Effect of lipid core material on characteristics of solid lipid nanoparticles designed for oral lymphatic delivery

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Nanomedicine: Nanotechnology, Biology, and Medicine 5 (2009) 184 – 191 www.nanomedjournal.com

Original Articles: Experimental Nanomedicine, Engineering

Effect of lipid core material on characteristics of solid lipid nanoparticles designed for oral lymphatic delivery Rishi Paliwal, MPharm, a Shivani Rai, MPharm, a Bhuvaneshwar Vaidya, MPharm, a Kapil Khatri, MPharm, a Amit K. Goyal, MPharm, a Neeraj Mishra, MPharm, a Abhinav Mehta, MPharm, a Suresh P. Vyas, PhD b,⁎ a

Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Dr. H.S. Gour Vishwavidyalaya, Sagar, India b Nanomedicine Research Centre, ISF College of Pharmacy, Moga, Punjab, India

Abstract

Key words:

Solid lipid nanoparticles (SLNs) are essentially composed of triglyceride(s) that orient to form a polar core with polar heads oriented toward the aqueous phase, resembling chylomicrons. The composition of such SLNs may alter the course of drug absorption predominantly to and through lymphatic route and regions, presumably following a transcellular path of lipid absorption, especially by enterocytes and polar epithelial cells of the intestine. SLNs were prepared using stearic acid, glycerol monostearate, tristearin, and Compritol 888 ATO by solvent diffusion method using demineralized double-distilled water as the dispersion medium. The SLNs were characterized for shape, size, zeta potential, and percentage drug content and its release. The characterization of SLNs suggests that Compritol 888 ATO–based nanoparticles were heterogeneous with better drug-loading and release characteristics as compared with the other formulations. The selected products were studied for in vivo absorption and hence bioavailability by measure of area under the blood plasma curve plotted as a function of time. Periodic lymphatic concentration of drug following oral administration of respective formulations was also determined by mesenteric duct cannulation and collection of samples. The comparative study conducted on methotrexate (MTX)-bearing SLNs revealed that the formulation based on Compritol 888 ATO could noticeably improve the oral bioavailability of MTX, presumably following SLNs constituting lipid digestion and co-absorption through lymphatic transport and route. © 2009 Elsevier Inc. All rights reserved. Solid lipid nanoparticles; Lipid-based carrier systems; Intestinal lymphatics; Methotrexate

Intestinal lymphatic regions have been routinely explored and used for site-specific oral absorption of peptides, proteins, drugs, and vaccines. The unique advantageous features are avoidance of hepatic first-pass metabolism (thus enhancing bioavailability of lipophilic drugs), stimulation of the immune response, and specifically targeting the drugs to the diseases of intestine-associated accessory biosystems

Received 12 August 2007; accepted 24 August 2008. Financial support by the University Grants Commission (UGC), New Delhi, INDIA, as JRF to one of the authors (Rishi Paliwal) is duly acknowledged. ⁎Corresponding author. E-mail address: [email protected] (S.P. Vyas). 1549-9634/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2008.08.003

such as lymph and lymphatic nodes, which includes cancer and acquired immunodeficiency syndrome.1-4 Several means and mechanisms of delivering bioactives to or through lymphatic regions following the oral route have been investigated and documented. These include paracellullar mechanism,5,6 exploitation of M cells for vaccine delivery,7,8 and transcellular mechanism9,10 of drug and nanocarrier absorption. The most promising mechanism is transcellular absorption, which operates for dietary lipids. The co-administration of lipid vehicle along with bioactives thus enhances the stimulation of chylomicron formation by enterocytes, which dissolve and assimilate lipophilic molecules into their nonpolar core and thus promote the absorption of water-insoluble drugs into

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intestinal lymphatics and organs. Moreover, the possibility of simple large-scale production at the industrial level and the nontoxicity of lipids are also advantageous features of lipid-based formulations. Variation in the lipid constituents may alter the bioavailability of drug or bioactives including vaccines.12 Lipid solutions, microemulsions, liposomes, self-emulsifying delivery systems, micellar solutions, and recently solid lipid nanoparticles (SLNs) have been exploited as probable possibilities as carriers for oral intestinal lymphatic delivery.13-17 Since 1991 SLNs have been explored extensively in drug delivery through various routes. The SLNs-based systems possess characteristics of conventional carriers as well as some additional characteristics that obviate the drawbacks associated and reported for conventional systems. Therefore, they are considered to be a better alternative than liposomes, polymeric nanoparticles, and microemulsion, among others. 18-20 SLNs have been documented as value-added carriers for successful delivery of peptides and anticancer drugs like doxorubicin through the oral intestinal route.21,22 SLNs reportedly enhanced the oral bioavailability of cyclosporine A,15 sustained release of such lipophilic drugs as camptothecin, 23 and for prolongation of the absorption kinetics of idarubicin after duodenal administration to rats. 24 Because SLNs are composed of a lipid core that may stimulate chylomicrons formation, which ultimately carries the carrier along with the co-entrapped drug by following the classical transcellular mechanism of lipid absorption.12 Such a role of SLNs has been studied earlier by Bargoni et al., who have estimated uptake and transport of SLNs to the intestinal lymphatics after intraduodenal administration. They reported that SLNs were found to be stable at gastric physiological pH, even after 180 minutes of administration, and suggested them as a suitable carrier system for lymphatic drug delivery.25 Most of the studies on SLNs aim at a direct assessment of the available dose of contained drug in the lymph after duodenal administration of the vehicle, which do not confirm or suggest the carrier behavior while it passes through acidic environment of the stomach. Furthermore, previous published reports have been on the chain length of glycerides and also the saturated and unsaturated state of the lipids and their effect on the lymphatic absorptions of the drug and hence the entrapped drug(s). In the present study SLNs consisting of various lipid cores (stearic acid, monostearin, tristearin, and Compritol 888 ATO [Colorcon Asia Ltd., Goa, India]) were developed. Methotrexate (MTX) was selected as the model drug. The optimized MTX-loaded SLNs formulations were studied for their size and shape to correlate their structural resemblance to chylomicrons, loading capacity, and in vitro release in simulated pH conditions encountered en route on oral administration, so as to assess the potential as oral formulation. The stability behavior in terms of size and entrapment efficiency of the formulations with time at

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various temperature and pH conditions were studied to evaluate the effect of the various pH environments of the gastrointestinal tract (GIT) on drug and carrier stability in vitro. The in vivo study was conducted to estimate both blood plasma and lymphatic concentration(s) of the MTX following oral administration of MTX-loaded SLNs.

Methods Materials MTX was obtained as a gift from Dabur Research Foundation (Ghaziabad, India). Compritol 888 ATO was generously provided by Colorcon Asia Ltd. Stearic acid, glycerol monostearate, tristearin, L-α-soya lecithin, and Sephadex G-50 were purchased from Sigma Chemicals Co. (St. Louis, Missouri). All other chemicals and reagents were of analytical grade unless otherwise specified. Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the official methods of the United States Pharmacopoeia (XXV; http://www.usp.org). Preparation of MTX-loaded SLNs MTX-loaded SLNs were prepared by the solvent diffusion method in aqueous system described elsewhere with slight modification.26 Briefly, 100 mg selected lipid (stearic acid, monostearin, tristearin, and Compritol 888 ATO) were dissolved completely in a 10-mL mixture of acetone and ethanol (1:1 v/v) in a water bath at 45°C. This lipid solution was poured into 200 mL of an acidic aqueous phase of the drug MTX (10% w/v) containing 1% w/v soya lecithin under continuous mechanical agitation (Remi Instruments, Mumbai, India) with 4000 rpm at room temperature (25°C-28°C) for 5 minutes. The pH value of the acidic aqueous phase was adjusted to 1.10 by careful addition of 0.1 M hydrochloric acid. The SLNs suspension was quickly produced. The dispersed system was centrifuged at 4000 rpm for 20 minutes (Remi Instruments, Mumbai, India) and resuspended in distilled water. The different concentrations of soya lecithin were used to prepare and optimize SLNs. Entrapment efficiency The entrapment efficiency of drug was estimated by measuring the concentration of unentrapped drug in the dispersion of SLNs as reported elsewhere.27 In brief, the dispersion of prepared SLNs was subjected to centrifugation. The amount of MTX in supernatant was determined by HPLC method as described by Moghbel et al.28 Size and zeta potential The average particle size, size distribution, and zeta potential were measured by photon correlation spectroscopy (ZS90 zeta sizer; Malvern Instruments, Worcestershire, UK) at 25°C. The samples were kept in polystyrene cuvettes, and observations were made at a 90-degree fixed angle every time.

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Transmission electron microscopy (TEM) Prepared SLNs were characterized for shapes by transmission electron microscope (JEOL, Tokyo, Japan) using a copper grid coated with carbon film and with phosphotungstic acid (1% w/v) as a negative stain. In vitro release studies In vitro release of MTX from SLNs was determined by dialysis membrane (molecular weight cutoff 10,000 Da). Briefly, the dialysis membrane was kept in double-distilled water for 24 hours before mounting into a Franz diffusion cell. MTX-loaded SLNs formulation (1 mL) was placed in the donor compartment, and the receptor compartment was filled with dialysis medium (10 mL of 0.1 M HCl or doubledistilled water or phosphate-buffered saline [PBS] pH 7.4) and stirred continuously at 100 rpm at 37°C. After regular time intervals samples were withdrawn from the receptor compartment through a side-arm tube. After each withdrawal of sample an equal volume of dialysis medium was added in the receptor compartment so as to maintain a constant volume throughout the study. Samples were analyzed for MTX concentration using ultraviolet spectrophotometry (Cintra, 10, G, GBC, Danetenong, Australia) at 259 nm. Storage stability studies All the MTX-SLNs formulations were studied for stability studies at various temperatures (4 ± 1°C, 25 ± 1°C) and pH conditions (SGF pH 1.2, SIF pH 7.5). These formulations were examined and estimated at regular time intervals for any change in particle size and drug content. In vivo studies In vivo 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. All the study protocols were approved by the animal ethical committee of the university, Sagar (MP), India. Albino rats of either sex were weighed (250–350 g) and divided into six groups comprising six animals in each. All animals were kept for overnight fasting but allowed free access to water. All the animals of group I were given an oral dose of MTX solution in PBS pH 7.4; group II was administered orally with a equivalent dose of MTX-loaded SLNs having stearic acid as core material (MTX-SA) formulation; the animals of group III received orally MTX-loaded monostearin-cored SLNs (MTX-MS); group IV was given MTX-loaded tristearin-cored SLNs (MTX-TS) orally, and group V was given MTX-loaded Compritol 888 ATO-cored SLNs (MTXCA) in equivalent doses of MTX orally, whereas the VI group served as control. All the animals were anesthetized by urethane injection (1.2 g/kg). The animals were dissected and the mesenteric duct was cannulated as described by Warshaw (1972).29 Simultaneously, the jugular vein was also cannulated. All the animals were kept in a supine

position and infused continuously with normal saline solution at the rate of 4 mL/hr/kg. Lymph and blood samples were collected periodically and analyzed for MTX content using high-performance liquid chromatography. Statistical analysis The results were expressed as mean ± standard deviation. Statistical analysis was carried out by using Student's t-test, and statistical significance was designated as Pb0.05.

Results The process of digestion and absorption of dietary or formulation lipids and co-administered drugs includes the assembly of lipoproteins within the enterocytes and the association of these lipoproteins with lymphatically transported drugs. This is a complex process of lipid absorption and transportation that operates under the control of a number of incompletely understood transporters and binding proteins. Passive delivery to the systemic circulation in association with lymph lipoproteins has been found to alter clearance and deposition profiles and might be expected to lead to altered activity and toxicity profiles.30 MTX has low oral bioavailability, indicating that a major fraction of the absorbed drug is either metabolized by the liver and or by other organs of the body. High-dose administration of MTX increases the chances of gastrointestinal toxicity. The present work was carried out with an aim to enhance the overall oral bioavailability of MTX and also to bypass the possible liver metabolism by promoting its lymphatic absorption using SLNs, the biomimics of chylomicrons. Method of preparation SLNs can be prepared by several methods reported earlier including hot homogenization, cold homogenization, microemulsion, and solvent-emulsification diffusion in aqueous medium. The solvent-emulsification diffusion method, being quick with simple laboratory setup, was used in the present study. Reproducibility of the results was also studied. Remarkably, high loading of MTX (72.4 ± 0.14% w/w) in optimized SLNs formulation was measured. It is likely that the sudden change in pH of dispersions to 1.2 followed by centrifugation might develop an SLNs-rich phase that on centrifugal separation results in SLNs with compressed intermolecular geometry and hence the interjoined pores. Entrapment efficiency The lipid core material was found to affect the extent of MTX loading in SLNs. As observed with MTX-SA, MTXMS, MTX-TS, and MTX-CA, the maximum loading was estimated to be 61.5 ± 0.44%, 66.2 ± 0.27%, 63.3 ± 0.22%, and 72.4 ± 0.14% (w/w) respectively. It was observed that all

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Table 1 Effect of concentration of soya lecithin on entrapment efficiency of MTXloaded SLNs

Table 2 Average size and zeta potential of MTX-loaded SLNs of different lipids (stabilized with 1% w/w soya lecithin)

Study no.

Formulation code

Size (nm) ⁎

Zeta potential (mV) ⁎

MTX-SA

131.0 ± 3.2

–28.3 ± 1.0

Formulation code

Entrapment efficiency (%) of MTX-loaded SLNs at various concentrations of soya lecithin ⁎ 0.5% w/w

1.0% w/w

1.5 % w/w

1

MTX-SA

58.2 ± 0.54

61.5 ± 0.44

57.3 ± 0.65

2

MTX-MS

62.1 ± 0.35

66.2 ± 0.27

65.5 ± 0.49

3

MTX-TS

57.8 ± 0.76

63.3 ± 0.22

60.9 ± 0.37

4

MTX-CA

66.1 ± 0.82

72.4 ± 0.14

69.2 ± 0.58

MTX-MS

166.2 ± 8.1

–14.4 ± 2.2

MTX-TS

140.4 ± 6.4

–10.7 ± 4.5

MTX-CA

120.1 ± 1.8

–12.0 ± 2.0

*Values are reported as mean ± SD (n = 4).

*Values are reported as mean ± SD (n = 4).

Figure 1. TEM photographs of different MTX-loaded SLNs formulations (stabilized by 1% (w/w) soya lecithin). (A) MTX-SA; (B) MTX-MS; (C) MTX-TS; (D) MTX-CA.

Figure 2. In vitro release studies in different time intervals of MTX-loaded SLNs in double-distilled water as dialysis medium.

Transmission electron microscopy the formulations containing an equal amount of the lipid, either in the form of free fatty acid (stearic acid) or in glycerides form (monostearin and tristearin), the amount of loaded MTX estimated was comparable (within ±5% variation of loaded drug). However, in the case of triglycerides with high chain length (Compritol 888 ATO) they showed nearly 10% higher drug loading than average loaded drug in formulations with stearic acid and its glycerides. This may be ascribed to the interchain intercalation of MTX offering intramolecular intercalation as well as intermolecular entrapment. Lipid-to-surfactant ratio can affect the loading efficiency of the formulations. The amount of soya lecithin was optimized in the range of 0.5% (w/w) to 1.5% (w/w) against a constant amount of lipid. It was observed that 1.0% (w/w) soya lecithin could entrap maximum drug, as shown in Table 1. The possible reason of this might be that at this concentration surfactant provided sufficient covering to the lipid core so as to minimize possible leaching of the drug.

Figure 1, A–D shows TEM photographs of MTX-loaded SLNs (MTX-SA, MTX-MS, MTX-TS, and MTX-CA, respectively). It was observed in the TEM photographs that particles were almost spherical with homogeneous shading for all formulations. Additionally, SLNs with Compritol 888 ATO core were more uniform in size and homogeneous in comparison to other formulations. Size and zeta potential Chylomicron-mimicking behavior of SLNs might be achieved when the size of SLNs is nearly equivalent. Zeta potential is an important criterion for study of the storage stability of lipid particles and their cellular behavior in drug release. Table 2 lists the sizes and zeta potentials of MTXloaded SLNs. All formulations possessed a mean size range from 120 nm to 167 nm. The highest negative zeta potential of the MTX-SA (–28.3 mV) formulation might be due to the highly negative charge distributed at the surface of the SLNs of stearic acid. Interestingly, the negative value of zeta potential decreased to –14.4 mV when stearic acid was

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Table 3 Effect of concentration of stabilizer (soya lecithin) on the in vitro release of MTX-CA SLNs Study no.

Time (hr)

Percentage release of MTX from the various MTX-CA SLNs formulated with different concentrations of soya lecithin ⁎ 0.5% w/w

1.0% w/w

1.5% w/w

1

1

0.8 ± 0.2

1.2 ± 0.1

1.0 ± 0.2

2

3

2.3 ± 0.4

4.8 ± 0.4

3.1 ± 0.5

3

6

3.0 ± 0.5

6.4 ± 0.7

4.4 ± 0.7

4

12

5.8 ± 1.1

11.7 ± 1.3

8.3 ± 0.9

5

24

8.8 ± 1.3

19.2 ± 1.5

13.5 ± 2.1

*Values are reported as mean ± SD (n = 4).

replaced with monostearin. Moreover, continuous reduction in the negative value of zeta potential was observed as the nature of the glycerides core changed to tristearin (–10.7 mV). The optimized formulations of MTX-CA showed lower value of zeta potential of (–12.0 mV) as compared with MTX-SA and MTX-MS but slightly higher than MTX-TS. In vitro release In vitro release studies were carried out using modified Franz diffusion cells with a dialysis membrane of molecular weight cutoff of 10,000 Da. Dialysis membranes retained SLNs and passed the MTX molecules, which were released over time into the receiver compartment of the model. Figure 2 shows the percentage release of the MTX in double-distilled water from various formulations coded as MTX-SA, MTX-MS, MTX-TS, and MTX-CA. These results revealed that the amount of MTX that was released in 24 hours from all formulations was less than 20% in all cases: MTX-SA (10.4%), MTX-MS (14.6%), MTX-TS (18.3%), and MTX-CA (19.0%). However, there is significant variation in the release rate. For example, MTX-CA release rate is about 90% higher than that of MTX-SA. It was also observed that any change in concentration of stabilizer (0.5%, 1.0%, and 1.5% w/w) also decreases the percentage release from all the formulations (Table 3). The slow release of the MTX from all formulations suggests homogeneous entrapment of the drug throughout the systems. To understand the behavior of SLNs in different pH media, the release pattern of all formulations was determined in 0.1 M HCl (for 2 hours) and in PBS pH 7.4 (for 4 hours). These results revealed that all formulations passing through the strong acidic environment of the stomach (as in 0.1 M HCl) tend to release a high amount of drug (81.1% with MTX-SA, 79.0% with MTX-MS, 75.8% with MTX-TS, and 70.4% with MTX-CA), thereby indicating significant instability (Pb0.05) in the gastric environment. All MTX-loaded SLNs formulations were found to release the MTX in a controlled manner in PBS pH 7.4. The maximum release up to 4 hours was 14.2%, 16.2%, 19.2%, and 22.3% for MTX-SA, MTX-MS, MTX-

Figure 3. Comparative release studies of MTX-loaded SLNs in various pH media in different time intervals (2 hours in 0.1 M HCl, 4 hours in PBS pH 7.4, 24 hours in double-distilled water).

SA, and MTX-CA formulations, respectively. Moreover, release of the MTX in double-distilled water up to 24 hours was less and is shown for comparison in Figure 3. The release of MTX was higher in the case of MTX-SA as compared with other formulations in all the cases. It is notable that nearly 60% to 75% of the drug was still associated with SLNs after 4 hours residence of SLNs in alkaline pH medium and thus would be available for its absorption through enterocytes. Storage stability studies All MTX-loaded SLNs were also tested for their stability at different temperatures (4 ± 1°C and 25 ± 1°C) and pH conditions (SGF pH 1.2 and SIF pH 7.4). MTX-loaded SLNs, when stored at refrigerated temperature (4 ± 1°C), were found to be more stable in terms of change in size and entrapment efficiency, in comparison to room temperature storage (Table 4). The net effect of pH on stability of MTXloaded SLNs was found to be remarkable (Table 5). All formulations were stable in SIF (pH 7.4) after 6 hours storage. When formulations were kept for 2 hours in SGF (pH 1.2), an increase in particle size along with high drug release was observed. This may be due to possible aggregation of lecithin-stabilized SLNs. The MTX-SA was observed to be highly unstable in SGF, in that it became clumpy on 2 hours storage. These results are in accordance with the previous reports.27 In vivo studies Several animal models have been used to study intestinal lymphatic transport. Mesenteric duct cannulation as a site for study is to be preferred over the thoracic duct, because the thoracic duct drains lymph from other areas of the body and sometimes may be misleading. An anesthetized rat model was used for the present study. The in vivo

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Table 4 Stability studies of MTX-loaded SLNs (stabilized by 1% w/w soya lecithin) at 4 ± 1°C and 25 ± 1°C after 2 months storage Formulations

Size (nm) ⁎

Entrapment efficiency (%) ⁎

0 day

After 60 days

RT ⁎⁎

0 day

4 ± 1°C

25 ± 1°C

RT ⁎⁎

After 60 days 4 ± 1°C

25 ± 1°C

MTX-SA

131.0 ± 3.2

135.1 ± 2.2

150.1 ± 0.7

61.5 ± 0.44

60.8 ± 0.81

58.8 ± 0.60

MTX-MS

166.2 ± 8.1

170.3 ± 1.0

180 ± 1.3

66.2 ± 0.27

66.0 ± 0.33

60.2 ± 0.32

MTX-TS

140.4 ± 6.4

145 ± 1.6

152 ± 0.6

63.3 ± 0.22

63.1 ± 0.41

59.2 ± 0.36

MTX-CA

120.1 ± 1.8

121.1 ± 0.6

131.2 ± 0.8

72.4 ± 0.14

71.9 ± 0.12

66.5 ± 0.45

*Values are reported as mean ± SD (n = 4). **Room temperature.

Table 5 Stability studies of various MTX-loaded SLNs (stabilized by 1% w/w soya lecithin) at different pH conditions Formulations

Size (nm) ⁎ 0 hr Initially

Entrapment efficiency (%) ⁎ 2 hr SGF (pH 1.2) †

6 hr SIF (pH 7.4)

0 hr Initially

150.4 ± 3.2

61.5 ± 0.44

2 hr SGF (pH 1.2) †

6 hr SIF (pH 7.4) 59.4 ± 0.22

MTX-SA

131.0 ± 3.2

MTX-MS

166.2 ± 8.1

245.3 ± 3.3

175.6 ± 0.8

66.2 ± 0.27

22.1 ± 0.88

60.3 ± 0.43

MTX-TS

140.4 ± 6.4

380.2 ± 2.1

148.2 ± 1.6

63.3 ± 0.22

28.2 ± 1.70

57.3 ± 0.82

MTX-CA

120.1 ± 1.8

260.6 ± 1.4

126.0 ± 0.9

72.4 ± 0.14

30.8 ± 1.44

60.2 ± 0.13

SGF, simulated gastric fluid; SIF, simulated intestinal fluid. *Values are reported as mean ± SD (n = 4). † No observation could be made as a result of clump formation.

performance of SLNs was evaluated via intraduodenal administration of the formulations, and the studies were conducted on six groups of albino rats of either sex. The rats were given an initial oral dose of soybean oil to swell the lymphatic duct to make it accessible for cannulation. Periodically collected plasma and lymph were estimated for MTX content. Figure 4 shows the plasma profile. The data obtained were subjected to appropriate statistical treatment, and bioavailability was determined by computing the area under the curve (AUC) of the drug plasma profile. The AUC was determined using the trapezoidal rule. The AUC recorded for the formulations of MTX as a plain solution in PBS pH 7.4, MTX-SA, MTX-MS, MTX-TS, and MTX-CA were 7.75 ± 0.93, 20.21 ± 1.52, 24.57 ± 2.23, 31.91 ± 1.62, and 52.84 ± 2.39 (μg·hr/mL), respectively. It is clear that a significantly higher bioavailability of MTX was found when administered in the form of SLNs as compared with plain MTX solution. Similarly, the bioavailability estimated for MTX-TS was higher than MTX-SA and MTX-MS, whereas it was highest in the case of MTX-CA. The time for attaining peak plasma drug concentration in the case of plain drug solution (1 hour) was less as compared with MTX-SLNs (4 hours). The lymphatic drug concentration profile reveals lymphatic absorption of the drug. This may be due to

co-absorption of drug and carrier lipid into chylomicrons and subsequent transportation and finally release of the drug at albuminal the site of the endothelium during reassembling of derivatized lipid(s). Thus, a small amount may remain associated yet to be carried by chylomicrons to the liver. The percentage lymphatic uptake of MTX at various time intervals was recorded and was found to be highest after 3 hours of administration. The comparative performance of MTX solution in PBS pH 7.4, MTX-SA, MTX-MS, MTX-TS, and MTX-CA were 1.2 ± 0.12%, 16.14 ± 1.52%, 18.46 ± 1.8%, 18.58 ± 2.2%, and 24.42 ± 2.6%, respectively (Figure 5). SLNs-based formulations showed many fold increase in MTX concentration in the lymphatic region as compared with MTX plain solution. The MTX-CA formulation has shown highest uptake of the lipid particles, which may be attributed to the small size of the particle and also to the solubilization characteristics of mono-glyceryl behenate, which was an important component of Compritol 888 ATO. It is clear from the above results that lymphatic uptake and welldefined transcellular mechanism of lipid processing played important roles in the increased oral bioavailability of MTX and thus may account for a delay in time to attain peak plasma concentration. The transport of intact SLNs or the stimulation of chylomicron formation may be other responsible factors. The latter one is a well-documented

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Figure 5. In vivo lymphatic uptake of various formulations.

instrumentation facility, Department of Anatomy, AIIMS, INDIA for electron microscopy. Figure 4. Plasma profile of MTX-loaded SLNs.

References process of lipid uptake.31-33 However, a well-defined study protocol is further required to establish the role of the lipid in chylomicron stimulation. Discussion Finally, it can be concluded from the above study that SLNs enhance the oral bioavailability of MTX and increase its concentration in the lymphatic region. Furthermore, SLNs developed from a highly lipophilic core material like Compritol 888 ATO showed more promising in vitro and in vivo results than other lipid core materials and thus may be used as a suitable carrier system for oral intestinal lymphatic delivery of bioactives. Results relating to size, shape, zeta potential, and entrapment efficiency of the drug revealed that MTX-CA was a better formulation as compared with MTX-SA, MTX-MS, and MTX-TS. Additionally, the method of solvent diffusion in aqueous medium seemed to be superior for promoting high drug loading in SLNs. Stability of MTX-loaded SLNs formulations was found to be higher under refrigerated conditions, whereas the instability of formulations in acidic medium suggested that administered formulations should also be protected from the harsh gastric environment of the stomach by some enteric formulation. The lipid nanoparticles are transported to the systemic pool via the intestinal lymphatics and thus bypass the hepatic metabolism—possibly resulting in the enhanced bioavailability of MTX. Acknowledgments The authors are thankful to Dabur Research Foundation (India) and M/s. Colorcon Asia Ltd. for providing the gift samples of MTX and Compritol 888 ATO, respectively. The authors duly acknowledge the sophisticated analytical

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