Solid lipid nanoparticles of diethylcarbamazine citrate for enhanced delivery to the lymphatics: in vitro and in vivo evaluation.

Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by University of Laval on 07/01/14 For personal use only.

Original Research

1.

Introduction

2.

Experimental section

3.

Results and discussion

4.

Conclusion

Solid lipid nanoparticles of diethylcarbamazine citrate for enhanced delivery to the lymphatics: in vitro and in vivo evaluation Karthik Siram, Vijaya Raghavan Chellan†, Tamilselvan Natarajan, Balakumar Krishnamoorthy, Habibur Rahman Mohamed Ebrahim, Vamshikrishna Karanam, Siva Selva Kumar Muthuswamy & Hari Prasad Ranganathan †

PSG College of Pharmacy, Department of Pharmaceutics, Coimbatore, Tamil Nadu, India

Objectives: The major objective is to target diethylcarbamazine citrate (DEC) to the lymphatics and to increase its retention time. The effect of various excipients on the physicochemical characteristics of the nanoparticles was also studied. Materials and methods: Solid lipid nanoparticles (SLNs) of DEC were prepared by ultrasonication by varying the concentrations of compritol 888 ATO, poloxamer 188 and soya lecithin. The SLNs were evaluated for size, shape, texture, surface charge, physical nature of the entrapped drug, entrapment efficiency and in vitro drug release. In vivo animal studies were carried out to estimate the pharmacokinetic parameters in blood and drug concentration in lymph after oral administration. Results: The size of the spherical particles was in the range of 27.25 ± 3.43 nm to 179 ± 3.08 nm and a maximum entrapment efficiency of 68.63 ± 1.53% was observed. In vitro release studies in pH 7.4 PBS displayed a rapid release and the maximum time taken for the complete drug to release was 150 min. In vivo studies indicated an enhancement in the amount of drug that reached lymphatics when administered via SLNs. Conclusion: Targeting of DEC to the lymphatics is possible through SLNs and the retention time in the lymphatics can also be enhanced. Keywords: diethylcarbamazine citrate, filariasis, lymphatic targeting, mesenteric lymphatic duct, poloxamer 188, solid lipid nanoparticles Expert Opin. Drug Deliv. [Early Online]

1.

Introduction

The lymphatic system is an important repository for some diseases like filariasis [1], tuberculosis [2], AIDS [3], metastatic cancers [4], edema [5] and so on and also plays a role in the inflammatory processes [6]. The lymphatic system majorly modulates the immune system, and because of the complex nature and complex roles of the lymphatics, targeting of drugs to the lymphatics is challenging [7]. Thus, in order to target the drugs to the lymphatics and to further increase the residence time in the lymphatics, the moieties apart from reaching the lymphatic system should evade opsonization by reticuloendothelial system (RES), which can be possible by preparing particles of size > 100 nm [8]. Lymphatic filariasis is a vector-borne parasitic disease caused by three kinds of thread-like, parasitic filarial worms: Wuchereria bancrofti, Brugia malayi or Brugia 10.1517/17425247.2014.915310 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

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K. Siram et al.

timori, and these worms are transmitted to the humans by the bites of infected mosquitoes. As per the available reports, over 120 million people in at least 80 countries throughout the tropics and subtropics are affected by lymphatic filariasis and > 1 billion people are at risk of acquiring the infection [9]. The largest numbers of infected people in the world are present in India (45.5 million) and sub-Saharan Africa (40 million). The World Health Organization has categorized this disease as one among the tropically neglected diseases. The microfilaria present in the blood of the affected patient are taken up by the mosquitoes and undergo a few moultings in the mosquitoes, which are later transferred to a new human as the larval form (L3). These L3 larvae migrate to the lymphatics and transform into adult filarial worms. These adult worms dwell in the lymphatics and lymph nodes and release their progeny, microfilaria into the blood [9,10]. Drugs like diethylcarbamazine citrate (DEC), albendazole and doxycycline are used for the treatment of lymphatic filariasis and are also administered orally in the endemic regions through annual mass drug administration as a preventive strategy. These drugs can effectively kill the adult filarial worms but fail to do so in the body when administered orally as they do not meet the criteria required for lymphatic uptake, namely, a log p value > 4.7 and good lipophilicity [11,12]. This leads to insufficient concentration of the drug in the lymphatics, where the adult worms reside. Hence, targeting the drugs to the lymphatics through oral route using a suitable carrier and increasing the residence time in the lymphatics will enable the drugs to kill the adult worms in an efficient manner. Works pertaining to enhance the lymphatic uptake of antifilarial drugs were not carried out extensively, and it might be because of the nonfatal nature of the disease and lower prevalence of filariasis in the developed countries where core research is usually carried out. The concept of lymphatic targeting has been attempted for many drugs using various carriers like liposomes [13], solid lipid nanoparticles (SLNs) [14], polymeric nanoparticles [15], dendrimers [16], self-emulsifying drug delivery systems [17] and so on. Oral administration through SLNs hold great advantage in achieving good lymphatic transport owing to the presence of phospholipids which not only enhance the production of chylomicrons but also facilitate the partitioning of drugs across various lymphatic structures present in the gut wall (Peyer’s patches, isolated lymphoid follicles, lymphocytefilled villi, colonic lymphatic tissue) to the mesenteric lymphatic duct in the intestinal region. Thus, through the mesenteric lymphatic duct, the drug can reach the lymphatics [18]. DEC is the primary drug of choice which is used in both prevention and treatment of filariasis. It has the potency to kill the microfilariae present in the blood as well as the adult worms present in the lymphatics. For a compound to enter the lymphatics, it should be sufficiently lipophilic with a log p value > 4.7 [11,12]. So, DEC with a log p value of 0.1 and high water solubility of nearly 64 mg/ml fails to enter the 2

lymphatics leading to ineffective killing of the adult filarial worms. Hence, an attempt was made to develop nanoparticles of DEC using a lipid carrier with size below 100 nm to target and to increase the retention time in the lymphatics. Compritol 888 ATO was used as the lipid carrier as it has been reported to have a good lymphatic uptake [19]. Poloxamer 188 was used as a surfactant because surface coating with poloxamer 188 prevents uptake by the RES [20]. Various factors affecting the size, entrapment efficiency and in vitro release have been discussed elaborately. Further, estimation of drug concentrations in blood and lymph has been carried out using an animal model. 2.

Experimental section

Materials DEC was obtained as a gift sample from SL Drugs, Hyderabad. Compritol 888 ATO was generously gifted by Gattefosse, France. Poloxamer 188 and soya lecithin were purchased from Sigma Aldrich, USA. Dialysis membrane with a molecular weight cutoff between 12,000 and 14,000 Da was purchased from HiMedia laboratories, Mumbai. 2.1

Formulation of DEC loaded SLNs SLNs of DEC were prepared by ultrasonication method with modifications [21]. An accurately weighed quantity of lipid was heated carefully on a water bath at 80 C in order to form a melted phase of the lipid. DEC was added to the melted lipid and heated until a clear homogeneous phase was formed. Simultaneously, a weighed quantity of the surfactant and cosurfactant was added to water to form an aqueous phase which was also heated to 80 C. The hot lipid phase was dispersed in the surfactant solution at 9700 r.p.m., 80 C for 2 min using a high shear homogenizer (POLYTRON PT 3100 D). The obtained pre-emulsion was ultrasonicated using an ultrasonic homogenizer (SONICS) at 50% amplitude for 2 min followed by cooling the dispersion in an ice bath to form SLNs. During homogenization, the production temperature was kept 5 C above the lipid’s melting point to prevent recrystallization. On the whole, 24 batches of DEC-loaded nanoparticles were prepared by varying the concentrations of lipid, surfactant and cosurfactant. The compositions of the various formulations are mentioned in Table 1. 2.2

Freeze drying Cryoprotector, trehalose (10%) was added to the formulations and kept in freezer for 4 h. Then, the formulations were freeze-dried using LYODEL freeze drier at a temperature and pressure of -30 C and -1 mbar, respectively, until the product dried [22]. The freeze-dried powder was used for differential scanning measurements. 2.3

Expert Opin. Drug Deliv. (2014) 11(8)

SLNs of DEC for enhanced delivery to the lymphatics


Table 1. Composition of SLN formulations. Formulation code

Drug (mg)

Lipid (%)

Surfactant (%)

Cosurfactant (%)

DS1 DS2 DS3 DS4 DS5 DS6 DS7 DS8 DS9 DS10 DS11 DS12 DS13 DS14 DS15 DS16 DS17 DS18 DS19 DS20 DS21 DS22 DS23 DS24

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

5 10 5 10 5 5 10 10 5 5 5 5 10 10 10 10 5 5 5 5 10 10 10 10

2.5 2.5 5 5 2.5 5 5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 5 5 5 5 5 5 5 5

----0.5 0.5 0.5 0.5 0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4

SLN: Solid lipid nanoparticle.

2.4

Differential scanning calorimetry measurements

The endothermic melting temperature for DEC, lipid and drug-loaded SLNs was measured by differential scanning calorimetry (DSC) (Universal V4.7A TA Instruments) to find out the physical state of the materials. Ten milligrams of the sample was placed on an aluminum pan and scanned between 25 and 80 C at a range of 5 C/min under nitrogen gas. Particle size and polydispersity index by photon correlation spectroscopy

2.5

The average diameter and polydispersity index were measured by photon correlation spectroscopy using a Malvern Zetasizer (Nano ZS90; Malvern Instruments) at 25 C. The samples were kept in polystyrene cuvette, and the readings were noted at 90 fixed angle.

were visualized using JEOL JEM 2100 High-Resolution Transmission Electron Microscope (HRTEM), Japan, at a voltage of 80 kV. The images were observed at a magnification value of 50,000. Atomic force microscopy A small aqueous drop (~ 10 µl) of the drug-loaded nanoparticles was adsorbed on the surface of silicon wafer and was allowed to dry at room temperature. The images were examined on Multimode Scanning Probe Microscope (NTMDT, NTEGRA prima, Russia) in semi-contact mode with a force constant range of 0.35 -- 6.06 N/m and a resonating frequency range of 47 -- 150 kHz. The phase image and topology images were used to determine the morphology of SLN. 2.8

Encapsulation efficiency For the estimation of encapsulation efficiency, 2 ml of the formulation was subjected to centrifugation at a speed of 20,000 r.p.m. for 30 min at 10 C following which the supernatant solution was collected and analyzed for the free drug using ultraviolet spectroscopy at 210 nm. Encapsulation efficiency was calculated by the following equation [23]: (1) 2.9

Zeta potential The electrophoretic mobility (zeta potential) measurements were made using Malvern Zetasizer (Nano ZS90; Malvern Instruments). The samples were placed in a glass cuvette (at 25 C), and a zeta dip cell was used to find out the potential. The samples were diluted suitably using Millipore water (pH = 5.5). 2.6

Transmission electron microscopy An aqueous drop (~ 10 µl) of drug-loaded nanoparticles was cast on top of a carbon-coated copper grid and was air dried at room temperature. The particle shape and morphology 2.7

Encapsulation efficiency (%) amount of drug added in the system − amount of drug added in the sup erna tan t ×100 = amount of drug added in the system

In vitro drug release studies The in vitro release studies were carried out by dialysis bag method with a molecular weight cutoff of 2.10


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K. Siram et al.


12,000 -- 14,000 Da (HiMedia Laboratories) in pH 7.4 PBS. Twelve hours prior usage, the dialysis membrane was kept in PBS to remove the preservatives. Precisely, 1 ml of the nanoparticle dispersion was placed in the dialysis bag by sealing both the ends with the help of clips. The dialysis bag was dipped in a 50-ml dissolution medium maintained at 37 ± 1 C and stirred at 50 r.p.m. using a magnetic stirrer. Two milliliters of the buffer solution is removed at an interval of 5, 15, 30, 45, 60, 75, 90, 120 and 150 min and was replaced by an equal amount of fresh buffer to maintain sink conditions [24]. The content of DEC in the samples was determined by ultraviolet spectroscopy at lmax of 210 nm. Stability studies Stability studies were carried out at 4 ± 1 C and 25 ± 1 C for a period of 3 months in amber-colored borosilicate glass bottles. Particle size and entrapment efficiency were calculated at regular time intervals [25]. 2.11

In vivo animal studies to estimate the amount of drug present in the plasma and lymph 2.12

In vivo animal studies were carried out to estimate the amount of drug that reaches the systemic circulation and to find out the uptake of the drug into the lymphatic vessels. The studies were carried out according to the guidelines of the Council for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India, and the study protocol was carried out after obtaining the consent from PSG Institutional Animal Ethical Committee with registration no: 158/99/CPCSEA. Male Sprague Dawley rats weighing 280 -- 310 g have been used for the study and were categorized into four groups with six rats in each group. The rats were fed ad libitum with rodents chow allowing free access to drinking water. Prior to the study, the animals were kept for overnight fasting but were allowed free access to water. Group 1 and Group 2 consisted of animals used for the estimation of pharmacokinetic parameters in blood, which were administered with SLN formulation and drug in pH 7.4 PBS, respectively. A dose equivalent to 30 mg/kg was administered orally using a gastric lavage needle, and the blood (0.3 ml) was collected by retro-orbital venous plexus puncture with the aid of a capillary tube at 0.5, 1, 2, 4, 8, 12 and 24 h post-dose. The samples were collected in a microcentrifuge tube containing 50 µl of trisodium citrate and centrifuged at 2500 r.p.m. for 10 min. The plasma was collected and stored at -80 C for quantitative analysis. Group 3 and Group 4 consist of animals used for the estimation of drug concentration in lymph and were administered with SLN formulation and drug in pH 7.4 PBS, respectively. The surgical procedure used for the cannulation of the mesenteric lymphatic vessel was followed as per the protocol developed by Boyd and co-workers [26]. One hour prior to the study, the animals were given an oral dose of 1-ml soya 4

bean oil for the mesenteric lymphatic duct to swell which will help in cannulation. A dose equivalent to 30 mg/kg was given orally by a gastric lavage needle, and the lymph (0.2 ml) was collected from the cannulated mesenteric lymphatic duct at 0.5, 1, 2, 4, 8, 12 and 24 h after dose. Lymph was collected in 2-ml centrifuge tubes containing 50 µl of trisodium citrate and stored at -80 C (after deproteining) until further analysis by HPLC. 2.13 Extraction of DEC from plasma and lymph samples

DEC was extracted from plasma and lymph using the principle of liquid extraction [27]. A 50 µl of the sample (either plasma or lymph) was transferred to a centrifuge tube containing 10 µl of quercetin solution (internal standard) and was allowed to mix well using a vortex machine for 30 s. To this mixture, 20 µl of 2 M sodium hydroxide and 4 ml of dichloromethane were added and mixed well using a vortex machine for 5 min. This solution was centrifuged at 10,000 r.p.m. for 10 min, and the organic layer was separated and dried. One hundred microliters of 80:20 of 10 mM potassium dihydrogen phosphate buffer (pH: 3.2) and acetonitrile were added to the dry residue and mixed well using a vortex machine for 1 min. Quantitative analysis of DEC by HPLC The concentration of DEC in plasma and lymph was estimated by HPLC with modification [28]. A Waters 515 HPLC along with an Atlantis, Water C18 column was used for the study, and the mobile phase consists of 10 mM potassium dihydrogen phosphate buffer (pH: 3.2) and acetonitrile at a ratio of 80:20. The flow rate was maintained at 0.5 ml/min, and 20 µl of the sample was injected using a rheodyne injector at every instance. An UV/visible detector was used for detection and the samples were analyzed at a wavelength of 210 nm. The method for estimation of DEC was validated using an internal standard, quercetin, in terms of specificity, linearity and reproducibility. The r2 value was found out to be 0.998, and the retention time for DEC and quercetin was found to be at 5.5 and 6.5 min, respectively. 2.14

Statistical analysis Statistical analysis was carried out with the help of GraphPad Prism (version 5). Results are expressed as mean ± standard deviation (three independent values of the same formulation). Statistical significance was determined for in vitro release data by two-way analysis of variance (ANOVA). Bonferroni posttest was also used to compare the p values of each column at various individual time points. Student’s t-test was used to carry out the statistical analysis for in vivo animal studies and for finding out a significant relationship between concentration of lipid and size. In all cases, p value < 0.05 was considered to be statistically significant. 2.15



20

Bulk compritol 888 ATO (A) Bulk DEC (B) DEC loaded SLNs (C) SLNs without DEC (D)

Heart flow (W/g)

10

A

B

0

-10

C D


-20

-30 -200 Exo Up

-100

0

100

200

300

Temperatire (°C)

400

500

Universal V4.7A TA Instruments

Figure 1. A. Overlay of DSC thermogram of bulk compritol 888 ATO. B. bulk DEC. C. DEC-loaded SLNs. D. SLNs without DEC. DEC: Diethylcarbamazine citrate; DSC: Differential scanning calorimetry; SLN: Solid lipid nanoparticle.

3.

Results and discussion

Formulation of DEC-loaded SLNs SLNs of DEC were efficiently prepared using a combination of high shear homogenization and ultrasonication method. Initially, the formulations were prepared at speeds of either 15,000 or 12,500 r.p.m. for 2 min followed by ultrasonication for 3 min to obtain smaller particles. High speed of 15,000 or 12,500 r.p.m. resulted in formation of froth and cream, and hence, high speed was found to be unsatisfactory for the preparation. This instability might be because of high shear forces induced due to high kinetic energy in the system which lead to the destabilization of the nanoparticulate system [29]. Further, this high speed also leads to partial damage of the surface of nanoparticles causing instability problems like frothing, creaming and aggregation and this phenomenon has been supported elsewhere [30]. Hence, the speed during the high shear homogenization was optimized to 9700 r.p.m. Compritol 888 ATO at concentrations of 5 and 10% was used as the lipid matrix for the preparation of SLNs. The concentration of the lipid was not allowed to exceed beyond 10% because an increase beyond 10% leads to an increase in the particle size. Compritol 888 ATO (glyceryl behenate) is a mixture of 12 -- 18% monoglycerides, 52 -- 54% diglycerides and 28 -- 32% triglycerides which will help in increasing the lymphatic uptake of DEC. Further, the ability of Compritol 888 ATO for lymphatic targeting has been well supported [19,31]. 3.1

DSC measurements DSC studies were performed to characterize the status of the material in the nanoparticles and for the identification of 3.2

melting points of the compounds [32]. Figure 1 represents the overlay of the DSC thermogram of bulk Compritol 888 ATO, DEC, formulation and blank nanoparticles. The thermogram for bulk Compritol 888 ATO showed a single endothermic peak at a melting point of 76.3 C. Compritol 888 ATO is a mixture of diglycerides and triglycerides, and hence, it exists either in bi or in b¢ form. The bi is an intermediate transition form between b and b¢ form, and it occurs because of the presence of triglycerides (b form) and diglycerides (b¢ form) which also prevents the transition to the stable b form. Hence, the observed melting point is due to the presence of bi or b¢ form [33]. The thermogram of the formulation reveals two peaks: one peak corresponds to Compritol 888 ATO at 70.74 C and the other peak at 53 C corresponds to that of poloxamer present on the surface of the nanoparticles. The thermogram of the blank nanoparticles also shows two peaks at 77.1 and 53.9 C, which corresponds to Compritol 888 ATO and poloxamer, respectively. The melting point of Compritol 888 ATO in the formulation was about 5.56 and 6.36 C lesser than the melting point of bulk Compritol 888 ATO and Compritol 888 ATO of blank nanoparticles which is because of the lattice imperfections caused by drug loading in the lipid matrix. Further, the decreased melting peak corresponds to a more unstable form, b¢. The absence of the drug peak in the thermogram of the formulation indicates that the entrapped drug in the nanoparticles is in amorphous state [34]. Particle size by photon correlation spectroscopy A particle size below 100 nm is a prerequisite for the nanoparticles to enter the lymphatics. Further, in order to remain in the lymphatics for a longer time by getting filtered at the lymph nodes and by evading RES uptake, a size below 100 nm is 3.3


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K. Siram et al.


Table 2. Physicochemical characteristics of DEC-loaded SLNs. Formulation code

Zeta average size (nm ± SD)

Polydispersive index (PI ± SD)

Zeta potential (mV ± SD)

Entrapment efficiency (% ± SD)

DS1 DS2 DS3 DS4 DS5 DS6 DS7 DS8 DS9 DS10 DS11 DS12 DS13 DS14 DS15 DS16 DS17 DS18 DS19 DS20 DS21 DS22 DS23 DS24

99.5 ± 4.14 60.10 ± 2.32 179.00 ± 3.08 142.13 ± 2.61 41.07 ± 1.94 98.06 ± 3.33 50.48 ± 2.08 27.25 ± 3.43 69.13 ± 4.58 56.82 ± 1.97 55.77 ± 1.20 49.72 ± 2.13 90.39 ± 2.11 104.78 ± 1.41 46.05 ± 1.60 34.76 ± 1.22 128.74 ± 1.2 132.99 ± 1.42 143.68 ± 1.46 140.72 ± 1.43 140.67 ± 1.73 134.61 ± 1.73 82.29 ± 1.13 69.71 ± 1.32

0.232 0.152 0.241 0.117 0.261 0.393 0.432 0.473 0.373 0.412 0.407 0.425 0.214 0.491 0.144 0.237 0.222 0.402 0.373 0.379 0.235 0.221 0.233 0.261

-0.23 ± 1.3 -0.12 ± 1.7 -1.10 ± 1.9 -0.98 ± 1.2 -9.55 ± 1.1 -8.92 ± 1.6 -9.50 ± 2.3 -11.3 ± 0.9 -4.48 ± 2.1 -0.037 ± 2.4 -0.52 ± 1.4 -1.14 ± 2.5 -2.93 ± 2.1 -2.62 ± 1.9 0.031 ± 1.7 -0.119 ± 2.1 0.101 ± 0.9 -1.23 ± 2.1 -1.456 ± 1.4 -2.18 ± 1.2 0.251 ± 1.2 -0.0546 ± 1.1 -2.14 ± 1.5 -2.34 ± 1.7

47.56 ± 0.95 64.61 ± 1.20 32.9 ± 1.10 43.1 ± 0.58 42.72 ± 0.69 40.34 ± 0.55 48.01 ± 1.20 48.9 ± 1.40 46.27 ± 1.20 46.79 ± 1.10 49.23 ± 1.70 49.61 ± 1.40 52.50 ± 0.99 62.33 ± 1.31 59.12 ± 1.22 54.12 ± 1.41 68.63 ± 1.53 36.31 ± 1.64 37.03 ± 1.91 43.20 ± 1.21 36.13 ± 1.83 49.14 ± 0.67 58.12 ± 1.24 53.74 ± 1.74

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.023 0.004 0.012 0.014 0.190 0.013 0.011 0.018 0.023 0.017 0.019 0.026 0.015 0.099 0.018 0.023 0.029 0.014 0.016 0.019 0.010 0.014 0.021 0.014

Values are expressed as mean ± standard deviation (n = 3). DEC: Diethylcarbamazine citrate; SLN: Solid lipid nanoparticles.

crucial [35,36]. Thus, attempts have been made using various concentrations of lipid, surfactant and cosurfactant to prepare nanoparticles which are intended to reach the lymphatics. The zeta average diameters of the formulations are mentioned in Table 2. The values show that the size of the SLNs ranged from 27.25 ± 3.43 (DS8) to 179 ± 3.08 nm (DS3). The effect of various concentrations of lipids, surfactants and cosurfactants on the size of the particles was studied. 3.3.1

Effect of lipid concentration on the size of the

SLNs

A relationship (p value > 0.05) was observed between the concentration of lipid and the size of the nanoparticles formed. An increase in lipid concentration from 5 to 10% resulted in nonlinear decrease in the size of the nanoparticles. Compritol 888 ATO contains surface active monoglycerides and diglycerides that are responsible for an increased emulsification of the system, leading to a decrease in the size of the formulations [29]. Effect of surfactant on the size of SLNs Generally, an increase in the amount of surfactant will lead to a decrease in size. But in the present study, increasing the amount of surfactant, poloxamer 188, from 2.5 to 5% in the formulations led to an increase in the size of the nanoparticles. Poloxamer is a neutrally charged compound and the absence of charges will cause the excess of poloxamer to accumulate on the 3.3.2

6

surface (poloxamer bridging) of the particles leading to an increase in the size of the particles. Poloxamer, a triblock polymer has a central hydrophobic polyoxypropylene oxide (PPO) and is flanked on both sides by hydrophilic polyoxyethylene oxide (PEO) chains (Figure 2A). The hydrophobic PPO moiety is attached to the lipid, and the PEO chains protrude into the aqueous environment. When lower amounts of poloxamer 188 are present (Figure 2B), the PEO chains fall along the particle surface and when higher amounts of poloxamer are added, the surface becomes more crowded causing an extension of PEO chains [37]. These extended PEO chains in the presence of higher amounts of poloxamer is the reason for the increased particle size (Figure 2C). This theory has been well explained by Stolnik et al. [37], who also showed an increase in the layer thickness with an increase in the amount of adsorbed poloxamer. Further, increase in concentrations of poloxamer beyond 2.5% will increase the cohesive forces among the poloxamer molecules which cause the poloxamer molecules to bind with each other. Poloxamer 188 in higher concentrations acts as a coating and gelling agent [30], which further corroborates the fact that an increase in concentration of poloxamer 188 beyond 2.5% will lead to an increase in the size of nanoparticles. Effect of cosurfactant concentration on the size of SLNs

3.3.3

A mixture of surfactants is generally desirable to use if a stable formulation is to be obtained. Hence, soya lecithin has been



A.

B.

C.

PEO

PEO

PEO

PEO

PEO

PPO

Nano particle

Nano particle

Small

Large

PEO


Figure 2. Diagrammatic representation of effect of poloxamer 188 on size of nanoparticles. A. Single Poloxamer 188 moiety. B. Lower concentration of poloxamer 188. C. Higher concentration of poloxamer 188.

used for the study as a cosurfactant and the effect of concentrations of 0.1, 0.2, 0.3, 04 and 0.5% soya lecithin on the size has been studied. Two kinds of effects have been prominently observed in the formulations. For formulations containing lipid and surfactant in the ratio of 2:1, an increase in the concentration of cosurfactant from 0.1 to 0.5% exhibited a linear decrease in the size of the particles. For formulations containing lipid and surfactant in the ratio of 4:1, 0.1 -- 0.2% concentrations of cosurfactant caused an initial increase in the size and an increase in concentration beyond 0.3% caused a decrease in the size of the particles. A similar kind of effect was observed for formulations containing lipid and surfactant in the ratio 1:1 but the amount of cosurfactant necessary to cause a reduction in the size of the particles is beyond 0.4%. Generally, addition of cosurfactants will lead to a decrease in the size of the particles and this fashion has been well observed for formulations containing lipid and surfactant in the ratio 2:1. But an initial increase in size followed by a decrease in the size of particles for formulations containing 4:1 and 1:1 ratios of lipid and surfactant indicates that an optimum level of cosurfactant is required to initiate the particle size reduction. Hence, it is well understood that for formulations containing 4:1 and 1:1 ratios of lipid and surfactant, a minimum level 0.3 and 0.4% of cosurfactant is necessary to initiate size reduction of the particles. Higher concentrations of lecithin provide additional surface area and good electrical barrier to the surface which promotes size reduction [38]. According to the used concentrations of excipients in the study for obtaining small particles, it is essential to use high amounts of lipid, low amount of surfactant and high amounts of cosurfactant and this principle was correlated for formulation DS8. Least particle size of 27.25 ± 3.43 nm was observed for formulation DS8, which had higher amount of lipid (10%), low amount of surfactant (2.5 %) and high amounts of soya lecithin (0.5%). Similarly, highest particle size of 179 ± 3.08 nm was observed for formulation DS3 which has low amount of lipid (5%), high amount of surfactant (5%) and no cosurfactant. Zeta potential The zeta potential reveals whether the particles in the system aggregate or remain stable over a long period. Generally, a 3.4

large positive or negative zeta potential is favorable for obtaining particles with better stability. The zeta potential of all the formulations varied between 0.101 ± 0.99 mV and -11.3 ± 0.98 mV and is mentioned in Table 2. In the absence of cosurfactant, soya lecithin, the zeta potential of the nanoparticles was around -0.12 to -1.1 mV. Poloxamer being a nonionic surfactant was not able to induce potential on the surface of the nanoparticles, and a partial negative charge observed was due to the behenate residues present in Compritol 888 ATO. In the presence of cosurfactant, soya lecithin, the potential reduced to a negative magnitude because soya lecithin is an anionic surfactant and hence it was able to induce some negative potential. The neutral zeta potential depicts instability problems upon long storage but because of osmotic force created by the coating of poloxamer around the nanoparticles surface, the system will remain stable [39]. Further, these neutral nanoparticles have added advantage of remaining in the lymphatics for a longer time by evading opsonization [40,41]. The components of lymph are nearly same as that of plasma and they vary mostly in the concentration of the components [35]. Thus, the poloxamer coating around the nanoparticles will increase the retention time in the lymphatics. Particle shape and surface morphology Transmission electron microscopy image in Figure 3 revealed that the particles possess a smooth texture and had a spherical morphology with an average size of 98.03 nm which coincides with the average size obtained by photon correlation spectroscopy. The center portion of the particle is dark in color, indicating the presence of lipid and the outer light surface indicates the presence of surfactant, poloxamer 188. Poloxamer induces a steric barrier on the particle surface by blocking the electrostatic and hydrophobic interactions with the opsonins and thus prevents the opsonization of the nanoparticles, leading to an increase in the retention time in the lymphatics. Analysis of the sample by AFM reveals that the particles are spherical in shape and possess a smooth topography. Further, the average roughness value of 6.42 nm indicates that the surface is smooth. The average particle size was found to be 3.5


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efficiency. In the presence of high amounts of lipids, a large amount of matrix is available for the drug to be entrapped. Hence, an increase in the lipid concentration shows an increase in the entrapment efficiency. Effect of surfactant concentration on the entrapment efficiency


3.6.2

98.03 nm

20 nm

An increase in the amount of surfactant, poloxamer 188, caused a decrease in the entrapment efficiency, and this can be justified with the increase in the solubility of drug in the aqueous phase with the addition of the surfactant. Further, high temperatures also favor the solubility of the drug in the aqueous phase. Hence, an increase in the surfactant concentration will lead to a decrease in the amount of drug present in the lipid matrix [34]. Thus, in order to obtain high entrapment efficiency with smaller particle size, it is favorable to use high amount of lipid and with low amount of surfactant at lower temperatures. In vitro drug release studies In vitro drug release studies have been carried out in pH 7.4 PBS using a dialysis bag with molecular weight 12,000 -- 14,000 Da. It has a pore size of 25 A which allows only the drug released from the nanoparticles to pass across the membrane. A comparison of the drug release patterns of the formulations have been displayed in Figures 5-8. In all the formulations, the total amount of DEC from the nanoparticles was released in < 150 min. Longer time of 150 min of drug release has been observed for formulation DS20 for the total amount of drug to be released. But formulation DS6 took only 60 min for the total amount of drug to be released which is because of the presence of higher amounts of surfactant and cosurfactant. The cumulative percentage values reveal that the drug release from nanoparticles followed a triphasic profile with an initial burst phase in which around 40% of the drug was released in initial 15 min, followed by a second fast phase during which around 65% of the total drug was released and finally a third slow phase is observed. The initial burst effect is due to the poor entrapment of the DEC in the lipid matrix which will cause the free drug in the solution to escape from the dialysis membrane to the buffer. During the second phase, the drug is released from the outer drug-enriched shell and further, the disruption of the soft outer surfactant layer on the particle surface due to the collisions among the nanoparticles will lead to fast release of the drug. The final sustained release phase of the drug is also not prolonged for longer hours because of the smaller size of the particles which provides a larger surface area for the drug-loaded particles. Further, the presence of hydrophilic surfactant and usage of high temperatures during homogenization facilitates faster release of drug from the nanoparticles. Mathematical modeling using DDSolver was performed to assess the drug release kinetics. The r2 (correlation coefficient) 3.7

Figure 3. TEM image of DEC-loaded SLN. DEC: Diethylcarbamazine citrate; SLN: Solid lipid nanoparticle.

in bounds with the values obtained with photon correlation spectroscopy and transmission electron microscopy. The AFM images in Figure 4 clearly show that the particles are well separated, ruling out the possibility of the aggregation of the particles. Encapsulation efficiency and drug loading The encapsulation efficiencies mentioned in Table 2 reveal that the drug is poorly encapsulated in all the formulations, and the values varied between a minimum of 32.9 ± 1.1% for formulation DS3 to a maximum of 68.63 ± 1.5% for formulation DS17. DEC being a hydrophilic drug has poor solubility in the lipid, Compritol 888 ATO, which causes some amount of the drug to repartition into the aqueous phase during cooling, leading to the formation of core shell model, drugenriched shell [42]. Thus, poor entrapment efficiencies are always confronted while incorporating hydrophilic drugs in lipid core material. Further, DSC studies confirmed the presence of DEC in amorphous form in the formulation, justifying the increased solubility in the aqueous phase which will in turn cause poor entrapment in the lipid phase. Also, DSC reports of formulation showed the existence of lipid in b¢/b form, which further promotes drug expulsion [43]. 3.6

Effect of lipid concentration on the encapsulation efficiency

3.6.1

For formulations containing similar concentrations of the drug and surfactant, an increase in the lipid concentration from 5 to 10% showed an increase in the entrapment 8



A.

B.

60 50 40 mm 30 20 10 0 3.0 2.5 3.50


2.0 mm

3.0

1.5

2.5 2.0

1.0

1.5

0.5 0 0

C.

mm

1.0 0.5

D.

35 30 mm

25 20 15 10 5

350

300

300

250 200 150 mm

100

50

50

100

150

200

400 350

250 mm

Figure 4. AFM images of DEC-loaded SLNs. A and C. Phase images. B and D. Topographic images. DEC: Diethylcarbamazine citrate; SLN: Solid lipid nanoparticle.

values indicate a first-order release pattern (r2 = 0.929 -0.996) for all the formulations. Statistical analysis was carried out using two-way ANOVA to check the significance of the release, and the p value obtained was < 0.05. Bonferroni post-tests carried out to obtain the p values at various individual time intervals indicated that some values were not significant. Stability studies Stability studies carried out at room temperature (25 ± 1 C) and refrigerated temperature (4 ± 1 C) showed significant difference in both entrapment efficiency and particle size (Table 3). The surfactant coating on the surface of the nanoparticles will create an osmotic force, which prevents aggregation of particles [39] at both room and refrigerated conditions. 3.8

An increase in the particle size was observed during storage at room temperature because the system gained energy in the form of light and heat from the surroundings (Brownian motion increases). This phenomenon was not observed at refrigerated conditions as the particles did not have the energy from the surroundings (light and heat). Drug leakage was observed in both refrigerated conditions (nearly 2.1%) and room temperature (nearly 8.3%). Initially, the lipid matrix in the formulation was in b¢ form and upon storage, it transformed to b form, which usually occurs with expulsion of drug from the lipid matrix. Hence, a mild decrease in the entrapment efficiencies has been observed during storage and has been supported by Mu¨ller et al. [44]. Hence, freeze drying of the SLN dispersion should be considered as a viable option in future studies while storing the formulations.


9

Cummulative percentage of drug released (%)

120 110 100 90 80 DS 1 DS 5 DS 9 DS 10 DS 11 DS 12

70 60 50 40 30 20 10 0 0

15

30

45

60 75 90 Time (minutes)

105

120

135

150

Figure 5. In vitro drug release profile of formulations DS1, DS5, DS9, DS10, DS11 and DS12 in PBS 7.4.



K. Siram et al.

120 110 100 90 80 70

DS 2 DS 8 DS 13 DS 14 DS 15 DS 16

60 50 40 30 20 10 0 0

15

30

45


105

120

135

150


3.9 Rationale behind selecting DS15 for in vivo animal study

3.10

In order to have a better uptake into the lymphatics and to avoid opsonization by RES, it is essential for the size to remain below 100 nm and the surface charge to be neutral. Apart from that good entrapment efficiency is also essential. Formulation DS15 has an average particle size of 46.05 ± 1.60 nm, a zeta potential of 0.031 ± 1.7 mV and an entrapment efficiency of 59.12 ± 1.2 %. Thus, DS15 was selected for in vivo studies.

Figure 9 displays the mesenteric lymphatic duct in rats.

10

In vivo animal studies for the estimation of DEC concentration in blood and lymph

Estimation of the pharmacokinetic parameters in blood was carried out only in noncannulated rats because cannulation of the lymphatic duct causes bifurcation of the mesenteric lymphatic duct into the collecting plastic tubing leading to a decrease in the amount of DEC reaching the systemic circulation. Thus by avoiding collection of blood from noncannulated groups, the actual amount of



120 110 100 90 80

DS 3 DS 6 DS 17 DS 18 DS 19 DS 20

70 60 50 40 30 20 10 0 0

15

30

45

60 75 90 105 120 Time (minutes)

135

150





120 110 100 90 DS 4 DS 7 DS 21 DS 22 DS 23 DS 24

80 70 60 50 40 30 20 10 0 0

15

30

45


105

120

135

150


drug reaching the systemic circulation can be quantified properly. Generally, drugs administered via SLNs have a tendency to enhance the bioavailability and a measure of pharmacokinetic parameters in blood in Table 4 reveals the same. Figure 10 represents a comparison of plasma concentration profiles of the DEC at different time intervals when administered orally via SLNs and as a solution in pH 7.4 PBS. But, the increase in the amount of drug reaching the blood when administered through SLNs is not significant (p value > 0.05) when

compared to DEC in pH 7.4 PBS. As DEC is a class I drug with good bioavailability, a significant difference in the amount of drug reaching the systemic circulation was not observed when administered via SLNs. A first-order profile was observed, and the pharmacokinetic parameters were estimated by noncompartmental method using PK solver 2.0. The Tmax of DEC increased from 1 to 4 h when administered via SLNs because the drug was initially absorbed through the lymphatics and a considerable amount of time has been consumed by the drug in reaching the systemic circulation


11

K. Siram et al.

Table 3. Particle size and entrapment efficiency of SLNs during stability study. Stability condition

Room temperature (25 C) Refrigerated temperature (3 -- 5 C)

Average particle size (nm)

Entrapment efficiency (%)

0 days

30 days

90 days

0 day

30 days

90 days

46.05 ± 1.60 46.05 ± 1.60

343.7 ± 7.92 50.93 ± 3.94

899.8 ± 5.83 52.8 ± 5.45

59.12 ± 1.22 59.12 ± 1.22

56.35 ± 2.3 57.64 ± 1.8

52.21 ± 3.1 56.83 ± 2.2


Values are expressed as mean ± standard deviation (n = 3). SLN: Solid lipid nanoparticle.

Mesenteric lymphatic duct

Figure 9. Mesenteric lymphatic duct in rat.

from the lymphatics. Although the main objective of the present work is to enhance the uptake of DEC to lymphatics (the residing place for adult filarial worms), the increased concentrations of DEC in blood is not futile as it will be beneficial in killing the microfilaria present in the blood. A comparison of concentration profiles of DEC in lymph over a period of 24 h upon oral administration via SLNs and as a solution in pH 7.4 PBS is depicted in Figure 11. It is obvious that the overall uptake of DEC has increased significantly (p value < 0.05) along with an increase in the retention time in the lymphatics when administered as SLNs. DEC when administered as SLNs has shown a maximum of 1318 ± 105.29 ng/ml at second hour in the lymph and plateau phase was observed at fourth hour with a concentration of 1464 ± 165.52 ng/ml. When administered as a solution in pH 7.4 PBS, only a maximum concentration of 354.67 ± 63.69 ng/ml of DEC was found in the lymph at second hour which shows that there is a good amount of increase in 12

the uptake of DEC to the lymphatics when administered as SLNs. The time taken for DEC SLNs to attain maximum concentration in blood and lymph is 4 and 2 h, respectively, indicating that DEC initially entered the lymphatics and then, it has reached the systemic circulation. At the end of 24th hour, the concentration of DEC in lymph was more when administered via SLNs, which suggests that the retention time for DEC in the lymphatics has also been increased. A mixture of long-chain and medium-chain fatty acids (12 -- 18 % monoglycerides, 52 -- 54% diglycerides and 28 -- 32% triglycerides) in Compritol 888 ATO allowed DEC to enter the lymphatics (by the production of chylomicrons) through various structures in the gut wall leading to an enhanced uptake into the lymphatics. Further, a particle size below 100 nm and a neutral charge on the nanoparticle surface (created by poloxamer) prevented opsonization by RES, leading to an increase in the retention time in the lymphatics. The role of compritol 888 ATO in enhancing the lymphatic uptake of methotrexate [19,45] and lopinavir [12] has been well supported. Apart from oral administration, compritol 888 ATO has shown enhanced lymphatic uptake when administered through pulmonary [46] and subcutaneous routes [47]. Thus, the composition of compritol 888 ATO makes it a potential lipid carrier for targeting various other antifilarial drugs and several potent anticancer drugs to prevent metastasis in the lymphatics.

4.

Conclusion

There are drugs that can kill the adult filarial worms, but upon administration, these drugs do not reach the lymphatics which causes the adult filarial worms to inhabit freely in the lymphatics. Surprisingly, there are no published research works to target the antifilarial drugs to the lymphatics. A solid lipid carrier has been developed for DEC with size < 100 nm which not only helps in reaching the lymphatics but also increases the residence time in the lymphatics by avoiding opsonization. In vivo experiments to quantify the drug concentrations in lymph using an animal model have shown promising results. With improved entrapment efficiency and stability, this carrier can serve well for the purpose. Further, trials in humans can be carried out to assess the efficacy of the nanoparticles against adult filarial worms.



Table 4. Pharmacokinetic parameters of DEC in blood. Group

Cmax (mcg/ml)

Tmax (h)

T1/2 (h)

AUC0 -- t (mcg/h/l)

AUC0 -- ` (mcg/h/l)

AUMC0 -- t (mcg/h/l)

AUMC0 -- ` (mcg/h/l)

CL (ml/min)

Vd (l)

1 (SLNs)

2.875 ± 0.18 2.698 ± 0.19

4

6.54 ± 0.34 5.82 ± 0.28

27.52 ± 2.32 16.7 ± 1.87

30.77 ± 3.20 17.88 ± 2.17

223.89 ± 24.87 98.59 ± 14.71

332.85 ± 50.58 137.01 ± 24.67

0.0009 ± 0.0001 0.0017 ± 0.0002

0.009 ± 0.0005 0.002 ± 0.0021

2 (PBS)

1

Concentration of DEC in blood (ng/ml)

3500 3000 2500 DEC SLNs DEC in pH 7.4 PBS

2000 1500 1000 500 0 0

2

4

6

8 10 12 14 16 18 20 22 24 Time (h)

Figure 10. Plasma concentration profile of DEC after administration of DEC-loaded SLNs and a solution of DEC in pH 7.4 PBS. DEC: Diethylcarbamazine citrate; SLN: Solid lipid nanoparticle.

2000 Concentration of DEC in lymph (ng/ml)


Values are expressed as mean ± standard deviation (n = 3). DEC: Diethylcarbamazine citrate; SLN: Solid lipid nanoparticle.

1500 DEC SLNs DEC in pH 7.4 PBS 1000

500

0 0

2

4

6

8

10 12 14 16 Time (h)

18

20 22

24 26

Figure 11. Lymph concentration profile of DEC after administration of DEC-loaded SLNs and a solution of DEC in pH 7.4 PBS. DEC: Diethylcarbamazine citrate; SLN: Solid lipid nanoparticle.


13

K. Siram et al.

Declaration of interest Animal Ethical committee approval number: 158/99/ CPCSEA. The authors have no relevant affiliations or financial involvement with any organization or entity with a Bibliography


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Affiliation

Karthik Siram1, Vijaya Raghavan Chellan†2, Tamilselvan Natarajan1, Balakumar Krishnamoorthy1, Habibur Rahman Mohamed Ebrahim1, Vamshikrishna Karanam1, Siva Selva Kumar Muthuswamy3 & Hari Prasad Ranganathan3 † Author for correspondence 1 PSG College of Pharmacy, Department of Pharmaceutics, Peelamedu, Coimbatore -- 641004, Tamil Nadu, India 2 Vice Principal and Head, PSG College of Pharmacy, Department of Pharmaceutics, Peelamedu, Coimbatore -- 641004, Tamil Nadu, India Tel: +91 9843128373; Fax: +91 0422 2594400; E-mail: [email protected] 3 PSG College of Pharmacy, Department of Pharmaceutical Analysis, Peelamedu, Coimbatore -- 641004, Tamil Nadu, India

15

Optimized nano-transfersomal films for enhanced sildenafil citrate transdermal delivery: ex vivo and in vivo evaluation.

Solid lipid nanoparticles for nose to brain delivery of haloperidol: in vitro drug release and pharmacokinetics evaluation.

In vivo pharmacokinetics and biodistribution of resveratrol-loaded solid lipid nanoparticles for brain delivery.

Physical characterization and in vitro skin permeation of solid lipid nanoparticles for transdermal delivery of quercetin.

Intranasal agomelatine solid lipid nanoparticles to enhance brain delivery: formulation, optimization and in vivo pharmacokinetics.

Surface-modified solid lipid nanoparticles for oral delivery of docetaxel: enhanced intestinal absorption and lymphatic uptake.

Solid lipid nanoparticles for potential doxorubicin delivery in glioblastoma treatment: preliminary in vitro studies.

Buparvaquone loaded solid lipid nanoparticles for targeted delivery in theleriosis.

Lipid nanoparticles for oral delivery of raloxifene: optimization, stability, in vivo evaluation and uptake mechanism.

In vitro and in vivo anti-cancer effects of targeting and photothermal sensitive solid lipid nanoparticles.

Development and evaluation of solid lipid nanoparticles of mometasone furoate for topical delivery.

Development and evaluation of solid lipid nanoparticles of raloxifene hydrochloride for enhanced bioavailability.

In vitro and in vivo trypanocidal activity of H2bdtc-loaded solid lipid nanoparticles.

Correction: in vitro and in vivo trypanocidal activity of h2bdtc-loaded solid lipid nanoparticles.

Enhanced oral bioavailability of efavirenz by solid lipid nanoparticles: in vitro drug release and pharmacokinetics studies.

Development and evaluation of coenzyme Q10 loaded solid lipid nanoparticle hydrogel for enhanced dermal delivery.

Candesartan cilexetil loaded solid lipid nanoparticles for oral delivery: characterization, pharmacokinetic and pharmacodynamic evaluation.

vivo studies of aclacinomycin A-loaded solid lipid nanoparticles.

Enhanced in Vitro Anti-Tumor Activity of 5-Azacytidine by Entrapment into Solid Lipid Nanoparticles.

Enhanced oral bioavailability of insulin-loaded solid lipid nanoparticles: pharmacokinetic bioavailability of insulin-loaded solid lipid nanoparticles in diabetic rats.

Formulation, in vitro and in vivo evaluation of halofantrine-loaded solid lipid microparticles.

Solid and liquid lipid-based binary solid lipid nanoparticles of diacerein: in vitro evaluation of sustained release, simultaneous loading of gold nanoparticles, and potential thermoresponsive behavior.

Enhanced skin delivery of aceclofenac via hydrogel-based solid lipid nanoparticles.

Cardiovascular effects of diethylcarbamazine citrate.