Journal of Drug Targeting, 2009; 17(10): 777–787

R E S E A R C H A R T I C L E

Mannosylated nanoparticulate carriers of rifabutin for alveolar targeting

Navneet Nimje1, Abhinav Agarwal1, Gaurav Kant Saraogi1, Narendra Lariya1, Gopal Rai1, Himanshu Agrawal2, and G. P. Agrawal1

1Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, India, and 2Pharmaceutics Research Laboratory, M. S. University of Baroda, Vadodara, India

Address for Correspondence: Abhinav Agarwal, Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, Madhya Pradesh 470003, India. E-mail: abhi_mph051@yahoo.co.in

(Received 22 April 2009; revised 04 June 2009; accepted 12 June 2009)

Introduction

Tuberculosis (TB) is a contagious, granulamatous communicable bacterial infectious disease caused by tubercle bacillus, which is an intracellular parasite, liv-ing and multiplying inside macrophages. TB is a disease that harbors in the pulmonary tissues of the lungs, and the bacteria primarily infects macrophages. Despite the fact that a number of potential treatments are available since almost half a decade, TB remains the leading cause of deaths among infectious diseases. The problem has been aggravated due to a number of other complicating factors like multidrug-resistant tuberculosis and HIV-coinfection (Starke Jeffrey, 1997; Peter, 1999; Iseman, 2002; Curvo et al., 2005).

In the context of drug delivery to the lung tissues, nanoparticles hold a tremendous prospective for the delivery of antitubercular drugs. Nanoparticles are solid, colloidal submicron particles in which the drug can be encapsulated or entrapped and/or on to which the drug can be adsorbed. Solid lipid nanopar-ticles (SLNs) are the class of nanoparticles which are biocompatible, bioerodible, and nontoxic in nature. While compared to liposomes, SLNs offer better pro-tection to drug against chemical degradation because there is little access of water to the inner core of lipid particles. In addition, the sterilization of SLNs is possible by autoclaving and gamma irradiation. The advantages of SLNs as compared to other biocompat-ible nanoparticles for lung targeting delivery include

ISSN 1061-186X print/ISSN 1029-2330 online © 2009 Informa UK LtdDOI: 10.3109/10611860903115308

AbstractThe objective of the present study was to evaluate the prospective of engineered nanoparticles for selec-tive delivery of an antituberculosis drug, rifabutin, to alveolar tissues. Drug-loaded solid lipid nanoparti-cles (SLNs) were synthesized and efficiently mannosylated. The formation of uncoated and coated SLNs was characterized by FTIR spectroscopy and SEM studies. A variety of physicochemical parameters such as drug loading, particle size, polydispersity index, zeta potential, and in vitro drug release were determined. The toxicity and targeting potential of the prepared formulation were assessed with alveolar macrophage uptake, hematological studies, and in vivo studies of uncoated and coated SLNs. Ex vivo cellular uptake studies of SLNs formulations in alveolar macrophages depicted almost six times enhanced uptake due to mannose coating. The hematological studies proved mannose-conjugated system to be less immunogenic and suitable for sustained delivery as evaluated against uncoated formulation. Further, the serum level and organ distribution studies demonstrated efficiency of the system for prolonged circulation and spatial delivery of rifabutin to alveolar tissues. Finally, it was concluded that mannose-conjugated SLNs can be exploited for effective and targeted delivery of rifabutin compared to its uncoated formulation and ulti-mately increasing the therapeutic margin of safety while reducing the side effects.

Keywords: Mannose; rifabutin; solid lipid nanoparticles; tuberculosis; alveolar macrophages; targeted drug delivery

http://www.informahealthcare.com/drt

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778 Navneet Nimje et al.

the absence of production of toxic metabolites; avoidance of organic solvents; and this provides lower cytotoxicity (Muller & Wallis, 1993; Muller et al., 1995; Zur Muhlen, Schwarz, & Mehnert, 1998). Coating of nanoparticles with a suitable biocompatible mol-ecule may help in their improved localization in the lung tissues probably because of preferred uptake by alveolar macrophages (Soppimath et al., 2001; Vyas & Khar, 2002; Bala, Hariharan, & Kumar, 2004; Gelperina et al., 2005).

Alveolar macrophages express cell surface lectin receptors that recognize glycoconjugates bearing terminal mannose residues with high binding affin-ity. Glycotargeting exploits the interaction of specific molecular signatures expressed on the surface of cells with ligands (mannose, galactose, fructose, lactose) (Davis & Robinson, 2002). Membrane lectin receptors bind with specific carbohydrate moieties followed by their receptor-mediated internalization. The conju-gation of mannose with various drug delivery vehi-cles (Kumar et al., 2006; Verma et al., 2008) has been extensively investigated and in many cases, these conjugates exhibited a high macrophage uptake, good activity, and fewer side effects. The results obtained may be ascribed to the preferential, selective uptake of mannose by alveolar macrophages. Consequently, the formulation of polysaccharide-tailored nanopar-ticulate formulations for the delivery of therapeutic bioactives can serve as a potential approach for the delivery to the alveolar tissues (Sharma, Sharma, & Khuller, 2004).

Rifabutin is an antitubercular drug having good anti-TB activity and holds the promise to circumvent cross-resistance with other rifamycins and interactions with antiretroviral agents (Onyebujoh et al., 2005). The drug inhibits bacterial DNA-dependent RNA polymer-ases (Barluenga et al., 2006) and posses a wide spectrum of antimycobacterial activity (O’Brien, Lyle, & Zinder, 1987; Saito, Sato, & Tomioka, 1988). At present, the drug is clinically used in the treatment of Mycobacterium avium complex, M. tuberculosis, and M. leprae (Grassi & Peona, 1996; Siegal, 1996; Maclean & Toulouse, 1997; Benson et al., 2000; Li et al., 2005) and was therefore selected for the present study.

The present study has been aimed to develop and investigate the potential of novel polysaccharide anchored SLNs for spatial drug delivery to the alveolar tissues. In the present study, mannosylated SLNs loaded with rifabutin were synthesized, characterized, and analyzed for drug release under various pH profiles. The formulation was then studied as vehicles for alveolar delivery using rifabutin as a model drug. The study also reveals the scope and efficacy of the mannosylated SLNs as an efficient drug delivery vehicle for selective locali-zation of the complex in lung tissues.

Materials and methods

Rifabutin was a gift from M/s Lupin laboratories (Aurangabad, India). d-Mannose, stearyl amine (SA), tristearin, and soya-lecithin (PC) were procured from Sigma Aldrich (Germany). Cellulose dialysis tubing (MWCO 1,000 Da), Cellulose dialysis bag (MWCO 12–14 kDa), and G-50 Sephadex were procured from Himedia (Mumbai, India). Nylon membrane filter (0.22 m) was obtained from Pall Gelman Sciences (USA). All other chemicals were of reagent grade and used without further modification.

Preparation of uncoated solid lipid nanoparticles (U-SLN-R)

SLNs were prepared by solvent injection method as reported previously by Schubert and Muller-Goymann (2003) with slight modifications. To summarize, rifabutin (0.1% w/v), tristearin (1% w/v), and soya-lecithin (PC; 0.3% w/v) were dissolved in ethanol (10 mL) at 70°C. SA was then added in the ratio of 10 mol% of PC into the mix-ture, and the temperature was maintained at 70°C. The melt was rapidly injected through a syringe at a flow rate of 5 mL/min into a stirred aqueous phase maintained at same temperature containing Tween 80 which was pre-viously dissolved in the aqueous phase. The suspension was stirred using electric stirrer (Remi, Mumbai, India) at 3000 rpm for 1 h and with subsequent sonication for 2 min. The resulting dispersion was then passed through membrane filter (0.22 µm) to remove any excess lipid. FTIR spectroscopic studies were performed after the adsorption of smaller amounts of U-SLN-R on KBr pellet by using an IR spectroscope (Perkin–Elmer, USA).

Preparation of mannosylated solid lipid nanoparticles (M-SLN-R)

Mannose coating was performed according to the method previously reported by Kumar and co-workers (2006). To summarize, mannose previously dissolved in sodium acetate buffer (pH 4.0) was added to uncoated SLNs and the mixture was continuously stirred on a magnetic stir-rer (Remi) maintained at room temperature for 72 h. The uncoated mannose and other impurities were removed by dialysis bag (MWCO 12–14 kDa; Himedia) in double distilled water for 24 h followed by lyophilization. FTIR spectroscopic (Perkin–Elmer) studies were performed to characterize the synthesis of mannosylated SLNs using the similar procedure as reported for uncoated SLNs.

Drug content determination

Drug entrapment of rifabutin in U-SLN-R and M-SLN-R was determined by using G-50 Sephadex mini column.

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Mannosylated nanoparticulate carriers of rifabutin 779

Separation of free rifabutin from U-SLN-R formulation was achieved by applying 0.2 mL of uncoated SLNs dispersion on the top of the Sephadex column and centrifugation at 2000 rpm for 2 min to expel and remove void volume containing SLNs into the centri-fuged tubes. The eluted SLNs dispersion was collected, lysed by 2% (v/v) Triton X-100, and the quantity of entrapped drug was analyzed spectrophotometrically (UV-1601 Shimadzu, Japan). Similar procedure was employed to determine the amount of drug entrapped in M-SLN-R.

Characterization of tristearin nanoparticles

MorphologySurface morphology of both U-SLN-R and M-SLN-R for-mulations were studied using scanning electron micros-copy (SEM) (LEO 435 VP, Eindhoven, Netherlands). The selected formulations were gently sprinkled on a double adhesive tape stuck on an aluminum stub. The stubs were then coated with gold by using a polaron sputter coater. The samples were examined at an acceleration voltage of 30 kV and photomicrographs were taken at suitable magnifications.

Particle size and polydispersity index determinationAverage particle size and polydispersity index (PDI) of the SLNs were determined by photon correlation spectroscopy using a Zetasizer (DTS Ver. 4.10; Malvern Instruments, England) after diluting with deionized water.

Zeta potentialThe zeta potential of the SLNs was determined by laser Doppler anemometry using a Malvern Zetasizer (DTS Ver. 4.10; Malvern Instruments). The instrument is a laser-based multiple angle particle electrophoresis ana-lyzer. Both U-SLN-R and M-SLN-R were dispersed in PBS (pH 7.4) and the zeta potential was determined.

In vitro release kinetic studyThe in vitro drug release of rifabutin entrapped in SLNs formulation was determined using dialysis tube diffusion technique. The prepared formulations (5 mL) were separately placed into the dialysis tube (MWCO 1000 Da; Hi Media, India), tied at both the ends, and suspended in different beakers each containing 40 mL of PBS (pH 7.4). The medium was magnetically stirred and the temperature was maintained at 37 ± 1°C throughout the procedure. Samples were withdrawn at definite time intervals and replaced with same volume of PBS. The samples were then spectrophotometri-cally (UV-1601 Shimadzu, Japan) analyzed for drug content.

Macrophage uptake studies

Macrophage cell lines “J774” were used for the present study using fluorescence activated cell sorter (FACS) instrument (BD Biosciences, FACS Aria, Germany). Aliquots (100 µL) containing J774 cell lines (1 × 103) were suspended in 0.9 mL of fresh RPMI-1640 medium (Himedia) supplemented with penicillin 10 U/mL, 10% fetal bovine serum, 100 g/mL streptomycin, 1 mM sodium pyruvate, and 10 mM HEPES medium (Alexandra et al., 2006). These were transferred into 24-well plates (Sigma, Germany) containing fresh medium and sus-pended for 12 h in a 37°C humidified incubator with 5% CO

2

atmosphere. Aliquots of 10 µL fluorescein isothiocyanate (FITC)-loaded U-SLN-R and M-SLN-R were suspended in separate wells containing J774 macrophage cell lines and incubated for 1 h. The cells were then washed with PBS (pH 7.4) to remove the SLNs adhering to the cell surface. Same procedure was followed to study the macrophage uptake after 2 h and 6 h. The cell-associated fluorescence was measured by FACS (Tomofumi et al., 2007).

Hematological studies

The studies were conducted with the permission and standard guiding principles of Institutional Animal Ethical Committee, Dr Hari Singh Gour University, Sagar (M.P.), India. Albino rats (Sprague–Dawley strain; Male) of uniform weight and size were used in the experimental study. Twelve albino rats were divided into four groups, each group having three rats. First group was adminis-tered with plain rifabutin (4.2 mg/kg) solution through tail vein. The formulation U-SLN-R and M-SLN-R dis-persions were administered through tail vein in a dose equivalent to 4.2 mg/kg of rifabutin to the second and third group, respectively, while the fourth group served as a control. After 7 days of treatment, blood samples were collected and analyzed for hemoglobin, RBC count, and WBC count (Agashe et al., 2006; Kumar et al., 2007) in pathology laboratory.

In vivo studies

Healthy albino rats (Sprague–Dawley strain), of uniform body weight and either sex, with no prior drug treatment were used for this experimental study.

Estimation of rifabutin in serum

Twelve albino rats were divided into four groups, each group having three rats. First group was treated with plain rifabutin (4.2 mg/kg) solution through i.v. route. The U-SLN-R and M-SLN-R dispersions were administered through tail vein in an equivalent dose of 4.2 mg/kg of rifabutin to the animals of second and third

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780 Navneet Nimje et al.

groups, respectively. The animals of fourth group did not receive any drug and served as control. The blood of ani-mals was collected from retro-orbital plexus of rat eye at 0.16 h, 0.5 h, 1 h, 2 h, 5 h, 8 h, 12 h, and 24 h intervals in anticlot vials (Himedia) and centrifuged at 5000 rpm for 10 min. Supernatant was collected and acetonitrile was added to precipitate the proteins. The precipitated proteins were settled by centrifugation at 5000 rpm for 10 min and supernatant was collected in HPLC vials (Himedia). An aliquot of 1 mL of collected supernatant was filtered through 0.22-µm membrane filter (Pall Gelman Sciences) and the rifabutin concentration was determined using HPLC.

Quantification of rifabutin in serum and tissues (lungs, liver, and spleen homogenate) was performed using HPLC (Shimadzu, C18, Japan) as reported by Gaspar et al. (2008) with slight modifications. The mobile phase was pumped from solvent reservoir to the column with a flow rate of 1 mL/min and at a pressure of 102/101 bars. The volume of each injection loop was 20 L; the reten-tion time was 10 min and the eluents were monitored at 275 nm (Gaspar et al., 2008).

Biodistribution studies

Twelve albino rats were divided into four groups, with three rats in each group. Each group was treated with

the same i.v. dose of rifabutin and formulations as given during the estimation of rifabutin in serum. The rats were killed after an interval of 1 h, 8 h, and 24 h; the organs (lungs, liver, and spleen) were excised and homog-enized in dichloromethane/isooctane (1:1) mixture. The extracts were separated by centrifugation at 1200 rpm for 10 min and then loaded on to HPLC for the estimation of drug content (Gaspar et al., 2008).

Results and discussion

Preparation of uncoated and mannosylated solid lipid nanoparticles

Preparation of plain SLNs was carried out by employing solvent injection method, which involves the rapid dif-fusion of solvent across the solvent-lipid phase into the aqueous phase (Scheme 1). SA was added to provide amine tailored surfaces on SLNs which play a crucial role in subsequent steps of mannosylation. Preparation of rifabutin-loaded SLNs (U-SLN-R) was confirmed using IR spectroscopy that depicted peaks of C=O stretching at 1736.7 cm−1 and O–H stretching of alcohol at 3417.1 cm−1. Strong N–H bending at 1574.5 cm−1 con-firmed the presence of amine functionalities of SA for attaching mannose (Figure 1).

Soya phosphatidycholine Tristearin/Stearyl amine Drug

Dissolved in ethanol (70°C)

Distilled water + Tween 80 (70°C)

Lipid suspension is formed

Solid lipid nanoparticles

Dispersed using syringe under constant stirring

Constant stirring

Sonicated under probe sonicator

Solvent injection method (Schubert et. al., 2003)

Lipid (solid)

Solid fat matrix

Monolayer of phospholipid

Scheme 1. Schematic representation of the method of preparation of solid lipid nanoparticles.

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Mannosylated nanoparticulate carriers of rifabutin 781

HO

HO

HO

HO

HO

HO

HO

HO

HO

HO

HO

HOOH

OH

OH

OH

OH

OHOH

OH

OHOH

OHOH

OH

OHOH

OH

OH

OH

OH

OH

OH

OH

OH

OHOH

OHOH

NH

NH NHNH

NH

SLN

SLN

NHNH

NH

OH

OH OHOH OH

OH OH

OHOH

OH

OH

O

O

Mannose

Sodium acetatebuffer, pH 4.0

Mannose isom-erization H

H

H

H H2N

H2N

NH2NH2

NH2

NH2NH2

Sodium acetatebuffer, pH4.0, incubatedfor 48 h

Scheme 2. Schematic representation of preparation of mannosylated solid lipid nanoparticles.

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0.004000 3500 3000 2500 2000 1500 1000 500cm-1

3417

.1

2917

.2 2852

.5

1736

.7

1462

.8

1351

.6

1247

.8

1101

.8

949.

0

841.

1

722.

4

573.

2

1642

.5 1574

.515

38.5

1960

.5

Figure 1. FTIR spectrum of rifabutin-loaded SLNs.

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782 Navneet Nimje et al.

Synthesis of mannosylated SLNs was confirmed by FTIR peaks. Mannose coating was done by ring opening reaction and subsequent reaction of aldehyde group of mannose in sodium acetate buffer (pH 4.0). This results in the formation of Schiff’s base (–N=CH–), which may then get reduced to secondary amine (–NH–CH

2–) and remain

in equilibrium with Schiff’s base (Scheme 2). Broad and intense O–H stretch and C–O stretch peaks of mannose around 3416.0 cm−1 and 1094.5 cm−1, respectively, and N–H deformation of secondary amine at 1412.9 cm−1 confirmed the Schiff base formation and some amide formation in linkage between aldehyde of mannose and amine termination of SLN as shown in Figures 2 and 3.

Drug content determination

The rifabutin entrapment efficiency of M-SLN-R and U-SLN-R was 82.6 ± 1.2% and 87.8 ± 1.2%, respectively (Table 1). Lower drug entrapment of mannosylated SLNs might possibly be because the mannose for coating pur-pose was dissolved in sodium acetate buffer (pH 4.0) in which the drug is more soluble and which might have led to the loss of surface adsorbed drug.

Characterization of tristearin nanoparticles

MorphologySEM micrographs show the spherical nature of both the coated and uncoated particles. The photographs of SLNs prove their nanometric size and that their size increase upon mannose coating (Figure 4).

Particle size determinationParticle size of mannosylated SLN was larger than uncoated SLN. The average particle size of the M-SLN-R and U-SLN-R was found to be 389 ± 2.3 nm with a PDI of 0.357 and 251 ± 5.1 nm with a PDI of 0.439, respectively. This variation may be due to the coating of mannose on the SLNs surface. The results are in accordance with the SEM studies which also revealed nanometric size of SLNs and an increase in size upon mannosylation (Table 1).

Zeta potentialThe zeta potential measurements of U-SLN-R reveal that these nanoparticles exhibit positive zeta potential (3.38 ± 0.3 mV) and M-SLN-R showed negative zeta potential (−11.7 ± 0.8 mV). Uncoated SLNs were posi-tively charged possibly because of the incorporation of amine functionalities via SA at the surface. Moreover, coating with mannose shields the positive charge and hence zeta potential may have been lowered (Table 1).

In vitro release kinetic studyIn vitro release studies of U-SLN-R showed a drug release profile of 89.21 ± 3.8% after 120 h in PBS (pH 7.4) whereas rifabutin-loaded mannosylated SLN showed a drug release profile of 65.23 ± 1.9% after 120 h in PBS (pH 7.4) (Figure 5). A significant decline in the release rate of rifabutin from M-SLN-R was observed in com-parison to U-SLN-R in PBS (pH 7.4). The results depict that coupling of mannose slows down the release of drug from the SLNs and thereby imparts a sustained

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0.004000 3500 3000 2500 2000 1500 1000 500cm-1

333.

7.7

2933

.1

1639

.3

1068

.1

960.

0

864.

1

720.

4778.

4

675.

861

1.0

1415

.6

1252

.8

1164

.5

Figure 2. IR spectrum of mannose.

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Mannosylated nanoparticulate carriers of rifabutin 783

release nature to SLNs. Similar results were obtained in the studies conducted by Agrawal, Gupta, and Jain (2007) and Bhadra, Bhadra, and Jain (2005).

Macrophage uptake studies

The data show that with the increase in time, mean FITC level increases in macrophage cell lines (J774) which

was more from mannosylated SLNs in comparison to uncoated SLN. After 6 h, the ratio of mean FITC level observed in coated and uncoated SLNs was 5.81. Specific cell surface receptors of mannose are found on alveo-lar macrophages (Kumar et al., 2006). This may lead to enhanced cellular uptake of FITC-loaded mannosylated

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4000 3500 3000 2500 2000 1500 1000 500cm-1

3461

.0

2916

.2 2850

.0

1552

.9

1468

.9

1644

.9

1736

.8

2133

.8

655.

6

1349

.014

12.9

1251

.3

1174

.3

1094

.5

950.

3

Figure 3. IR spectrum of mannosylated solid lipid nanoparticles.

A B

Figure 4. SEM photomicrographs of (A) uncoated SLNs and (B) mannosylated SLNs.

Table 2. Hematological studies of various formulations.

FormulationsHb content (g/100 mL)

Average RBC count no./cmm

Average WBC count no./cmm

Control 14.89 ± 0.3 7.82 ± 0.4 4200 ± 0.3

Plain rifabutin solution

12.06 ± 0.2 5.96 ± 0.3 2900 ± 0.2

U-SLN-R 12.78 ± 0.2 6.13 ± 0.6 4000 ± 0.5

M-SLN-R 13.57 ± 0.4 6.61 ± 0.1 4300 ± 0.4

Mean ± SD (n = 3); P ≤ 0.05.

Table 1. Comparison of % drug entrapped, zeta potential, particle size, and polydispersity index (PDI).

Code% Drug

entrappedZeta potential

(mV)Particle size

(nm) PDI

U-SLN-R 87.8 ± 1.2 3.38 251 ± 5.1 0.439

M-SLN-R 82.6 ± 1.2 −11.7 389 ± 2.3 0.357

Mean ± SD (n = 3); P ≤ 0.05.

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784 Navneet Nimje et al.

SLNs. The result illustrates that with M-SLN-R, high rifabutin concentration can be achieved in alveolar macrophages due to phagocytic uptake of mannosylated formulation (Figure 6). Similar results were observed by Kumar et al. (2006) upon mannosylation of polypropyl-ene imine dendrimers.

Hematological studies

The hematological study was performed to study the effect of mannosylation in comparison to plain SLNs and free drug on various blood components. The results indicated that hemoglobin (Hb) content,

RBC count, and WBC count were undergoing a devia-tion from normal value. The Hb content of free drug, U-SLN-R, and M-SLN-R was found to decrease from 14.89 ± 0.3 g/100 mL to 12.06 ± 0.2 g/100 mL, 12.78 ± 0.2 g/100 mL, and 13.57 ± 0.4 g/100 mL, respec-tively. This may be due to higher cytotoxicity and hemolytic toxicity of free drug and plain SLNs that had been tailored with amine terminal groups induced by SA. Mannosylation shields the drug and amine terminal functionalities and may therefore lead to lowest decrease in Hb content. In a similar manner, M-SLN-R formulation upon administration was found to decrease RBC count and WBC count to the low-est degree in comparison to free drug and U-SLN-R (Table 2). The results are in accordance with the results obtained previously by Duncan, Dimitrijevic, and Eyagororou (1996), Veronese et al. (1989), and Bhadra et al. (2003, 2005).

In vivo studies

Rat serum was used to determine the concentration of drug in blood samples at various time intervals so as to explore the effect of mannosylation on the release profile of drug and retention of SLNs formulation in systemic circulation (Figure 7). The data revealed that U-SLN-R possessed slightly lower C

max and M-SLN-R depicted the

least Cmax

as compared to intravenous administration of free drug. The serum concentration of free drug was detected till 48 h in trace quantities, and thereafter no drug was found. However, C

max duration of rifabutin in the

formulation was found to be prolonged, that is, from 48 h to 78 h and from 48 h to 96 h for U-SLN-R and M-SLN-R (Figure 7). This was probably due to slower release of drug from U-SLN-R and slowest release from M-SLN-R. The release rate in the case of coated formulation was found to be lowest possibly due to decreased rate of dif-fusion of drug upon mannosylation. Consequently, the blood level of the drug was sustained to a greater extent in the case of coated formulation than that of an uncoated system due to slower release rate of rifabutin. The results are in agreement with the in vitro drug release studies. A difference in the extent of drug estimated in vivo was observed which may possibly be due to biological effects on the drug that predominate its biodistribution pattern.

Biodistribution studies

Biodistribution studies were performed to assess the amount of drug reaching the lungs, liver, and kid-ney. The free rifabutin was found to have greatest access to kidney, where up to 25.4 ± 1.9% µg/g and 6.2 ± 0.08% µg/g of drug was localized after 2 h and 24 h of administration, respectively. However, after 24 h, 9.8 ± 1.04% µg/g and 5.2 ± 0.09% µg/g of rifabutin

00.5

11.5

22.5

33.5

44.5

5

0 20 40 60 80 100 120Time (h)

Rifa

butin

con

cent

ratio

n(µ

g/m

L)

Free drug U-SLN-R M-SLN-R

Figure 7. Serum concentration of plain drug, uncoated, and man-nosylated formulation.

0102030405060708090

100

0 20 40 60 80 100 120 140Time (h)

% D

rug

rele

ase

U-SLN-R M-SLN-R

Figure 5. In vitro drug release in phosphate buffer (pH 7.4).

0100200300400500600700800

1hSD ± mean (n=3)

2h 6hTime (h)

Mea

n FI

TC le

vel (

ng) U-SLN-R M-SLN-R

Figure 6. Cellular uptake of FITC-loaded mannosylated solid lipid nanoparticles (Mean ± SD, n = 3).

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Mannosylated nanoparticulate carriers of rifabutin 785

concentrations were found in liver and lungs, respec-tively. The drug concentrations in the case of U-SLN-R formulation were detected to be 11.5 ± 0.11% µg/g, 17.4 ± 0.54% µg/g, 15.7 ± 0.21% µg/g in kidney, liver, and lungs, respectively, after 24 h of administration. With M-SLN-R, only 4.2 ± 0.07% µg/g rifabutin was found in kidneys after 24 h of administration, whereas its con-centration in liver and lungs was 20.1 ± 0.54% µg/g and 23.6 ± 0.41% µg/g, respectively, after 24 h of administra-tion of the formulation (Figure 8).

The biodistribution pattern and quantity of drug in various body organs depends on its pharmacokinetic parameters (absorption, distribution, metabolism, and excretion). Initially, the free rifabutin content was found to be highest in the kidney and secondarily in liver and lungs. However, the free drug concentration in various organs was observed to decrease rapidly as compared to formulations (U-SLN-R, M-SLN-R). In the case of uncoated formulation, the concentration of drug was found to be highest in the liver as compared to other organs. On the contrary, the concentration of drug in the mannosylated formulation was found to be great-est in lungs and lower in liver and kidney (Figure 8). The above data suggested that although the kidney is a crucial organ for the clearance of bioactives, the man-nosylation possibly helped to bypass renal elimination and is finally eliminated after the formulation yielded to metabolism in the liver. Sufficiently, high concentra-tion of drug was detected in liver in the case of U-SLN-R probably due to preferred uptake by mononuclear phagocytic system.

A higher rifabutin level was observed in lung tis-sues with mannosylated nanoparticulate formulation as compared to plain drug (Figure 8). This may be explained on the basis of preferred uptake of the for-mulation by alveolar macrophages, which promotes selective entry in lungs and helps to preclude the entry

to other organs. M-SLN-R was found to have attained much better concentration in lungs as against U-SLN-R and free rifabutin (Figure 8). This illustrates the pref-erential targeting of M-SLN-R to lung tissue followed by a sustained release in the vicinity leading to a high concentration of drug in the lungs as compared to plain drug solution and U-SLN-R. The drug levels in liver were increased with time in the case of uncoated SLNs possi-bly because of uptake of uncoated SLNs by mononuclear phagocytic system. However, the drug level in lung was also increased with time in the case of uncoated SLNs because of indirect action of enhanced residence time of the drug loaded in uncoated SLNs formulation in the body that promotes distribution of drug to various body organs. But the effect was not so pronounced in com-parison to coated SLNs because of small size of SLNs that may possibly have led to the rapid elimination of uncoated SLNs from lungs and absence of targeting moiety prevented specific and selected uptake. The drug level in lung was increased with time in the case of coated SLNs due to the presence of mannose recep-tors on alveolar tissues and the size of SLNs that favored enhanced dwell time of coated SLNs in lung tissues. The drug level in liver was increased with time in the case of coated SLNs and was possibly due to the presence of mannose receptors also on macrophages that promoted macrophagic uptake. However, the extent of increment was less in comparison to distribution of SLNs to lung tissues probably because the mannose conjugations shielded the hydrophobic nature of SLNs and thus pre-vented specific uptake by RES.

The SEM studies also support the results obtained. An increase in average particle size from 251 ± 5.1 nm to 389 ± 2.3 nm was observed upon mannosylation from SEM studies. Particles which are very small are rapidly cleared from alveolar tissues and the larger ones may undergo preferential uptake by mononuclear phagocytic

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Kidney (free drug) Liver (free drug) Lung (free drug)Kidney (U-SLN-R) Liver (U-SLN-R) Lung (U-SLN-R)Kidney (M-SLN-R) Liver (M-SLN-R) Lung (M-SLN-R)

Figure 8. Biodistribution of free drug and formulations attained at various time intervals in different tissues.

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system (Garnett et al., 1999; Stahl et al., 2002). Thus, the size of M-SLN-R may be considered to be optimum and favoring alveolar targetability. Zeta potential of M-SLN-R was found to be negative possibly because mannosyla-tion shields the amine terminal functionalities. However, the expression of mannose receptors on alveolar tissues helps to overcome this drawback and promotes specific targeting of M-SLN-R to lungs and thus augments the lung targeting index. The presence of mannose recep-tors provide a unique platform so that multiple copies of ligands can be attached to it and facilitate targeting. In addition, the presence of positively charged amine func-tionalities imparts cytotoxicity and hemolytic toxicity to uncoated SLNs and consequently mannosylation adds to the biocompatibility to the SLNs.

The outcomes of the study point toward the localized action, augmented bioavailability, and higher reten-tion potential of the formulation in lung tissues, subse-quently enhancing the therapeutic efficacy by providing the prospective to lower the dose of therapeutic moiety as well as nanoparticles required for its delivery.

Conclusion

Nanoparticles-based vehicles for controlled, sustained, and targeted delivery hold a considerable potential for the delivery of antitubercular drug. SLNs are the class of carrier systems which have been widely explored for the delivery of various pharmacological moieties. The present study reveals the efficacy and suitability of man-nosylated nanoparticles for the delivery of rifabutin. In vitro studies depict the sustained release nature of the formulation. At the same time, mannosylation promotes their selective uptake by lung tissues. This promotes site-specific delivery, thereby enhancing the therapeu-tic efficacy and reducing the untoward side effects. Thus, the formulation holds a promising prospective for ferry-ing large doses of drug, providing targeted delivery with the minimal side effects. The prospective of this carrier system in a variety of categories of antitubercular drugs for site-specific delivery is under study.

Acknowledgements

Declaration of interest: The authors report no conflicts of interest.

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