coenzyme Q10 dual delivery

Journal of Drug Delivery Science and Technology 41 (2017) 239e250

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Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst

Lipid nanocarriers for tamoxifen citrate/coenzyme Q10 dual delivery Eman S. El-Leithy a, b, Rania S. Abdel-Rashid b, * a b

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA), Cairo, Egypt Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Helwan University, Ain Helwan, Cairo 11795, Egypt

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 February 2017 Received in revised form 19 June 2017 Accepted 27 July 2017 Available online 29 July 2017

Nanotechnology based combinatorial therapy has emerged as an effective strategy for cancer treatment due to its synergistic activity, suppression of multi-drug resistance and successful delivery to target site. The objective of this work was to develop and characterize tamoxifen citrate (TC) and coenzyme Q10 (CoQ10) lipid nanocarriers for oral breast cancer chemotherapy. Stearic acid (2%w/v) and poloxamer188 (3%w/v) were selected as optimal lipid matrix and surfactant for development of solid lipid nanocarriers (SLNs). Incorporation of lecithin into lipid matrix (SLN9) significantly reduced particle size to 180 nm and increased %EE of CoQ10 (45%). Nanostructured lipid carriers containing 10% Labrafac oil showed further decrease in particle size reaching only 81 nm and increased %EE up to 94% and 56% for TC and CoQ10, respectively. Lipid nanocapsules showed more prominent effect on decreasing particle size (36 nm). Lipid nanocarriers offered controlled drug release profiles. The study showed that lipid carriers significantly improved drugs permeation through rabbit intestinal mucosa and suggested them as potential delivery systems for improving the bioavailability of TC/CoQ10 therapeutic molecules. Subsequent studies will be performed in order to elucidate the cytotoxicity and genotoxicity of selected lipid nanocarriers formulas on MCF-7 (adenocarcinoma breast cancer cells) versus normal cells (WISH cell line). © 2017 Elsevier B.V. All rights reserved.

Keywords: Tamoxifen citrate Coenzyme Q10 Solid lipid nanoparticles Nanostructured lipid carriers Lipid nanocapsules

1. Introduction Significant efforts have been made to develop novel targeted delivery systems for breast cancer therapy that can provide higher specificity to cancerous cells with no/minimal effect on normal cells [1]. Combination therapy has evolved as a rationale strategy to increase response and to decrease resistance. The advantages attributed to combination chemotherapy include improved patient compliance due to the reduced number of administrations, ability to overcome or delay Multi drug resistance, and reduction of drug dose with consequent diminishing of toxicity to healthy tissues [2]. Combination therapy may include: additive or synergistic drug combinations of the same mechanism or connected mechanisms of action, synthetic lethality pairings, and the addition of a second agent with a different mechanistic activity to reverse resistance mechanisms [3]. Much evidence has arisen about antioxidant supplementation with cancer chemotherapy suggesting that certain antioxidant supplements may reduce adverse reactions and toxicities. Significant reductions in toxicity may alleviate dose-

* Corresponding author. Tel.: 00201156995596; fax: 002025553487. E-mail address: [email protected] (R.S. Abdel-Rashid). http://dx.doi.org/10.1016/j.jddst.2017.07.020 1773-2247/© 2017 Elsevier B.V. All rights reserved.

limiting toxicities so that more patients are able to complete prescribed chemotherapy regimens and thus, improve the potential for success in terms of tumor response and survival [4]. Oral administration of delivery systems containing lipids is believed to enhance bioavailability of drugs by enhancing transport through intestinal epithelial layer and protecting them against the hostile environment of GIT [5]. Co-administration of drug with lipid is also a successful approach to target drug through intestinal lymphatic pathway [6,7] which shows numerous advantages viz; protection against hepatic first-pass metabolism, increased bioavailability, reduced hepatotoxicity and decrease systemic toxicity of drug [8]. Lipid nanoparticles have unique characteristics such as small size, high surface area, high drug loading (both hydrophilic and lipophilic drugs), long blood circulation time and high capacity for tissue selectivity and drug targeting [9e11]. Solid lipid nanoparticles (SLNs) were developed to entrap drugs within biocompatible lipid core and a surfactant at the outer shell. They are stable in both lipophilic and hydrophilic environments for carrying and liberating drugs [12]. Different methods for SLNs preparation showed high physical stability, low aggregation, nonexpensive large-scale production and can be modulated for the desired drug release and drug targeting [13]. However, SLNs reported some disadvantages including; limited drug loading and

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drug expulsion during storage due to lipid matrix crystallization or lipid polymorphism [6]. Thus, nanostructured lipid carriers (NLCs) prepared with lipid and oil mixture were developed as a second generation to overcome limitations of SLNs. They allowed for higher drug loading capacity with controlled release due to drug dissolution in the oil and imperfections in solid lipid [14,15]. Later, Lipid nanocapsules (LNCs) were also presented as an excellent alternative to microemulsion for pharmaceutical delivery of hydrophobic drugs [16,17]. They consist of an oil-filled core allowing efficient encapsulation of several anti-cancer drugs with a surrounding polymer shell [18,19]. Efficacy of LNCs for controlling release, selective cytotoxicity, minimizing serious side effects of cancer drugs and preventing damage to healthy tissues has been recorded [20,21]. Tamoxifen citrate (TC) is a poorly soluble nonsteroidal estrogen antagonist that adheres to all stages of estrogen receptor positive breast cancer treatment, as well as, long-term prophylactic therapy in high-risk and post-menopausal women [22]. Long-term clinical application of TC revealed its extensive presystemic clearance through hepatic first-pass metabolism leading to high dose-dependent side effects such as oxidative stress, hepatotoxicity, endometrial cancer development, carcinoma, sarcoma, vaginal hemorrhage and liquid retention in postmenopausal breast cancer patients [23e25]. Coenzyme Q10 (CoQ10) is a potent antioxidant that protects cells from damage attributed to free radicals [26,27]. Prescribing vitamins (C and E), essential fatty acids, and CoQ10 for patients receiving TC therapy, showed cancer enhancing treatment outcomes and improved chemotherapy tolerability. Moreover, TC induced disruptions in the lipid profiles were reverted to near-normal levels after 90 days of treatment with TC plus CoQ10 combination therapy [28]. Increasing bioavailability of oral anticancer drugs to the therapeutically acceptable level is still a challenging goal where, poor solubility, membrane permeability, high molecular weight, and high doses-side effects have remained unsolved issues. To accomplish patients' compliance and increase healthcare system sustainability the study was focused on developing different types of lipid nanocarriers loaded with TC/CoQ10 dual therapy. The effect of formulation variables including; lipid type, lipid and surfactant concentrations, presence of lecithin in the matrix, and difference in nature of lipid nanocarrier systems were studied. The effect of lyophilization process was also studied in absence and presence of cryoprotectant. Selected formulas were subjected for in-vitro dissolution, ex-vivo permeation through rabbit intestinal mucosa and kinetic analysis to estimate the potential of the developed systems for delivering TC and CoQ10 for preliminary clinical application. 2. Materials and methods

2.2. Methods 2.2.1. Simultaneous spectrophotometric estimation of TC and CoQ10 in different lipid nanocarriers Two simultaneous equations for estimating TC and CoQ10 concentrations in their binary mixtures or pharmaceutical dosage forms at different media were previously developed by El-Leithy and AbdelRashid 2016 [29]. Equations (1)e(4) were previously developed for measuring concentrations of both drugs in methanol and simplified simulated intestinal fluid (SSIF), respectively using the drugs molar absorptivity coefficients. Simplified simulated intestinal fluid (SSIF) developed by Taupitz and Klein, 2010 [30] with certain modifications was chosen as dissolution medium to ensure complete solubility of both drugs, especially the highly lipophilic drug CoQ10. The SSIF (pH 6.5) was prepared by dissolving 3.438 g NaH2PO4 and 6.186 g of NaCl in 900 mL of distilled water. The pH was adjusted by addition of 0.1 N NaOH solution. The SSIF was reached by adding 0.25% (w/v) SLS and 0.25% (w/v) Tween 20 and the volume was completed to 1000 ml by small volume of isopropyl alcohol. The isopropyl alcohol was added to adjust pH and enhance drugs solubility [31]. TC has solubility in water 3 mg/ml, while coQ10 is completely insoluble and the sink conditions were thoroughly considered in both entrapment and release experiments. The buffer solution was placed in an ultrasonic bath for 15 min and finally stirred for another 15 min on a magnetic stirrer before use. The SSIF was stored at 25
C. Absorbance of both drugs was measured at previously identified l max 236 nm and 275 nm (JASCO V-530 double beam UV-VIS spectrophotometer connected to a computer loaded with Spectra Manager Program, Japan). The correlation between samples concentrations and their absorbencies complied with Beer's law as illustrated by high values of regression coefficients (R2 z 0.999) and small values of intercepts. The low values of SD and %RSD (<2%) are another confirmatory parameters for high precision, reproducibility and accuracy of proposed method [29]. Simultaneous equations for measuring TC and CoQ10in methanol were,

CTC ¼

76714A1  30340A2 855181848

CCoQ10 ¼

15332:12A2  10580A1 855181848

(1)

(2)

Simultaneous equations for measuring TC and CoQ10in SSIF were,

CTC ¼

68389A1  28749A2 1137279743

(3)

2.1. Materials Tamoxifen citrate(m.wt: 563.62) was generously given as a gift by Medical United Pharmaceutical Company, Cairo, Egypt. Coenzyme Q10 (m.wt: 863.34) and soybean lecithin (Phospholipon 90G, 90% w/w of phosphatidyl choline) were given as a gift from Amriya Company for Pharmaceutical Industries, Rushdie, Alexandria, Egypt. Labrafac (mixture of capric and caprylic acid triglyceride) was kindly donated from Gattefosse (Cedex, France). Stearic acid, sodium lauryl sulphate (SLS), methanol, sodium dihydrogen phosphate, isopropyl alcohol, Tween 20, sucrose and sodium chloride were purchased from EL-Gomhoria Company, Cairo, Egypt. Palmitic acid and Poloxamer 188 (P188) were purchased from Sigma Aldrich. Cellulosic membrane; Spectra/pore No.2, 12-14000D was purchased from Spectrum Laboratories, Inc., USA. All other chemicals were of analytical grade. Distilled water was used throughout the study.

CCoQ10 ¼

21128A2  10701A2 1137279743

(4)

The CTC and CCoQ10 are concentrations of TC and CoQ10, respectively in their sample solutions (mole/L) and A1 and A2 are absorbance of measured samples at 236 nm and 275 nm, respectively. 2.2.2. Development of lipid nanoparticles 2.2.2.1. Preparation of SLNs. The SLNs were prepared by meltemulsion/ultrasonication method [32e34]. Accurately weighed 15 mg of each TC and CoQ10 (Analytical balance Setra BL-410 S, USA) were firstly dissolved in 3 ml methanol to be sure that both drugs are in molecular dimension. Solution of both drugs was poured into lipid melt at 70
C to obtain clear drug-lipid mixture. Poloxamer (P188) was reported as good emulsifying agent [35,36]. The drug-lipid mixture was emulsified into30 ml preheated

E.S. El-Leithy, R.S. Abdel-Rashid / Journal of Drug Delivery Science and Technology 41 (2017) 239e250

aqueous solution of p188 at 10
C above melting point of lipid using hot plate stirrer (Jenway 1000, U.K) and mechanically stirred under 6000 rpm (IKA Works, Asia Sdn. Bhd., Malaysia)to obtain a crude emulsion. The crude emulsion was subsequently ultra-sonicated by probe ultrasonic processor Ultrasonic processor (UP50H, Hielscher, Germany) at water bath (90
C) for 10 min [37]. The formulations were allowed to cool at room temperature to obtain congealed SLNs followed by freezing of 0.5 ml at 20
c to prevent aggregation of particles to be used in particle size analysis. The effect of formulation variables including; lipid type and concentrations (stearic acid and palmitic acid at concentration levels of 2e3.5%w/v), surfactant concentrations (2e4%w/v) and incorporation of soy lecithin (amphiphilic co-surfactant) at concentration 10e30%w/w of lipid matrix was studied. Table 1 presented composition of different formulations based on the different screened variables. 2.2.2.2. Preparation of nanostructured lipid carriers. The NLCs of TC/ CoQ10 bitherapy were prepared by pre-emulsion/ultrasonication technique using oil/lipid mixture [38]. Selected lipid matrix at concentration 2%w/v was allowed to melt before addition of Labrafac® oil at concentrations of 10, 20 and 30%w/w of total lipid. Both drugs dissolved in 3 ml methanol were added into hot oil/lipid blend and then dispersed into 30 ml preheated aqueous solution of 3%w/v p188 at 70
C to form crude emulsion. The previously described procedures for precipitating lipid nanoparticles were carried out. The formulas were coded NLC1-3, respectively according to surfactant concentration. 2.2.2.3. Preparation of lipid nanocapsules. Lipid nanocapsules (LNCs) were formulated using soy lecithin and Labrafac® oil as lipid matrix at 2%w/v. The LNCs were prepared using different concentrations of Labrafac® oil (10, 20 and 30%w/w of total lipid content). Accurately weighed15 mg of each TC and CoQ10 was dissolved in Labrafac® oil. The oily solution was then mechanically stirred for 10 min into aqueous solution of lecithin and p188 (3%w/v) at 70
C until the crude emulsion was formed. Procedures previously described under SLNs preparation were also followed. The formulae were coded LNC1-3, respectively. 2.2.3. Criteria for selection of optimum dosage form Percentage yield (%yield), drug loading (%DL), entrapment efficiency (%EE) and particle size were monitored for selecting optimum formula. Data were expressed as mean of triplicate ± SD. 2.2.3.1. Determination of percentage yield of SLN formulations. Solid lipid nanocarriers suspensions were centrifuged at 13,000 rpm for 30 min at 4
C using high speed refrigerated centrifuge (XCHR20, Bio Lion, and USA). Isolated particles were weighed and referred to initial weights of solid components (equation (5)).

Percentage Yield ¼

241

SLNs weight  100 Total initial solids weight

(5)

2.2.3.2. Determination of percentage drug loading and entrapment efficiency. Percent drug loading (%DL) is the percentage of entrapped drug relative to weight of lipids incorporated in SLNs equation (6).

%DL ¼

Amount of drug entrapped in SLNs  100 Total weight of lipids incorporated in SLNs

(6)

Percent entrapment efficiency (%EE) is referred to percentage of entrapped drug relative to total drug content. The %EE was reported to be determined either by direct [39] or indirect method [40]. For accurate quantification of both drugs especially for CoQ10, which is a highly lipophilic drug and may not be easily detected in aqueous supernatant, both methods were applied for estimating TC or CoQ10 concentrations in formulated lipid nanocarriers.  Direct determination of %EE. Five milligrams of isolated lipid nanocarriers were dissolved in 25 ml methanol. The absorbance of clear solutions was measured spectrophotometrically (JASCO, Japan) at 236 nm and 275 nm. The concentrations of both drugs were calculated using the above described simultaneous equations (eq. (1) and (2)) and %EE was then calculated using (equation (7a)).

%EE ¼

Amount of drug actually present  100 Total amount of drug added

(7a)

 Indirect determination of %EE: Concentration of free drugs was measured in aqueous supernatant solution after separation of lipid nanoparticles by centrifugation at 13, 000 rpm for 30 min at 4
C in high speed refrigerated centrifuge. The absorbance of solution was measured spectrophotometrically at 236 nm and 275 nm and concentrations of both drugs were calculated (eq.(1) and (2)) and %EE was determined (equation (7b)).

%EE ¼

Total amount of drug  amount of unbound drug  100 Total amount of drug (7b)

Table 1 Composition of SLNs formulations related to different screened variables. Screened variable

Formula code

Lipid type

Lipid conc. (%w/v)

Surfactant (p188) conc. (%w/v)

Lecithin to total lipid weight. (%w/w)

Lipid type

SLN1 SLN2 SLN3 SLN4 SLN5 SLN6 SLN7 SLN8 SLN9 SLN10 SLN11

Palmitic acid

2 3.5 2 3.5 2

2

10 20 30

Surfactant concentrations

Presence of Lecithin

Stearic acid Palmitic acid Stearic acid Stearic acid

2

3 4 3 4 3

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2.2.3.3. Determination of particle size, PDI and zeta potential. Particle size, PDI and zeta potential of developed formulas were measured by photon correlation spectroscopy (Malvern zetasizer, UK). One ml sample of isolated SLNs was diluted in 10 ml double distilled water. The sample was vortexed prior to size determination. All measurements were taken as average of triplicate. 2.2.4. Effect of freeze drying process as a secondary production step with or without cryoprotectant From the above experimental evaluation tests, optimum formulation from each category of lipid nanoparticles was selected for investigating effect of freeze drying in absence or presence of cryoprotectant. The suspension of lipid nanoparticles was primary dried at 0
C for 5 h followed by 10
C for 2.5 h and 15
C for 2 h and secondary drying at 25
C for 2.5 h (Freeze drier Alpha 1e2 LD Martin- Christ- Germany). The chamber pressure and cold trap temperature was maintained at 20 Pa and 50
C, respectively for entire process (24 h). The process was performed with and without addition of 5%w/v sucrose as cryoprotectant, based on the results published by Shete and Patravale, 2013 [12]. Freeze dried formulations were characterized for their particle size, PDI, and entrapment efficiency. The Angle of repose for freeze dried powder was also measured as an indication of nanoparticles flowability. The fixed funnel method was used for estimating the angle of repose for different formulation, tan q ¼ H/r, where q is angle of repose, r is the radius, and h is the height. 2.2.5. Physicochemical characterization of optimum selected formulations Formulas that fulfill optimum criteria of highest %yield, %DE, % EE and lowest particle size were subjected to further investigation and characterization. 2.2.5.1. Compatibility studies. Differential scanning calorimetry (DSC) and Fourier transform infra-red spectroscopy (FTIR) were proposed as rapid methods for evaluating physicochemical interactions between the formula components [41]. Samples of pure excipients, pure TC and CoQ10, physical mixture or freeze dried TC/ CoQ10formulated nanocarriers were investigated for compatibility. i. Differential scanning calorimetry (DSC). Each sample was encapsulated and sealed in flat bottomed aluminum pan with crimped on lid. The sample was heated in an atmosphere of nitrogen over a temperature range 5e250
C with a constant heating rate of 10
C/min (Shimadzu DSC- 50, Japan). ii. Fourier transformation infrared spectroscopy (FTIR). The sample was mixed with KBr (IR grade) at ratio 100: 1 and scanned over a wave range of 4000e400 cm1(Shimadzu IR/FTIR spectrophotometer (435 U-O4), Japan). 2.2.5.2. Transmission electron microscope (TEM). The TEM analysis was performed to examine morphological characteristics of selected optimum lipid nanoparticles (JEOL, JEM-1230, Japan). The sample was diluted appropriately with 0.1 M phosphate buffer and placed on Cu grid stained with 2%w/v phosphotungstic acid solution to capture the particles image [37]. 2.2.5.3. In-vitro drug release study. Drug release from selected optimum formulas was investigated in in-vitro dissolution medium of SSIF buffer solution (pH 6.5), using a modified USP apparatus-I dissolution tester (Hanson research dissolution tester,

Chatsworth, USA). A sample weight equivalent to 5 mg TC was placed in an opened plastic tube wrapped at the base with cellophane membrane. The tube was attached from its open end to the bottom of the shaft replacing the basket to sink in 100 ml SSIF solution at 37
C and 100 rpm stirring speed. At predetermined time intervals, 1 ml/h of dissolution medium was withdrawn and replaced by fresh media. All samples were diluted to 10 ml with SSIF solution before spectrophotometric measurement. The release study was performed in triplicate for each sample for a period of 8hr. The concentration of TC and CoQ10 was calculated using simultaneous equations (eq. (3) and (4)). Calculated average values of %cumulative drug release were used for constructing drugs release profiles. 2.2.5.4. Kinetic analysis of release data. To investigate kinetics and mechanisms of drug release form developed nanoparticles, the release data were fitted to zero-order, first-order and Higuchi's square root time models. Correlation coefficient (R2) values was calculated by plotting Q vs. t for zero-order, log (Q0 e Q) vs. t for first-order and Q vs. t1/2for Higuchi. Where Q, is the amount of drug released at time t, and (Q0 e Q) is the amount of drug remained at time t [42]. 2.2.5.5. Ex-vivo drug permeability study. The potential of optimum formulations to allow drug permeation through GIT mucous membrane was investigated using intestinal rabbit tissue. This work was carried out according to the international guidelines for care and use of laboratory animals. The experimental protocol was ethically approved by the Animal Care Committee, Faculty of Pharmacy, Helwan University. Experimental procedures were followed according to Mukherjee et al., 2009 [43]. Male rabbits weighed 2 kg were allowed to fast overnight before starting the experiment. The anaesthetized rabbits were incised then intestinal tissue was cut off into small pieces of 5 cm length and washed with Tyrode's solution. One end of tissue was tied with surgical suture. A specified weight of each formula equivalent to5mg TC was suspended in 2 ml SSIF and introduced into ileum tissue using a syringe. The other end of the tissue was tied carefully and hanged in a beaker contains 40 ml of SSIF. The experiment was carried out at 37
C for 6 h under aeration and constant stirring rate (Organ Bath, RUMO, Egypt). At different time intervals, 2 ml sample aliquot was withdrawn from release medium and replaced with fresh one to maintain sink conditions. Samples were measured spectrophotometrically at 236 nm and 275 nm after appropriate dilution and simultaneous equations (eq. (3) and (4)) were applied for calculating drugs concentrations. Percentage drug permeated through intestinal mucosa was calculated and plotted against time in compassion to those of free drugs. The study was carried out in triplicate for all formulas. 2.2.6. Statistical analysis Data were analyzed by using SPSS 16.0 program (SPSS Inc., Chicago, IL, USA) with paired sample T-test at least significant difference at P < 0.05. 3. Results 3.1. Screening effect of lipid types and concentrations Effect of lipid type and concentration on %yield, %DL, %EE, particle size, PDI, and zeta potential of developed SLNs was studied in formulas coded SLN1-4 as illustrated in Table 2. The results showed that SLNs formulated with stearic acid at different concentration levels (SLN 3 and 4) gave higher %yield (90e93%) compared to those prepared with palmitic acid (%yield 85e87%).

E.S. El-Leithy, R.S. Abdel-Rashid / Journal of Drug Delivery Science and Technology 41 (2017) 239e250

243

Table 2 Effect of lipid types and concentrations, surfactant concentrations, and lecithin concentration on physical characteristics of SLNs. Formula code

%yield

TC

CoQ10

%(W/W)

SLN1 SLN2 SLN3 SLN4 SLN5 SLN6 SLN7 SLN8 SLN9 SLN10 SLN11

85±1.2 87±0.7 93±0.9 93±2.3 89±1.6 89±2.3 95±1.7 95±0.9 94±0.97 94.3±1.2 95±0.86

DL

EE

DL

EE

1.02±0.0 1.23±0.0 2.1±0.07 2.2±0.03 1.10±0.06 1.13±0.05 2.20±0.03 2.20±0.05 2±0.03 2.1±0.02 2.1±0.03

41±0.5 49±0.9 84±1.1 88±1.7 44±0.6 45±1.2 87±2.9 86±1.2 82±1.3 83±1.5 83±1.2

0.60±0.1 0.7±0.06 0.93±0.0 1.05±0.0 0.55±0.04 0.60±0.02 0.95±0.05 0.95±0.04 1.1±0.1 1±0.04 0.9±0.03

23±0.3 28±3.2 37±0.2 42±0.9 22±0.3 24±0.1 38±0.1 38±0.1 45±1.1 40±0.8 43±0.9

Average particle size (nm)

PDI

Zeta potential (mV)

116±2.0 211±3.5 325±3.6 526±3.9 120±2.6 117±1.9 266±5 266±5.3 180±1.8 199±1.7 198±2.4

0.59±0.01 0.623±0.01 0.621±0.01 0.619±0.01 0.617±0.01 0.547±0.00 0.541±0.01 0.614±0.01 0.418±0.02 0.374±0.01 0.414±0.01

-18.5±0.06 -21.3±0.21 -22.4±1.1 -21.9±0.78 -22.7±0.98 -25.6±0.27 -27.5±0.99 -29.3±0.63 -31.5±0.82 -28.7±0.25 -29.2±1.02

3.3. Screening effect of lecithin incorporation into lipid matrix

Changing lipid type and increasing its concentration had obvious effect on SLNs particle size. Stearic acid at both concentration levels (2 and 3.5%w/v) showed highly significant increase in SLNs particle size (325 and 526 nm, for SLN 3and 4, respectively). However, using palmitic acid as solid lipid significantly decreased particle size of SLNs to 116 and 211 nm forSLN1 and 2, respectively, (p < 0.001). In formulas SLN1-4, the PDI was above 0.5indicated non-homogenity and polydispersity [44]. By calculating %DL and % EE, stearic acid SLNs showed significantly efficient drugs encapsulation where, the %EE of SLN3 and 4 was 84, 88% for TC and 37, 42% for CoQ10, respectively (p ¼ 0.003), reaching nearly two-fold greater the %EE offered by palmitic acid formulas (41, 49% for TC and 23, 28% for CoQ10, respectively). Zeta potential of all developed SLNs was in range of 18.5 to 22.4 mV. The results clearly showed non-significant effect for lipid concentration on the ability of SLNs to encapsulate drugs.

The effect of lecithin incorporation at different concentrations into SLNs at constant lipid type (stearic acid), concentration (2%w/ v), and surfactant (P188 at 3% w/v) was investigated as shown in Table 2. The results showed %yield 94e95%, indicating minimum loss of excipients and high accuracy of preparation technique. Table 2, also demonstrated the significant effect of lecithin on increasing %DL and %EE of CoQ10 reaching 1.13 and 45%, respectively for formula SLN9 compared to 0.95 and 38%, respectively for formula SLN7. The results also showed highly significant reduction in particle size and PDI of SLNs (p < 0.001). Formula SLN9 showed particle size of 180 nm and 0.4 PDI compared to 266 nm and 0.54 for SLN7 (Table 2). The results also showed that increasing lecithin concentration from 10 to 30%w/w of lipid matrix had no remarkable effect on properties of developed SLNs.

3.2. Screening of surfactant concentrations

3.4. Effect of changing the nature of lipid system

Increasing P188 concentration showed no obvious effect on physicochemical properties of SLNs prepared with palmitic acid (Table 2). A contrary result was recorded with those prepared with stearic acid. Increasing P188 concentration from 2 to 3 %w/v had extremely significant effect on decreasing particle size and PDI of SLNs (p ¼ 0.005). Particle size was decreased from 325 nm at 2%w/ v p188 (SLN3) to 266 nm at 3%w/v p188 (SLN7). Further increase in p188 concentration up to 4%w/v had no effect on SLNs particle size. Thus, 3%w/v P188 was taken as the maximum concentration of surfactant for SLNs preparations.

The %yield of NLCs was nearly 96% for all formulations (Table 3). The %DL and %EE of both drugs were significantly increased compared to previously formulated dosage forms SLN7 and SLN9. All prepared NLCs (1e3) increased %EE of TC to be 91e94%, respectively and CoQ10 to be 56e60% respectively. The NLCs also proved potential power in decreasing particle size and PDI to less than half the values recorded with SLNs 7 and 9 (p < 0.01). The particle size was ranged from 80 to 110 nm, while PDI was 0.324e0.4, which was considered promising for effective oral absorption. All NLCs formulations showed optimum zeta potential

Table 3 Physical characterization of nanostructured lipid carriers and lipid nanocapsules. Nanostructured lipid carriers Formula code

Lipid type (%w/w)

TC

CoQ10

%w/w

NLC1 NLC2 NLC3

Stearic acid

Labrafac oil

Yield

DL

EE

DL

EE

90 80 70

10 20 30

96±0.99 95.6±1.3 95±1.4

2.4±0.2 2.3±0.1 2.3±0.1

94 ±1.3 91±1.5 92 ±0.9

1.4±0.02 1.4±0.01 1.5±0.02

56 ±1.1 57 ±0.9 60 ±0.7

Average particle size (nm)

PDI

Zeta potential (mV)

80±1.6 110±0.8 108±2.3

0.32 ±0.04 0.33 ±0.02 0.41 ±0.04

-31.3±0.9 -32.8±0.2 -32.6±0.5

Z-average (nm)

PDI

Zeta potential (mV)

28±1.0 36±0.9 42±3.4

0.623±0.02 0.600±0.05 0.630±0.03

-28.3±0.29 -27.8±0.67 -28.7±0.29

Lipid nanocapsules Formula code

LNC1 LNC2 LNC3

Lipid type(%w/w)

%w/w

Lecithin

Labrafac oil

Yield

DL

EE

DL

EE

90 80 70

10 20 30

87±0.6 92±1.3 93±1.8

1.1±0.07 1.15±0.1 1.3±0.06

40±2.0 48±3.1 50±2.4

0.38±0.03 0.75±0.07 0.85±0.04

15±0.8 30±1.2 34±1.6

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of > 30 mv. Among all prepared NLCs, formula NLC1 was selected as an optimum formula for subsequent investigation. Table 3, presented also the characteristics of LNCs containing lecithin (90, 80 and 70%w/w) and Labrafac® oil (10, 20 and 30%w/w) reaching 2% w/v total lipid matrix. The results revealed that all formulations were successfully prepared with 87e93 %yield. Comparing to previously formulated SLNs and NLCs, the LNCs showed lower capacity to encapsulate both drugs. The %EE was 40e50%w/w for TC and 15e34%w/w for CoQ10. Increasing oil content from 10 to 20%w/w increased %EE of TC from 40 to 48% and doubling amount of encapsulated CoQ10 from 15 to 30%. Further increase in Labrafac® concentration to 30%w/w, had slight effect on increasing %EE and % DL of both drugs. The LNCs showed outstandingly significant effect on decreasing particle size reaching 28e42 nm (half the size of NLCs and 5e10 fold less than SLNs). Increasing oil concentration led to slight increase in particle size of LNCs (28, 36 and 42 nm at 10, 20 and 30% w/w, respectively). Table 3, revealed absolute zeta potential values for LNCs formulas around 28mv. From this category of lipid nanocarriers, formula LNC2 was chosen as the optimum one for subsequent investigation.

aef). Fig. 1-a shows characteristics bands of TC at 3000 cm1 for COOH bond, 1750 cm1 for C¼O, 1477 cm1 for C¼C ring stretching and 3180 cm1 for NH2 group [46]. Fig. 1-b presents characteristic peaks for COQ10. A small broad peak appeared at 3500 cm1 for OH groups, in addition to a very high intensity one at 3000 cm1for double bonds. Peaks at 1900 to 1000 cm1 for aliphatic CH bonds, and at 1000 - 629 cm1for ¼ CeH bond were also recorded. Fig. 1cef illustrated spectra of the four optimum TC/CoQ10 loaded lipid nanocarriers. The IR spectra showed great superimposition between all formulations. The characteristic peaks of both TC and CoQ10 were clearly seen at their respective wave numbers, indicating high compatibility and absence of any chemical or ionic interaction between drugs and excipients. Different DSC thermograms of pure drugs, excipients and lipid nanocarries were shown in Fig. 2. Two well defined endothermic peaks at 149
C for TC and at54
C for CoQ10 were clearly seen (Fig. 2 a and b). Lecithin had unrecognizable peak (Fig. 2c) and stearic acid showed very sharp endothermic peak at 70
C (Fig. 2d). Lipid nanocarries thermograms showed a slight shift, broadening and decrease in intensity of TC peak >150
C. The CoQ10 peak was clearly appeared at same position with complete disappearance of stearic acid peak (Fig. 2e, f, g and h).

3.5. Physicochemical characterization of selected lipid nanocarriers 3.6. Morphological examination of different lipid nanocarriers 3.5.1. Effect of freeze drying process as a secondary production step with or without cryoprotectant The particle size, PDI, and %EE of SLN7, SLN9, NLC1, and LNC2 before and after freeze drying (with and without cryoprotectant) were presented in Table 4. It was found out that lyophilization process caused no significant damage on %drugs encapsulated in the lipid nanoparticles, in presence or absence of cryoprotectant. Lyophilization process induced a high significant increase (p < 0.001) in particle mean diameter of all formulae in absence of cryoprotectant with an increase in PDI (Table 4), indicating wide size distribution or particles aggregation. On the contrary, addition of 5 %w/v sucrose significantly reduced particle size growth and PDI after lyophilization, which was in agreement with Xia et al., 2011 [45]. It could be also observed that SLN9 and LNC2 showed nearly the same particle size before and after lyophilization in presence of cryoprotectant (180, 173 for SLN 9, respectively and 36, 39 for NLC2, respectively). The effect of cryoprotectant was clearly observed on the flowability of the produced powdered lipid nanoparticles. It was found that formulations lyophilized in presence of cryoprotectant showed angle of repose ranging from 32 to 38
compared to angle of repose ranging from 42 to 53
for those freeze dried without cryoprotectant (Sucrose 5%w/v). 3.5.2. Compatibility studies Compatibility of selected nanocarriers was regarded by observing changes in position of characteristic bands obtained by FTIR in comparison to those of pure drugs and excipients (Fig. 1

Fig. 3aed demonstrated TEM micrographs of SLN7, SLN9, NLC1 and LNC2 respectively. All micrographs showed successful formation of nanosized lipid nanoparticles with distinct morphological characteristics. Fig. 3a, showed smooth non-porous surface, spherical and uniform particles of SLN7. Fig. 3b showed a welldefined disc like shaped nanoparticles for SLN9 formula. The NLC1 also appeared as dark circular discs (Fig. 3-c). In Fig. 3-d, the structure of LNC2 clearly visualized with an oil core enveloped within thick coat of lecithin. 3.7. In-vitro drug release study Release profiles from the optimum four formulations of lipid nanocarriers (SLN 7 and 9, NLC2 and LNC1) were presented in Figs. 4 and 5 for TC and CoQ10, respectively. Both figures markedly revealed high capabilities of the developed dosage forms in controlling the release of both drugs. Pure TC profile showed fastest and highest %cumulative release (79.2% after 8hr). The slowest release profiles for TC were observed with SLN7 and SLN9. Both formulations illustrated biphasic release pattern. During first hr, initial burst release of 11 and 19% TC cumulative release was recorded followed by continuous slow release that reached 41 and 53.5%TC cumulative release form SLN7 and 9, respectively after 8 h. Both NLC1 and LNC2 showed a more prominent controllable effect on drug release patterns. A continuous slow release of TC was observed (46.7 and 35.8% cumulative release for LNC2 and NLC1, respectively after 8 h).

Table 4 Effect of freeze drying with and without cryoprotectant on physical characteristics of selected lipid nanocarriers. In absence of 5%w/v Sucrose Formula Code SLN7 SLN9 NLC1 LNC2

Particle size(nm)

350±9.8 246±6.4 199±8.1 42±3.2

In presence of 5%w/v sucrose

PDI

1±0.3 1±0.2 0.72±0.1 0.81±0.2

%EE

Particle size(nm)

TC

CoQ10

85±2.1 82±1.5 92±1.8 44±0.9

38±0.7 43±1.1 56±1.9 30±0.5

275±3.7 173±1.9 105±2.2 39±0.7

PDI

0.526±0.04 0.457±0.03 0.431±0.01 0.392±0.01

%EE TC

CoQ10

85±0.54 82±1.2 92±2 44±0.9

38±1.2 43±0.26 56±0.35 30±0.08

E.S. El-Leithy, R.S. Abdel-Rashid / Journal of Drug Delivery Science and Technology 41 (2017) 239e250

Fig. 1. FTIR spectral analysis results (aef). a-TC b- COQ10 c- SLN7 d- SLN9 e NLC1 f- LNC2.

245

246

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Fig. 2. DSC thermograms of pure drugs, excipients and prepared lipid nanocarriers (a-TC, b-Co-COQ10, c-lecithin, d-stearic acid, e-SLN7, f-SLN9, g-NLC1, h-LNC2).

Fig. 3. TEM micrographs of lipid nanocarriers.

E.S. El-Leithy, R.S. Abdel-Rashid / Journal of Drug Delivery Science and Technology 41 (2017) 239e250 Table 6 Fitting of CoQ10 in-vitro release data to kinetic models.

90 %CumulaƟve TC release

80

Release Profile

70 60 50

Pure CoQ10 SLN7 SLN9 NLC1 LNC2

40 30

20 10 0 0

2

4

6

8

10

Time (hr)

SLN7

SLN9

NLC1

LNC2

TC

Fig. 4. Release profiles of TC from lipid nanocarriers compared to free drug.

50

%cumulaƟve COQ10 release

247

45 40 35 30 25

Zero order (Q¼kt)

First order (log Qº-Q¼kt)

Higuchi (Q¼kt1/2)

R2

K

R2

K

R2

K

0.9299 0.9648 0.9768 0.9392 0.9622

0.097 0.065 0.054 0.065 0.052

0.9740 0.9911 0.9921 0.9775 0.9865

-0.012 -0.021 -0.028 -0.019 -0.028

0.9707 0.9946 0.9926 0.9870 0.9960

4.00 5.03 3.67 4.88 2.35

release behavior of drugs was highly dependent on drug dissolution and nature of formula matrix. Both pure TC and SLN7 showed the same release behavior that followed first order kinetic model. The R2 was 0.9912 and 0.9851 for TC and SLN7, respectively. The SLN7 showed a more prolonged (slower) drug release behavior (K changed from 0.08 for pure TC to 0.026 for SLN7). However, both SLN9 and NLC1 showed good fitting to Higuchi model kinetic with a prominent effect for formula NLC1 (K value increased from 4.4 with the formula SLN7 to 5.7 with NLC1). Increasing percentage of Labrafac® oil to 20 %w/w of lipid matrix (LNC2) showed a shift in the kinetic release behavior to zero-order that allowed sustained drug release. The robust effect of the delivery systems on improving the release behavior of CoQ10 became markedly obvious. The lipid nanocarriers has changed the CoQ10 release profile to fit Higuchi model.

20 15

3.9. Ex-vivodrug permeability

10 5 0

0

2 SLN7

4 Time (hr) SLN9

6

NLC1

8 LNC2

10 CoQ10

Fig. 5. Release profiles of CoQ10 from lipid nanocarriers compared to free drug.

Fig. 4, showed the high potential of developed drug delivery systems on improving solubility and dissolution of highly lipophilic CoQ10 which demonstrated a very low release pattern in its pure form. About5% cumulative drug release only was obtained during first 5 h and reached about17.5 %cumulative drug release after 8 h. Release profiles of all lipid nanocarries showed an obvious increase in release patterns of CoQ10 (33.3, 42.5, 29.5, and 38.8% for SLN7, SLN9, NLC1, and LNC2, respectively after 8 h). The CoQ10 release from different formulations also superimposed with that obtained with TC release profiles.

The %cumulative TC and CoQ10 permeated through rabbit intestine mucous membrane were presented in Figs. 6 and 7, respectively. Both figures markedly confirmed the effectiveness of different delivery systems on improving drug permeation through GIT membrane. The %cumulative TC permeated was arranged in descending order as 85.1 > 80.9> 65.8 > 50.1% for SLN9, LNC2, SLN7 and NLC1, respectively in comparison to pure TC pure that allowed about 14.4% cumulative permeated drug after 6hr (Fig. 6). The same permeation patterns were recorded with CoQ10 (Fig. 7). Only 4% of pure drug was permeated during 6hr in comparison to 41e79 % cumulative drug permeated from lipid nanocarriers formulations. Experimental results highly recommend the use of lipid nanocarriers as delivery systems for TC or CoQ10 due to their robust effect on improving %permeation of highly hydrophobic drug (CoQ10) that reached up to 10 folds than unformulated drug. 4. Discussion The %EE was considered as potential indication for selecting

3.8. Kinetic analysis of in-vitro release data

Table 5 Fitting of TC in-vitro release data to kinetic models. Release Profile

Pure TC SLN7 SLN9 NLC1 LNC2

Zero order (Q¼kt)

First order (log Qº-Q¼kt)

Higuchi (Q¼kt1/2)

R2

K

R2

K

R2

K

0.9591 0.9731 0.9761 0.9689 0.9930

0.057 0.050 0.057 0.045 0.035

0.9912 0.9851 0.9909 0.9942 0.9841

-0.080 -0.026 -0.036 -0.025 -0.034

0.9840 0.9541 0.9992 0.9961 0.9528

3.26 5.70 4.47 5.71 9.17

%CumulaƟve TC permeability

100

Release data were subjected to kinetic analysis whereas correlation coefficient (R2) and release constant (K) were presented in Tables 5 and 6 for TC and CoQ10, respectively. It could be seen that

90

80 70 60 50 40 30 20 10 0 0

1

2

3

4

5

6

Time (hr) SLN7

SLN9

NLC1

LNC2

TC

Fig. 6. Permeation behavior of TC from lipid nanocarriers versus pure drug.

7

248

E.S. El-Leithy, R.S. Abdel-Rashid / Journal of Drug Delivery Science and Technology 41 (2017) 239e250

%CumulaƟve CoQ10 permeability

80 70 60 50 40 30 20 10 0 0

1

SLN7

2

3

SLN9

Time (hr) NLC1

4

5

LNC2

6

7

CoQ10

Fig. 7. Permeability behavior of CoQ10 from lipid nanocarriers versus pure drug.

optimum lipid type and concentration. Among estimated solid lipid substance, stearic acid was selected as the optimum lipid matrix at concentration 2%w/v due to its highest loading capacity for both drugs compared to palmitic acid. This result was attributed to presence of long chain fatty acids attached to triglycerides that create a less ordered lipid matrix and leaves enough space to accommodate drug molecules [47e49]. At the same time, high melting point of stearic acid and its viscosity would reduce drug escaping into external phase, thus ensuring highest %EE [47]. The results showed significant reduction in particle size by increasing P188 concentration from 2 to 3%w/v which may be due to decreasing interfacial tension between lipid matrix and dispersion medium during emulsification process. Also high concentration of surfactant allowed sufficient coverage for nanoparticles surfaces with surfactant molecules that, prevented particles agglomeration and allowed SLNs formation with smaller particle size [50]. Lecithin's amphiphilic nature strongly suggested it as dispersing, emulsifying, permeation enhancer, and stabilizing agent for SLNs formation [40,51]. Lecithin at concentration of 10% w/w of lipid matrix (SLN) was recorded to decrease particle size of SLNs was that could probably be explained by the large oil/water interface that provide an additional interfacial area [52]. Incorporation of liquid lipids into lipid matrix (NLCs) resulted in drug-loading capacity enhancement due to crystal lattice imperfections [53]. Presences of Labrafac® oil among NLC composition also increased solubility of highly hydrophobic CoQ10, thus resulted in marked increase in %EE of CoQ10 from 45% in SLNs to approximately 60% in NLCs. Changing the nature of lipid nanocarriers from SLNs to NLCs significantly affected particle size and PDI (p < 0.01). The smaller particle size of NLCs was related to oil content inside of NLCs that decreases the viscosity of lipid mixture, and therefore, could reduce the surface tension to form smaller and smoother surface particles comparing with SLNs [54]. The NLCs Zeta potential < -30mv was sufficient to prevent colloidal particles aggregations [55]. Lipid nanocapsules took great attention due to combining advantage of lecithin's biocompatibility and permeation enhancing effect along with its physicochemical properties that increase antitumor effects of chemotherapeutic drugs [56,57]. Comparing % EE of all prepared lipid nanocarriers, LNCs had the lowest %EE, which was attributed to low solubility of TC or CoQ10 in lecithin. The advantage of using lecithin as oily phase was more obvious on particle size however LNCs showed lowest particle size (36 nm) which may be related to low viscosity of lipid phase. The results also indicated the necessity of cryoprotectant during

freeze drying to avoid particles aggregation and Polydispersity and enhance powder flowability. On the other hand, it was proven that different generations of lipid nanoparticles had great potential in preventing freeze drying damages on encapsulated drugs even in the absence of a cryoprotectant which coincided with results of Lopes et al., 2012 [51]. Compatibility and complete absence of chemical or ionic interaction was recorded among both drugs and excipients for all lipid nanocarriers and was attributed to the decrease in lipid crystallinity within formulated nanocarriers in comparison to its pure form [58]. Analyzing the mechanisms of drug release revealed robust effect of delivery system formulations on changing release behavior of drugs. Selection of SSIF was based on its ease, inexpensive preparation and its performance for determining drugs in vitro release through 8hr only instead of 36hr commonly recorded in literature for most of lipid nanoparticles using phosphate buffer as dissolution medium [40,45]. The release profiles of tested lipid nanocarriers were arranged in descending order as follow; SLN9 > LNC2 > SLN7> NLC1. The high dissolution rate recorded with SLN 9 and LNC 2 was attributed to presence amphiphilic phospholipids that impart certain hydrophilic properties to the lipid nanocarriers and increase matrix wettability. On the contrary, formulations containing pure stearic acid (SLN7) or stearic acid and 10% Labrafac® (NLC1) showed lower drug release due to hydrophobic characteristics of their matrix. Significant difference in TC burst release from SLN7 (11 ± 0.9%) and SLN9 (19 ± 0.7%) (P < 0.05) was related to difference in particle size and surface area exposed to dissolution medium (266 nm for SLN7 compared to 180 nm for SLN9) [59]. Presence of lecithin in SLN9 may also affect surface active properties of the matrix [60]. On the other hand, effect of NLC1 formula on delaying TC release was attributed to its solubility in the viscous oil phase and good association within lipid matrix that lead to slower diffusion from non-homogenous oil/lipid matrix [61]. First order release kinetic recorded for pure drugs and SLN9 formula was related to direct proportionality between drug release and amount of remaining drug. Changing drug release of SLN9 and NLC1 formulations to be best-fitted Higuchi model was attributed to molecular structure of lecithin phospholipid that induces certain porosity to lipid matrix and allows drug diffusion or due to matrix erosion and lipids degradation [13]. Zero order release kinetics of LNC formula was attributed to morphological structure of nanocapsules that behaves as reservoir-type device with oily core surrounded with inert coat of phospholipid molecules, which in turn functions as a rate-controlling membrane. Numerous literature reports results supported our results that showed high permeability enhancement for different lipid nanocarriers with more prominent effect for those containing lecithin [62,63]. Our hypothesis and justification recommending them as optimum delivery systems were SLN9 and NLC1 for dual targeted drug delivery of TC/CoQ10. 5. Conclusion Development of lipid nanocarriers containing antitumor/antioxidant biotherapy present a revolutionary step in accomplishing a successful cancer therapy. Lipid nanocarriers were loaded with TC and CoQ10 with very reasonable physical characteristics, %EE and controlled release pattern. Increasing ex-vivo intestinal permeability of CoQ10 up to 10 folds than unformulated drug highlighted the importance of lipid nanocarriers in facing the challenge for improving solubility and bioavailability of poorly water soluble drugs. The study greatly suggested use of optimum selected TC/ CoQ10 lipid nanocarriers formulas for a cytotoxicity and genotoxicity assay conducted on MCF-7 (adenocarcinoma breast cancer

E.S. El-Leithy, R.S. Abdel-Rashid / Journal of Drug Delivery Science and Technology 41 (2017) 239e250

cells) in comparison with WISH (normal amniotic cells) accompanied with examining cytopathology and oxidative status that will be published in separate research article.

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