel matrix), Cur dispersed into Poloxamer 407 and, the one originally of interest CurSLN (G50/13-PX407). It is illustrated in (Figure 2 A, B) that HPMC and polycarbophil sponges loaded with curcumin powder showed perfect porous structure. It is

445

worth mentioning that sponges loaded with curcumin dissolved in Poloxamer showed different structure than that without Poloxamer. It has been shown that the incorporation of poloxamer into HPMC matrix resulted in stuffed and dense matrix compared to HPMC sponges with Cur which showed to be more porous in structure. On the other hand

450

incorporation of poloxamer into polycarbophil did not affect much the porosity of the sponge, on the contrary it showed smooth surface (Figure 2C, D). By higher magnification, SEM micrographs showed 19

Cur crystals aggregates popping out of the sponge surfaces in a needle like shape. The results showed that more aggregates were formed in 455

case of using HPMC as a matrix (Figure 2 E, F) Loading of CurSLN also affected the sponge morphology where, SEM micrographs showed that polycarbophil sponges possess more porous structure and smoother texture than the HPMC sponges. As an overall, incorporation of SLN affected the sorting and lamellar structure of the

460

sponge. It was obvious that SLN interfered with the networking formed in between the polymer chains during freeze drying process (Figure 2G, H). Interestingly, CurSLN loaded into a sponge showed significantly different behaviour, according to the polymer used. SEM micrographs for HPMC sponges showed that SLN were deposited on

465

the surface. This supports the observation taken during preparation where the consistency of HPMC gel differed upon addition of CurSLN, and a sort of phase separation was detected which indicated that lipid particles floated onto the surface of HPMC gel, and upon freeze drying lipid particles deposited on the surface.

470

On the other hand, the SEM micrographs of polycarobophil sponge showed no SLN on its surface, some nodules covered with a layer of polymer were observed protruding of the sponge surface. A cross section of the sponge filaments found nodule like filling, indicating that the SLN are embedded into the gel matrix. This could be due to

475

the utter compatibility of lipid, with the gel used and Poloxamer (Figure 2 I, J).

20

480

4.4.2. Effect of mannitol

It is worth mentioning that the incorporation of mannitol affected the arrangement of sponge lattice. Mannitol as a cryoprotectant helps the solution to maintain some flexibility in a glassy state [Wang 2000]. The interposing of plasticizer and cryoprotectant between the polymer 485

chains, and their interaction with functional groups of polymers result in reduced intermolecular cohesive forces between polymer chains. This could also improve the mucoadhesive of the formulated polymer due to less rigidity, ease of hydration and consequent degradation (Figure 3).

490 4.5.

Transmission electron microscopy (TEM)

TEM examination was done to confirm that the sponge formation process did not affect the integrity of the SLN particles; the sponges 495

were dispersed in water to liberate SLN. As shown from the TEM micrographs, SLN kept their integrity in the sponge matrix indicating that the whole process of preparation and lyophilization did not affect the SLN structure (Figure 4). In addition, TEM examination supports the results obtained upon SEM

500

examination, where SLN were free and not bounded to any polymer residual matrix in case of HPMC sponge (Figure 2 I), which previously appeared to be floating on the surface. On the other hand, SLN liberated from polycarbophil sponges showed to be bound to the gel matrix surrounding its surface all around, which 21

505

is a further confirmation for micrographs obtained by SEM showing nanoparticles embedded into the gel matrix (Figure 2 J)

4.6.

510

Moisture content

Determination of moisture content was carried out to investigate the efficiency of freeze drying process to eliminate water content of the formulations. The values are presented in Table 1. It is shown that HPMC sponges generally kept moisture traces ranging from 1.8 to 3.5%, while for polycarbophil sponges, most of formulae showed no

515

moisture traces except for cases where higher amount of glycerol was used, (0.4, 0.8%). The formula (PCurSLNGL20M1) containing 20% glycerol showed 7.7% residual amount of water which could be due to the hygroscopic nature of glycerol, in addition, this particular concentration hindered the lyophilization process where glycerol

520

interfered with the icing process.

4.7. Porosity During the freeze drying stage, porous structure is generated as after water removal, the remained space that was originally occupied by 525

the solvent becomes pores offering the sponge structure. By investigation, the porosity of polycarbophil sponge (PCurSLN GL1M1) and HPMC (H

CurSLN

GL1M1) were calculated to be 82.77 %, and

51.53% respectively. These findings are further confirmation for the micrographs obtained by SEM, which was previously attributed to 530

the interaction of HPMC with Poloxamer 407 lowering its ability to 22

swell adequately upon formulation prior freeze drying step, resulting in lower amount of water in between spaces , thus leading to a more condense and less porous structure. 4.8.

535

Hydration capacity

Adequate hydration capacity of a buccal adhesive system is a critical

property for uniform and controlled release of the

therapeutic agent and effective mucoadhesion [Ismail, Napaporn et al. 2000]. The hydration capacity was observed to be higher in case of polycarbophil than HPMC. The cross section of both sponges 540

showed that the polycarbophil sponges have more porous structure than those of HPMC. The maximum hydration capacities of the optimized samples formulae obtained were within 200 min of incubation for polycarbophil sponge (PCurSLN GL1M1 ) while 120 min for HPMC sponge (HCurSLN GL1M1). This could be due to the

545

more porous structure formed allowing more water absorption (Figure 5)

4.9.

Fourier –Transformed Infrared spectroscopy(FT-IR)

A behavioural change of HPMC when concomitantly used with 550

poloxamer 407 was observed. This change was presented in a form of loose gel, and a less porous sponge upon freeze drying. It was also observed upon SEM topography investigation that HPMC sponge was unable to entrap SLN.

Accordingly, FTIR was carried out to

23

investigate the possible interaction between HPMC and Poloxamer 555

407. The infrared spectra of HPMC4000, Poloxamer 407, and their aqueous co-precipitate are shown in (Figure 6). The IR spectra showed slight shifting in the broad OH band of HPMC (3556.24-3368.3) cm-1 to (3394.14) cm-1.

560

This could be attributed to hydrogen bonding

formation between HPMC and ether oxygen of polyoxyethylene of poloxamer [Kondo and Sawatari 1996]. This possible bonding could be an acceptable justification for the HPMC behaviour in the presence of poloxamer, where HPMC did not show the same gelling behaviour when dispersed into water.

565 4.10. In-vivo residence time

In-vivo residence times of the selected formulae (HCurSLNGL1M1), and (PCurSLNGL1M1) were 4 ±1 h, and 15 ±2.5 h respectively. Several theories have been proposed to describe mucoadhesion, it is generally 570

accepted that the adhesive interaction between the polymeric system and the mucus covered substrate is facilitated by initial wetting of the dosage

form on the

biological

substrate,

followed

by the

interpenetration of the polymeric chains into the mucus layer [Smart 2005]. Then a secondary bond formation (e.g. van der Waals/hydrogen 575

bonding) maintain the intimate contact between the two surfaces. Selection of the polymeric components can modify the mucoadhesive properties of formulations.

24

It has been reported [Woolfson, Malcolm et al. 2002] that the mucoadhesive properties of dosage forms are, in part, dependent on the 580

state of the mucoadhesive polymer within the final product. Consequently, the mucoadhesive properties of formulations in which the polymers are fully hydrated, (e.g. solutions, dilute gels) will be markedly less than those in which the mucoadhesive component is dispersed within dosage forms which are solid in nature, e.g. films or

585

compacts. Accordingly, mucoadhesive hydrated systems show relatively lower mucoadhesive interactions and limited polymer diffusion into mucin. Conversely, in systems where the mucoadhesive components are presented as solids or in systems where there is limited aqueous fluid (e.g. semi-solids, films), contact with mucin facilitates

590

polymer chain hydration and expansion, leading to deep penetration into and interaction with mucin [Jones, Bruschi et al. 2009]. The significant difference between both sponges tested could also be attributed to the hydration capacity of both, where polycarbophil showed more hydration capacity which allowed more swelling and

595

more interaction with mucin. In addition, the lower residence time shown by HPMC sponges could also be due to the deposition of lipid onto the surface lowering the adhesion property to the oral mucosa. While, for polycarbophil sponges, lipids where embedded into the matrix thus not interfering with adhesion characters of polycarbophil.

600

25

4.11. In-vitro mucoadhesion studies

Agar, a carbohydrate based gel, as the representative mucosal substrate 605

due to the presence of mucin (cysteine rich) that augmented the adhesion process was used for this investigation. The mucoadhesion properties were tested by displacement method represented as downwards

movement

of

sponges

under

investigation.

The

displacement is inversely proportion with mucoadhesion property of 610

the tested sponge. The experiment was set to measure the distance covered by sponge upon inclination of 30o. Both formulae showed no displacement or detachment over 3 hours. It is worth noting that the polycarbophil sponge (PCurSLN GL1 M1) kept its structure throughout the whole experiment even when observed after

615

24 hrs but it showed to be softer. On the other hand, the HPMC-sponge (HCurSLN GL1 M1) formed a gel mass around the spot of application but did not show any displacement as well. The rapid and strong adherent of the sponge to the mucin-agar surface could be due to the hygroscopic character of both glycerol and mannitol. This could have

620

aided in the stronger initial attachment of the formulations to the model mucoadhesive surface. 4.12. In-vitro release testing

In-vitro release testing using dialysis method was conducted to the chosen formulae (HCurSLN GL1 M1, PCurSLN GL1 M1) in comparison to 625

their corresponding sponges loaded with curcumin in poloxamer

26

(HCurpx407GL1

M1,

PCurpx407GL1

M1).

CurSLN

(G50/13-PX407)

dispersion was also tested for comparison. Generally, the results showed that incorporation of Cur, CurSLN into a sponge decreased the amount of drug release compared to that from 630

CurSLN dispersion. Upon sponge hydration, the polymer regains its gel structure leading to increase in diffusional path length of the drug which may delay the release. Furthermore, the thick gel layer formed on the swollen sponge surface is capable of preventing matrix disintegration and controlling additional water penetration hence

635

retarding the drug release[Rodriguez CF, Bruneau N et al. 2000]. Figure 7 shows that the percent drug release from the different curcumin formulations under study was in the following order; CurSLN > HCurSLN GL1 M1> PCurpx407GL1 M1 , HCurpx407GL1 M1> PCurSLN GL1 M1.

640

Figure 7 shows that the amount of drug released from (HCurSLN GL1 M1) was much higher than its corresponding sponge containing curcumin in poloxamer407 (HCurpx407GL1 M1,) with a similarity factor f2 =35, as in this case, CurSLN were observed to be deposited on to the sponge surface upon investigation using SEM, so the gel matrix effect on the

645

drug release was minimal. For the same reason, the amount of drug release from (HCurSLN GL1 M1) was also higher than that from the tested polycarbophil sponge (PCurSLN GL1M1) with a similarity factor f2 =30 in which the SLN were embedded into the matrix which required more time to be released into the dissolution medium.

27

650

On the other hand, as expected, the amount of curcumin released from (PCurSLN GL1M1) was lower than that its corresponding formulation containing curcumin in poloxamer (PCurpx407GL1 M1) with f2 =59. This could be attributed to the nature of dispersed curcumin into the sponge, where (PCurSLNGL1M1) contains curcumin encapsulated into lipid

655

nanoparticles, while (PCurpx407GL1 M1) contains curcumin dispersion in poloxamer 407.

4.13. In-vivo release

The in-vivo Cur concentrations released from optimized mucoadhesive 660

sponges (PCurSLNGL1M1), (HCurSLNGL1 M1). The mean release curve demonstrates a striking difference in release rate between different sponges (Figure 8). Detectable Cur concentrations were present in saliva even after the complete erosion of the sponge (14, 15 hrs). It is clear that the mucoadhesive sponge has a greater ability to sustain an

665

elevated Cur concentration in saliva. This could be attributed to the higher in-vivo residence time imparted by polycarbophil sponge matrix

The high standard deviation values obtained from in-vivo release can be justified by the difference in salivary flow between volunteers, 670

which in turn had an impact on sponge hydration, hence drug release. Needless to say that salivary flow and cheek movement during normal day activity as talking and food intake could play an important role in the adhesion time and drug release from the buccal mucoadhesive formulation. The fluctuation in the release peaks might be due to the 28

675

daily activity as no restriction was added to patients. Also, it could be due to food intake decreasing the amount recovered in saliva.

Conclusion In the light of described results it could be concluded that, CurSLN can 680

be efficiently and successfully delivered in solid dosage form that keeps its integrity. Freeze drying process can be efficiently adopted to develop sponges. Incorporation of 1% of each glycerol, and mannitol provided flexible and elegant sponge. The physical nature of drug significantly

685

affects

the

prepared

sponge

structure.

Using

polycarbophil as a gel matrix to form sponge was found superior to HPMC regarding, structure, in-vivo residence time, and in-vivo release. Polycarbophil sponge can provide a sustained release over 14-15 hrs in the buccal cavity, providing a steady concentration of curcumin thus no need for several dosing.

690

695

700 29

References:

705

710

715

720

725

730

735

740

745

Association., W. M. (2001). " World medical association declaration of helsinki, ethical principles for medical research involving human subjects. Bull World Health Organ " 79(4): 373-374. Ayensu, I., J. C. Mitchell, et al. (2012). "Development and physico-mechanical characterisation of lyophilised chitosan wafers as potential protein drug delivery systems via the buccal mucosa." Colloids and surfaces b: biointerfaces 91: 258-265. Boateng, J. S., H. N. Stevens, et al. (2009). "Development and mechanical characterization of solvent-cast polymeric films as potential drug delivery systems to mucosal surfaces." Drug Development and Industrial Pharmacy 35(8): 986-996. Bonifácio, B. V., P. B. da Silva, et al. (2014). "Nanotechnology-based drug delivery systems and herbal medicines: a review." International Journal of Nanomedicine 9: 1-15. Bruschi, M. L. and O. de Freitas (2005). "Oral bioadhesive drug delivery systems." Drug Development and Industrial Pharmacy 31(3): 293-310. Dai, M., X. Zheng, et al. (2009). "Chitosan-alginate sponge: preparation and application in curcumin delivery for dermal wound healing in rat." BioMed Research International 2009: 1-8. Di Tommaso, C., C. Como, et al. (2010). "Investigations on the lyophilisation of MPEG–hexPLA micelle based pharmaceutical formulations." European Journal of Pharmaceutical Sciences 40(1): 38-47. El-Refaie, W. M., Y. S. Elnaggar, et al. (2015). "Novel Self-assembled, Gel-core Hyaluosomes for Non-invasive Management of Osteoarthritis: In-vitro Optimization, Ex-vivo and In-vivo Permeation." Pharmaceutical Research: 111. Farid, R. M., M. A. Etman, et al. (2013). "Formulation and in vitro evaluation of salbutamol sulphate in situ gelling nasal inserts." AAPS PharmSciTech 14(2): 712-718. Gupta, S. C., J. H. Kim, et al. (2010). "Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals." Cancer and Metastasis Reviews 29(3): 405-434. Hazzah HA, Farid RM, et al. (2015). "Curcumin: The golden spice for precancerous oral lesions treatment." Fourth Euro-Mediterranean conference of Natural Products and Drug Discovery: Back to Mother Nature (BioNat-IV) Hazzah HA, Farid RM, et al. (2015). "Treatment of Precanacerous Lesions : Curcumin a Non surgical Promising Option " 17th International Conference on Pharmaceutics , Paris Ho, M.-H., P.-Y. Kuo, et al. (2004). "Preparation of porous scaffolds by using freezeextraction and freeze-gelation methods." Biomaterials 25(1): 129-138. Ismail, F. A., J. Napaporn, et al. (2000). "In situ gel formulations for gene delivery: release and myotoxicity studies." Pharmaceutical Development and Technology 5(3): 391-397. Jones, D. S., M. L. Bruschi, et al. (2009). "Rheological, mechanical and mucoadhesive properties of thermoresponsive, bioadhesive binary mixtures composed of poloxamer 407 and carbopol 974P designed as platforms for 30

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implantable drug delivery systems for use in the oral cavity." International Journal of Pharmaceutics 372(1): 49-58. Kasper, J. C. and W. Friess (2011). "The freezing step in lyophilization: physicochemical fundamentals, freezing methods and consequences on process performance and quality attributes of biopharmaceuticals." European Journal of Pharmaceutics and Biopharmaceutics 78(2): 248-263. Kassem, M. A., A. N. ElMeshad, et al. (2014). "Lyophilized Sustained Release Mucoadhesive Chitosan Sponges for Buccal Buspirone Hydrochloride Delivery: Formulation and In Vitro Evaluation." AAPS PharmSciTech: 1-11. Khairy, H. M. (2014). Develpment and evaluation of some drug delivery systems to the oral cavity [Master Thesis], Univeristy of Alexandria;. Pharmaceutics. Masters: 140. Kondo, T. and C. Sawatari (1996). "A Fourier transform infra-red spectroscopic analysis of the character of hydrogen bonds in amorphous cellulose." Polymer 37(3): 393-399. Lai, H. L., A. Abu'Khalil, et al. (2003). "The preparation and characterisation of drugloaded alginate and chitosan sponges." International Journal of Pharmaceutics 251(1): 175-181. Mazzarino, L., C. Travelet, et al. (2012). "Elaboration of chitosan-coated nanoparticles loaded with curcumin for mucoadhesive applications." Journal of Colloid and Interface Science 370(1): 58-66. Negi, P., G. Jayaprakasha, et al. (1999). "Antibacterial activity of turmeric oil: a byproduct from curcumin manufacture." Journal of Agricultural and Food Chemistry 47(10): 4297-4300. Peng, S.-F., C.-Y. Lee, et al. (2014). "Curcumin-loaded nanoparticles enhance apoptotic cell death of U2OS human osteosarcoma cells through the Akt-Bad signaling pathway." International Journal of Oncology 44(1): 238-246. Portero, A., D. Teijeiro-Osorio, et al. (2007). "Development of chitosan sponges for buccal administration of insulin." Carbohydrate Polymers 68(4): 617-625. Prasad, S., A. K. Tyagi, et al. (2014). "Recent Developments in Delivery, Bioavailability, Absorption and Metabolism of Curcumin: the Golden Pigment from Golden Spice." Cancer Research and Treatment 46(1): 2-18. Refai, H. and R. Tag (2011). "Development and characterization of sponge-like acyclovir ocular minitablets." Drug Delivery 18(1): 38-45. Rodriguez CF, Bruneau N, et al. (2000). Hydrophilic cellulose derivatives as drug delivery carriers: influence of the substitution type on the properties of compressed matrix tablets in "Handbook of pharmaceutical controlled release technology". New York: , CRC Press. Rossi, S., M. Marciello, et al. (2012). "Development of sponge-like dressings for mucosal/transmucosal drug delivery into vaginal cavity." Pharmaceutical Development and Technology 17(2): 219-226. Sandri, G., M. C. Bonferoni, et al. (2013). "Wound dressings based on silver sulfadiazine solid lipid nanoparticles for tissue repairing." European Journal of Pharmaceutics and Biopharmaceutics 84(1): 84-90. Shah, R., D. Eldridge, et al. (2014). "Optimisation and Stability Assessment of Solid Lipid Nanoparticles using Particle Size and Zeta Potential." Journal of Physical Science 25(1): 59-75. Sharma, O. (1976). "Antioxidant activity of curcumin and related compounds." Biochemical Pharmacology 25(15): 1811-1812. 31

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Smart, J. D. (2005). "The basics and underlying mechanisms of mucoadhesion." Advanced Drug Delivery Reviews 57(11): 1556-1568. Sun, J., C. Bi, et al. (2013). "Curcumin-loaded solid lipid nanoparticles have prolonged in vitro antitumour activity, cellular uptake and improved in vivo bioavailability." Colloids and Surfaces b: biointerfaces 111: 367-375. Tønnesen, H. H., J. Karlsen, et al. (1986). "Studies on curcumin and curcuminoids VIII. Photochemical stability of curcumin." Zeitschrift für LebensmittelUntersuchung und Forschung 183(2): 116-122. Wang, W. (2000). "Lyophilization and development of solid protein pharmaceuticals." International Journal of Pharmaceutics 203(1): 1-60. Woolfson, A., R. Malcolm, et al. (2002). Bioadhesive drug delivery systems in : "Polymeric biomaterials" New York, Marcel Dekker.

815

820

825

830

835

840

845

32

Figure 1: DSC thermograms of A) Curcumin, B) Gelucire 50/13, C) Poloxamer 407, 850

and D) CurSLN (G50/13-PX407). Figure 2: SEM micrographs of cross section of curcumin loaded sponges of different matrices; A) HPMC matrix, B) polycarbophil matrix. C) HPMC sponges loaded with Cur in poloxamer (lower magnification), D polycarbophil sponges loaded with Cur in poloxamer (lower magnification), E) HPMC sponges loaded with Cur in poloxamer

855

(higher magnification), F) polycarbophil sponges loaded with Cur in poloxamer (higher magnification). G)CurSLN loaded HPMC sponge, H) CurSLN loaded polycarbophil sponge. I) surface topography in HPMC loaded Sponge, J) Cross section of a filament in Polycarbophil sponge showing nodule like filling. Figure 3: SEM micrographs of cross section into polycarbophil sponges loaded with

860

CurSLN (curcumin solid lipid nanoparticles) containing 1% mg glycerol and different amounts of mannitol: A) no mannitol, B) 0.5% mannitol, C) 1% mg mannitol Figure 4: TEM micrographs for CurSLN liberated upon dispersion of sponges in water obtained from A) CurSLN –HPMC sponges, B) CurSLN – Polycarbophil sponges. C) CurSLN dispersion (control)

865

Figure 5: Hydration capacity of selected formulae PCurSLNGL1M1, and HCurSLNGL1M1 in phosphate buffer (pH6.8) at 25 OC. Figure 6: FT-IR spectra for A) HPMC4000, B) Poloxamer 407, C) HPMC – poloxamer407 Co-precipitate (1:4)

Figure 7: In-vitro release profiles of different sponges (HPMC, Polycarbophil) loaded 870

with curcumin as solid lipid nanoparticles or cur dispersion in poloxamer407 by dialysis method (dissolution medium: 10 ml Ethanol :Water (1:1)) at 37 oC. Figure 8: In-Vivo release profile of selected formulae: PCurSLNGL1M1, HCurSLNGL1 M1.

33

875

Fig. 1

880

885

34

Fig. 2

35

890

Fig. 3

36

Fig. 4 895

37

Fig. 5

38

900

Fig .6

39

905 Fig. 7

910

Fig. 8

Table 1: Composition of different sponge formulations using 2 915

%w/w HPMC, or 2 % w/w Polycarbophil with 0.6%w/w curcumin loading, and the moisture content of the sponge %w/w.

40

Formulae Code

920

925

Glycerol (%)**

Mannitol (%)**

Moisture content(%)

0.25 0 0.5 0 1 0 1 1

2.4 3.5 2.5 1.8 2.2 ND ND ND

0.25 0 0.5 0 1 0 1 1 1 1 1

0 0 0 0 0.4 0.8 ND ND 7.7 ND ND

HPMC (2%) based Sponges 0.25 HCurSLN GL 0.25 M0.25 0.5 HCurSLN GL0.5 M0 0.5 HCurSLN GL0.5 M0.5 1 HCurSLN GL1 M0 1 HCurSLN GL1 M1 2 HCurSLN GL2 M0 1 HCur-px407 GL1 M1 1 HCur GL1 M1 Polycarbophil (2%) based Sponges 0.25 PCurSLN GL0.25 M0.25 0.5 PCurSLN GL0.5 M0 0.5 PCurSLN GL0.5 M0.5 1 PCurSLN GL1 M0 1 PCurSLN GL1 M1 2 PCurSLN GL2 M0 5 PCurSLN GL5 M1 10 PCurSLN GL10 M1 20 PCurSLN GL20 M1 1 PCur-px407GL1 M1 1 PCur GL1 M1

*P= polycarbophil, H=HPMC, GL= Glycerol, M =Mannitol

** subscripts represent the type of dispersed curcumin, and concentration of glycerol and mannitol in % of original gel formula)

41