Galactose engineered solid lipid nanoparticles for targeted delivery of doxorubicin

Colloids and Surfaces B: Biointerfaces 134 (2015) 47–58

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Galactose engineered solid lipid nanoparticles for targeted delivery of doxorubicin Ashay Jain a,b , Prashant Kesharwani b,c,∗ , Neeraj K. Garg a,b , Atul Jain a,b , Som Akshay Jain b , Amit Kumar Jain b , Pradip Nirbhavane a , Raksha Ghanghoria b , Rajeev Kumar Tyagi d,e , Om Prakash Katare a,∗∗ a Drug Delivery Research Group, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Studies, Panjab University, Chandigarh 160014, India b Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar 470003, MP, India c Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA d Department of Periodontics, College of Dental Medicine Georgia Regents University, 1120 15th Street, Augusta, GA 30912, USA e Biosafety Support Unit, Regional Centre for Biotechnology-DBT, C.G.O. Complex, Lodhi Road, New Delhi 110003, India

a r t i c l e

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Article history: Received 28 February 2015 Received in revised form 17 May 2015 Accepted 11 June 2015 Available online 19 June 2015 Keywords: Galactose Solid lipid nanoparticles Doxorubicin Cytotoxicity Lectin Targeting

a b s t r a c t The present investigation reports the preparation, optimization, and characterization of surface engineered solid lipid nanoparticles (SLNs) encapsulated with doxorubicin (DOX). Salient features such as biocompatibility, controlled release, target competency, potential of penetration, improved physical stability, low cost and ease of scaling-up make SLNs viable alternative to liposomes for effective drug delivery. Galactosylation of SLNs instructs some gratifying characteristic, which leads to the evolution of promising delivery vehicles. The impendence of lectin receptors on different cell surfaces makes the galactosylated carriers admirable for targeted delivery of drugs to ameliorate their therapeutic index. Active participation of some lectin receptors in immune responses to antigen overlaid the application of galactosylated carriers in delivery of antigen and immunotherapy for treatment of maladies like cancer. These advantages revealed the promising potential of galactosylated carriers in each perspective of drug delivery. The developed DOX loaded galactosylated SLNs formulation was found to have particle size 239 ± 2.40 nm, PDI 0.307 ± 0.004, entrapment efficiency 72.3 ± 0.9%. Higher cellular uptake, cytotoxicity, and nuclear localization of galactosylated SLNs against A549 cells revealed higher efficiency of the formulation. In a nutshell, the galactosylation strategy with SLNs could be a promising approach in improving the delivery of DOX for cancer therapy. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Neoplastic cells express the number of receptors on the surface which encompass very high empathy for carbohydrate molecules; these receptors are known as membrane lectins and because of these receptors different carbohydrates may be used as ligand to target the therapeutic agents [1–5]. Lectin receptors mediated targeting uses interaction of endogenous ligands with different sugar moieties like galactose, mannose, fucose, fructose and

∗ Corresponding author at: Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar 470003, India. Tel.: +91 7582 244432; fax: +91 7582 244432. ∗∗ Corresponding author. E-mail addresses: prashant [email protected], [email protected] (P. Kesharwani), [email protected] (O.P. Katare). http://dx.doi.org/10.1016/j.colsurfb.2015.06.027 0927-7765/© 2015 Elsevier B.V. All rights reserved.

lactose [5,6]. When these carbohydrate moieties are anchored to different drug vehicles the resultant glycosylated carriers having carbohydrate as stratum ligands are acknowledged and endocytosed by lectin receptors. Lectin receptors are highly expressed on the alveolar macrophages, liver endothelial Kupffer cells, splenic macrophages, peritoneal macrophages, macrophages of brain, illustrate a quick internalization of galactose-terminated glycoproteins via receptor-mediated endocytosis [7–9]. Consequently, the expansion of polysaccharide galactose-tagged drug delivery vehicles may emerge as a prospective strategy for the selective delivery of anti-cancer agents to the tumor tissues [10]. In this sequel transport of anticancer bioactives upon encapsulation in different delivery vehicles has been comprehensively investigated. Amidst of these vehicles vis a vis microspheres [11], micelles [12,13], liposomes [14,15], nanoparticles [16–18], and dendrimers [19–21]. SLNs have materialized as the most

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rationalized carriers which composed of nontoxic, bio-acceptable, biodegradable lipids components with a mean particle size between 50 and 1000 nm [22–24]. Physical stability (several years), more flexibility in modulating the release of the drug and minimized chemical degradation of entrapped drug are very decisive issues that score SLNs as an alternate colloidal drug delivery system to liposomes, micelles, emulsions and polymeric nanoparticles [25]. These vehicles serve as circulating reservoir of cytotoxic agents and thus prevent direct admittance of drug entity to healthy tissues/cells [26]. However, in absence of a marker (target) nonselective delivery is still visualized. In the context of drug delivery in treatment of cancer, anthracyclins are the most widely used anti-neoplastic agents and doxorubicin (DOX) is the leading drug of this class [27]. DOX has been demonstrated activity against a variety of tumors. Its antineoplastic value is predominantly ascribed to direct intercalation with DNA [28] or DNA topoisomerase II [29] leading to the arrest of cell cycle. However, use of DOX accompanies probability of cardiac dysfunctioning including congestive heart failure, arrhythmias, dilated cardiomyopathy and subsequent death [30,31]. Studies have revealed that the narrow therapeutic index, acute and chronic toxicity associated with the use of free drug is a topic of enormous concern [32]. Hence the fundamental prerequisite in the drug delivery is spatial and sustained delivery of doxorubicin. The present study discussed the preparation and optimization of galactose appended SLNs and evaluated their effectiveness for cellular localization and cytotoxicity. Galactose anchored SLNs encapsulating DOX were prepared and characterized in terms of size, uniformity and zeta potential. The formulations were then analyzed for in vivo pharmacokinetics and organ distribution, in vitro cellular uptake and cytotoxicity against A549 cells and compared with free DOX. 2. Materials and methods 2.1. Materials Doxorubicin HCl was provided as a generous gift by Sun Pharma Advanced Research Laboratories, Vadodara, India. Hydrogenated soya phosphatidyl choline (HSPC), was generously gifted by Lipoid, Ludwigshafen, Germany. Glyceryl mono stearate, stearyl amine (SA), galactose, dextran sulfate (DS) and cellulose dialysis tubing (MWCO 1000 Da) were purchased from Sigma Aldrich (Germany). Cellulose dialysis bags (MWCO 12 kDa) were acquired from Himedia (Mumbai, India). Nylon membrane filter (0.45 ␮m) was procured from Pall Gelman Sciences (USA). All other chemicals and solvents were of reagent grade and used as such without further modification. 2.2. Fabrication of DOX loaded SLNs (SLN-D) The SLNs were fabricated using solvent injection method [33]. Briefly, the organic phase was prepared by dissolving glyceryl mono stearate (GMS) (1%, w/v) along with soya–lecithin (PC; 0.3%, w/v) in a 10-mL blend of acetone and ethanol (1:1, v/v) at 60 ◦ C under constant stirring at 1000 rpm for 15 min. Stearyl amine (SA) was added in the ratio of 10 mol% of PC into the lipid mixture. The molten lipid phase was then rapidly added drop wise through a syringe at a flow rate of 2 mL/min into a preheated aqueous phase (with 0.2%, w/v Tween 80, containing 0.1%, w/v DOX and 0.05%, w/v DS which were previously dissolved in the aqueous phase) under constant stirring and further allowed to stir for 1 h at 3000 rpm. This leads to the formation of lipoidal suspension which has further undergone to probe sonication (2 min; 30% amplitude) to allow size reduction. The resulting suspension was filtered through

Table 1 Optimization of parameters and different process. Optimization parameters

Variables

Remark

Lecithin/lipid ratio (mg)

1:0.5 1:1 1:1.5 1:2

Drug:lipid 1:10 Surfactant 1% Lipid:stearylamine 100:1 Stirring speed 3000 rpm Stirring time 60 min

Drug:lipid

5:100 10:100 15:100 20:100

Surfactant 1% Lipid:stearylamine 100:1 Stirring speed 3000 rpm Stirring time 60 min Lecithin/lipid ratio (mg)

Lipid:stearylamine

100:0.5 100:1.0 100:1.5 100:2.0

Surfactant 1% Stirring speed 3000 rpm Stirring time 60 min Lecithin/lipid ratio (mg) Drug:lipid 1:10

Surfactant

0.5 1.0 1.5 2.0

Stirring speed 3000 rpm Stirring time 60 min Lecithin/lipid ratio (mg) Drug:lipid 1:10 Lipid:stearylamine 100:1

Stirring speed

1000 2000 3000 4000

Stirring time 60 min Lecithin/lipid ratio (mg) Drug:lipid 1:10 Lipid:stearylamine 100:1 Surfactant 1%

Stirring time

15 30 45 60

Lecithin/lipid ratio (mg) Drug:lipid 1:10 Lipid:stearylamine 100:1 Surfactant 1% Stirring speed 3000 rpm

membrane filter (0.45 ␮m) to remove excess lipid and subsequently dialyzed (MWCO 10 kDa) to remove any unentrapped or free drug. Various process variables for the preparation of SLN-D (Table 1) were optimized to acquire a final formulation with small particle size (<200 nm), narrow polydispersity index [(PDI); <0.5], and maximum drug loading with high entrapment efficiency (>60%). Finally, the SLN-D dispersion was lyophilized (VirTis AdVantage) and stored. 2.3. Preparation of galactosylated DOX loaded SLNs (G-SLN-D) Galactose coating was conceded in accordance to the technique described elsewhere [34]. The method associates ring opening of galactose followed by reaction of its aldehyde group with free amino groups present at the facade of prepared SLN-D (Fig. 1). Firstly d-galactose (8 ␮M) was dissolved in 0.1 M sodium acetate buffer (pH 4.0) and afterward added to uncoated SLNs (SLN-D) dispersion. The mixture was allowed to continuously be agitated on a magnetic stirrer (Remi, Mumbai, India) maintained at an ambient temperature for 3 days to make sure the completion of reaction. Galactosylated SLNs (G-SLN-D) were subjected to extensive dialysis against double distilled water (DDW) in a dialysis tube (dialysis bag; MWCO 12 kDa, Himedia, India) for 30 min to remove free galactose and other impurities, followed by lyophilization (Heto Drywinner, Denmark, Germany). The G-SLN-D was characterized by FTIR using a Perkin-Elmer IR spectroscope (Waltham, Massachusetts). 2.4. Preparation of fluorescent SLNs (SLN-F) and galactosylated fluorescent SLNs (G-SLN-F) SLN-F was prepared adding 3.4 × 10−3 mmol of FITC and kept the other components of the formulations fixed. The carboxylic ( COOH) and isothiocyanate ( NCS) group of FITC react with amine

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Fig. 1. Optimization of (a) lipids ratio with respect to particle size (b) drug/lipid ratio (c) surfactant concentration (d) lipid/stearylamine ratio (e) stirring speed and (f) stirring time with respect to particle size and %drug entrapment efficiency. S.D. ± mean (n = 3).

terminal functionalities of plain SLNs and hydroxyl group of galactosylated SLNs respectively [35]. 2.5. Characterization of nanoparticles Prepared nanoparticles were characterized for particle size, PDI, zeta potential and surface characteristics [transmission electron microscopy (TEM)]. 2.6. Drug content determination 10 mg of lyophilized SLN-D was dispersed in DDW, and filled in cellulose dialysis bag (MWCO 10 kDa, Sigma, Germany). Further, un-entrapped drug (if any) was removed from the formulation by extensively dialysis of the dispersion against DDW under sink conditions for 10 min while agitating with magnetic stirring (50 rpm; Remi, India). Subsequently, 0.2 mL of protamine sulfate solution (5 mg/mL) was added to cumulate the SLN-D in the dispersion and ultracentrifuged at 20,000 × g for 30 min. The pellet obtained was washed with DDW and lyophilized. 2.5 mg of lyophilized powder

of SLN-D was added in a mixture of methanol–chloroform (1:1, v/v), vortexed for 1 min followed by centrifugation at 3000 rpm for 10 min [36] The supernatant solution was filtered through a 0.45-␮m membrane, transferred to HPLC vials (Himedia, India), loaded on HPLC system and analyzed for amount of DOX entrapped within the SLN-D. Same procedure was employed to enumerate the DOX content in G-SLN-D. DOX was quantified as per the method suggested by Reddy et al. with slight modifications using HPLC (Shimadzu, C18, Japan). The flow rate of the mobile phase (ammonium hydrogen phosphate buffer (pH 4.0)/acetonitrile/methanol (60:24:16, v/v/v)) was maintained at 1.5 mL/min at pressure of 102/101 bars, the run time of DOX was obtained to be 21 min. Peaks of the eluents were monitored at 480.2 nm [37]. 2.7. In vitro release study In vitro release patterns of both SLNs formulations were evaluated using dialysis tube diffusion technique in phosphate buffered saline (PBS) pH 7.4, under sink condition. Briefly, freeze-dried SLND and G-SLN-D (equivalent to 2 mg of DOX) was filled in the dialysis

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tube bag (MWCO 12 kDa; Himedia, India) and suspended in 25 mL of PBS in a shaker water bath (Lab Tech, India) maintained at 37 ◦ C. Aliquots of 200 ␮L of sample were withdrawn and replaced with the fresh release medium. The amounts of DOX released were analyzed by the validated HPLC method.

2.8. Hemolytic toxicity Whole human blood samples were collected from a healthy person (with the kind permission) and heparinized in HiAnticlot blood collection vials (Himedia, India). Subsequently, the red blood cells (RBCs) suspension was centrifuged and re-suspended in normal saline. Two milliliter of the RBCs suspension was separately dispersed in normal saline solution producing no hemolysis (served as control) and in distilled water considered as producing 100% hemolysis. The RBC suspension was mixed with double distilled water, which was considered as producing 100% hemolysis, and normal saline producing no hemolysis, hence acting as blank. One microliter of adequately diluted DOX, SLN-D and G-SLN-D dispersions were incubated individually with 2 mL RBCs suspension following making up the volume up to 10 mL with normal saline. The formulations were taken in such amount that the resultant final concentration of DOX was equivalent in every case so as to facilitate the assessment of extent of hemolysis. Further, the formulations were allowed to incubate at 37 ± 1 ◦ C for 30 min, followed by centrifugation at 4000 rpm for 10 min. Subsequently, supernatants were analyzed spectrophotometrically (Schimadzu, 1601 Japan) at 540 nm [38,39].

2.9. Cell cultures and viability assay A549 cells were seeded at 3 × 104 cells/mL in different 96 well plates (Sigma, Germany) and treated for 72 h with various concentrations (100.0–0.01 ␮M) of DOX (plain DOX, SLN-D and G-SLN-D, galactose [1 mg/mL] + G-SLN-D) simultaneously under controlled environment. Sequentially, MTT solution (10 mg/mL; w/v) was added to each well and the cells were allowed to incubate for 4 h at 37 ◦ C. MTT was reduced by mitochondrial dehydrogenase enzymes present in viable cells, and identified by the formation of water insoluble purple formazan crystals. The formazan crystals were dissolved in DMSO and the absorbance of each well was taken at 570 nm via an ELISA plate reader at 25.8 ◦ C [40,41].

2.11. In vivo pharmacokinetic and biodistribution studies 2.11.1. Plasma profile In vivo pharmacokinetics of G-SLN-D was evaluated in albino rats (either sex) of Sprague Dawley strain (120 ± 5 g). The animals were adequately marked and randomly divided into 4 groups each containing 5 animals. Different groups of rats administered intravenously free DOX (5 mg/kg), SLN-D (equivalent to 5 mg/kg free DOX), and G-SLN-D (equivalent to 5 mg/kg free DOX). All formulations were dispersed in saline and administered through tail vein [36]. The animals of fourth group were served as control. The blood samples (approximately 0.3 mL) were collected at predetermined time intervals (0.16, 0.5, 1.0, 1.5, 2.0, 3.0, 5, 8, 12, 24 and 48 h) from the retro-orbital plexus under mild anesthesia into heparinized microcentrifuge tubes. After each withdrawal, 1 mL of dextrose normal saline was interposed to partially compensate the electrolyte level and central compartment volume. Plasma was separated by centrifuging the blood samples at 10,000 rpm for 5 min. One hundred ␮L of 10% (w/v) trichloroacetic acid (TCA) was added to 100 ␮L of sample. The contents were vortexed for 1 min and 5 mL methanol was added. The mixture was vortexed (Superfit vortexer, India) for 10 min and centrifuged at 3000 rpm for 15 min. The supernatant was filtered through a 0.22 ␮m membrane, collected in HPLC vials (Himedia, India) and quantified for DOX by HPLC [37]. 2.11.2. Biodistribution studies The experiments were performed on tumor-induced rats (either sex) of Sprague Dawley strain (120 ± 5 g). The tumors were generated on the right flank using A549 cells [44]. The tumor-induced albino rats were randomly divided into 4 groups each containing 12 animals. Different groups of rats administered with the same i.v. dose of the free DOX, SLN-D and G-SLN-D via, tail vein as in the case of pharmacokinetic studies. Four animals from each group were sacrificed at intervals of 2, 8 and 24 h. After sacrificing the rats, the organs viz. liver, spleen, kidney, heart and tumor were carefully excised, weighed and stored under freeze condition. Weighed organ samples were homogenized in normal saline to form 20% tissue homogenate, vortexed for 60 s, and kept aside for 45 min. The tissue homogenates were then treated with 100 ␮L of 10% TCA solution, vortexed for 1 min and then 5 mL methanol was added. The mixture was extracted (Superfit vortexer, India) for 10 min and centrifuged at 4000 rpm for 10 min. The supernatant was filtered through a 0.22 ␮m membrane, collected in HPLC vials (Himedia, India) and analyzed for DOX content by HPLC [37]. 2.12. Statistical analysis

2.10. Cellular uptake study A549 cells were seeded at 2 × 106 cells/mL in 6 well plates (Sigma, Germany) containing fresh DMEM and kept in humidified incubator at 37 ◦ C with 5% CO2 atmosphere for 12 h. The cellular uptake efficacy as a function of conjugation of the ligand was estimated by in vitro incubation of A549 cells with fluorescein isothiocyanate (FITC), FITC labeled SLNs (SLN-F), galactosylated FITC labeled SLNs (G-SLN-F) and galactose [1 mg/mL] + G-SLN-F in separate wells for 1 h. Each well was washed twice with PBS (pH ∼7.4) for complete removal of the SLNs adhering on the cell surface and re-suspended in DMEM culture media. Subsequently, the cells were trypsinized (0.1%, w/v), then subjected to centrifugation (1000 rpm) to remove the trypsin and cells were re-suspended in PBS (pH ∼7.4) and fluorescence was measured by FACS instrument [42,43]. Additionally, treated with SLN-F, GSLN-F and galactose [1 mg/mL] + G-SLN-F were washed with PBS (pH 7.4) after 6 h cells and fixed using 3.7% paraformaldehyde. The slides were mounted on fluorescent microscope (Nikon, Japan) and visualized for nanoparticle binding/uptake.

The results were expressed as mean ±SD and the statistical analysis was done by analysis of variance (ANOVA). A probability level of p < 0.05 was considered to be significant. 3. Results and discussion 3.1. Fabrication of DOX loaded SLNs (SLN-D) SLN-D was fabricated using solvent injection technique which involves the intense diffusion of solvent transversely the solvent–lipid phase into the surrounding aqueous phase followed by evaporation of the organic solvent that leads to rigidization of lipid particles. For ensuring the nano range particles, high shear homogenization was used (Scheme 1). Prior to homogenization, high speed stirring was deployed to acquire a pre-emulsion phase. Lipid employed in the production of SLNs was first subjected to optimization by varying the ratio of GMS:lecithin from 1:0.5 to 1:2 while keeping GMS quantity as constant. The prepared formulation was further characterized on the basis of particle size and PDI.

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Scheme 1. Schematic representation of synthesis of galactosylated drug loaded solid lipid nanoparticles.

Formulation with 1:1 lipid/lecithin ratio was selected for further optimization process variables as it showed an optimum size of 161 ± 3.71 nm and a PDI of 0.224 (Fig. 1a). SLN formulations except the one prepared with GMS:lecithin – 1:1 ratios, an increase in the size and PDI was observed with an increase in lecithin concentration. This may be due to the formation of vesicles as excess of PC forms bilayers in the lipid. It was observed that on increasing the amount of DOX, the entrapment efficiency increased up to drug:lipid ratio of 10:100 while on further increasing drug concentration in the lipid, the entrapment efficiency gradually decreased (Fig. 1b). This could be due to the saturation of lipid with the drug. The greater entrapment efficiency (83.90 ± 1.9%) of SLN-D was found at 1% surfactant concentration (Fig. 1c). However, further increase or decrease in surfactant concentration has shown a loss of entrapment efficiency and increased PDI [45]. Tween-80 minimizes the surface tension between aqueous and organic phases that perhaps allows the configuration of initially tiny droplets of solvent at the site of solvent injection and causes the decreased particle size. Moreover the Tween-80 might also help to stabilize the recently generated surfaces and particle aggregation [46]. Stearyl amine was incorporated to provide amine terminated functionalities on SLN-D which play an essential role in galactosylation. Stearyl amine (SA) ratio was optimized keeping lipid ratio constant and it was observed that particle size tends to increase with an increase in the amount of SA. Drug entrapment increased on increasing the amount of stearyl amine up to 1% of lipid (Fig. 1d). SLN formulations showed varied effects on varying stirring speed. Particle size decreased on increasing the stirring speed but after a period, particle size was increased. On varying stirring speed, particles also showed variation in drug entrapment (Fig. 1e). Moreover, the addition of DS (counter ion) forms an interaction with drug. The interaction was largely govern by a long range electrostatic forces between ion pairing agent DS and drug, which ultimately results in increased loading of the drug due to relative increased lypophilicity of the drug [46]. Despite the presence of DS in the SLN formulation,

the average particle size monotonically decreased. This was as a result of higher amphiphilic properties and reduced interfacial tension between interfaces [47]. 3.2. Preparation and characterization of DOX loaded galactosylated SLNs (G-SLN-D) Galactosylation of the SLN-D involved the ring opening of dgalactose and subsequent reaction of its aldehyde group with free amino functionalities expressed over the surface of SLND in sodium acetate buffer (pH 4.0). The above process lead to the formation of Schiff’s base ( N CH ). The Schiff’s base may probably get reduced to secondary amine ( NH CH2 ) and reside in equilibrium with Schiff’s base (Scheme 1). FTIR analysis revealed the formation of Schiff’s base and secondary amine ( NH CH2 ) linkage between aldehyde groups of d-galactose and amine termination of SLN-D (Fig. 2a). Broad and intense O H stretch and C O stretch peaks of galactose 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 group of dgalactose and amine termination of SLN-D (Fig. 1). 3.2.1. Particle size determination The average size of all formulations was in range between 140 and 320 nm range (Fig. 1). The component ratio and Tween 80 concentration dependent changes (deviation in particle size) also showed an impact on PDI and entrapment efficiency of optimized formulation (Fig. 1c). The formulation with average particle size (148.3 ± 2.5 nm) (Table 2a) and highest EE (83.90 ± 1.2) was considered optimized formulation as compared with other formulations. The average particle size of G-SLN-D was found to be larger than that of SLN-D (Table 2a). The disparity in size may be due to galactose conjugation in case of G-SLN-D. The data are in harmony with TEM studies (Fig. 2b). We notice that the sizes obtained by TEM are differing from those determined by DLS. The difference in sizes

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Fig. 2. (a) IR spectrum of G-SLN-D; (b) TEM photomicrographs of galactosylated SLNs; (c) in vitro drug release of DOX in PBS, pH 7.4 from SLN formulations [n = 6; p ≤ 0.05 (significant difference between SLN-D and G-SLN-D)].

should be attributed to the fact that the TEM reveals the morphological size of the nanoparticle in the solid state; however zetasizer measure the hydrodynamic diameter of the nanoparticle in aqueous solutions. Our results are well in accordance with the previous reports [48,49]. Another reason might be due to the operation principal of both instruments while zetasizer measured the average particle size while TEM measured the size of individual particles. The PDI were less than 0.4 for both the optimized formulation

(SLN-D and G-SLN-D) which is an indicative of homogeneity of size of SLNs. 3.2.2. Zeta potential The zeta potential of SLN-D formulation was observed to be 9.1 ± 0.25 mV possibly because of positively charged amine functionalities distributed at the surface of the uncoated SLNs (Table 2a). However, the zeta potential lowered to 6.2 ± 0.12 mV upon

A. Jain et al. / Colloids and Surfaces B: Biointerfaces 134 (2015) 47–58 Table 2 (a) Zeta potential, particle size, PDI and % drug entrapped of optimized formulation. (b) Different pharmacokinetic parameters of free drug, and formulation in serum of albino rats. (a) Code

Zeta potential (mV)

Particle size (nm)

% drug entrapped

PDI

SLN-D G-SLN-D

9.1 ± 0.25 6.2 ± 0.12

148.3 ± 2.5 239 ± 2.4

83.9 ± 1.2% 72.3 ± 0.9%

0.224 ± 0.005 0.307 ± 0.004

(b) Parameter

Free DOX

SLN-D

G-SLN-D

Cmax Kel Cl AUC0–t AUC0–inf AUMC0–t AUMC0–inf t½ MRT

4.17 0.434 43.6 11.0846 11.4676 24.6362 28.564 1.5691 2.49

3.85 0.119 13.6 35.0693 36.2502 256.983 294.041 5.11589 8.11143

3.7 0.047 8.02 58.1509 63.7005 925.919 1306.37 14.247 20.5079

galactose conjugation (G-SLN-D) may be due to a shielding of the positively charged amine groups that were present on the surface on uncoated SLNs. The higher positive value of zeta potential provides repulsive interaction between SLNs, and thus prevents aggregation of nanoparticles. In addition, Tween 80, used in the formulation also provided stearic stability to achieve stable formulation.

3.3. Drug content determination The greater entrapment efficiency of DOX (83.9 ± 1.2%) in SLN-D was found with 1:1 lecithin/lipid (w/w in mg), 1:10 drug:lipids ratio (w/w in mg) and 1% surfactant concentration (Table 2a). Moreover, the addition of DS forms a drug–polymer complex which leads to increase the partitioning of drug between both the phases which ultimately results high EE of SLN formulations. However, the DOX entrapment efficiency of G-SLN-D was 72.3 ± 0.9% (Table 1). Slightly lesser drug entrapment of G-SLN-D might possibly be because of the dissolution and subsequent loss of surface adsorbed drug while galactosylation in sodium acetate buffer (pH 4.0) which was used as a medium for galactosylation of SLN-D. The results are in accordance with previous findings [50].

3.4. In vitro release study Formulations displayed a biphasic sustained release pattern and an initial burst release viz. 21.98 ± 1.25% and 28.71 ± 1.26% of DOX was obtained from G-SLN-D and SLN-D, respectively in the PBS until the end of 8th hour (Fig. 2c). A possible reason that may be accounted is the fast release of drug adsorbed on the surface of the SLNs or entrapped in the outermost stratum. Further, the cumulative DOX release from G-SLN-D and SLN-D was 76.2 ± 2.26% and 93.38 ± 2.12%, respectively at the end of 144 h. This sustained and prolonged release demeanor of drug molecules observed was mainly due to diffusion of drug through the lipoidal matrix of the SLNs. As evident from the inset graph, significantly higher (p < 0.05) release of DOX from SLN-D as compared to that of G-SLN-D was observed at the all time points (up to 144 h). This may possibly be due to structural integrity conferred by galactose thus providing a diffuse double layer barricade and offering a steric barrier to diffusion of drug. The outcomes are in accordance to antecedently reported data [51].

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3.5. Hemolytic toxicity DOX, SLN-D, G-SLN-D (0.1 ␮M equivalent of DOX) were exhibited hemolytic toxicity 17.1 ± 0.3%, 10. ± 0.2%, and 5.2 ± 0.2% respectively (Fig. 3a). DOX exhibited highest hemolytic toxicity amongst all formulations, might be due to direct contact to RBC cells. The toxicity of SLN-D and G-SLN-D formulations was less in comparison to plain drug due to the shielding of drug in a biocompatible lipidic environment. However, the hemolytic toxicity of SLNs with functionalities at the surface is a major limitation in the use of such systems and was enough to preclude its use as vehicles for drug delivery. Galactose coating to the SLN-D significantly decreased the hemolysis of the RBCs probably due to inhibition of interaction of RBCs with the anticancer moiety associated with the surface of the SLNs.

3.6. Cytotoxicity study MTT assay was performed against A549 cells to investigate the concentration and time dependent cytotoxic response (percent growth inhibition) of DOX loaded SLNs formulations (SLN-D and GSLN-D) as compared to free DOX. According to reported literatures, cell viability of soya lecithin and GMS were both well tolerated as the lipid matrices of SLN. Soya lecithin, a natural bio-acceptable surfactant, is principally located on the SLN surface. Previously Muller and coworkers [52,53] observed that the cytotoxicity of lipidic nanocarriers was lower compared to polymeric nanoparticles; in particular SLN consisting lecithin had no cytotoxic effect, allowing their utilize as colloidal drug carriers in vivo. They also narrated that the nature of the lipid matrix had no effect on cell viability. Experiments undoubtedly suggested a significant augmented % cellular inhibition or death of A549 cells with increasing concentration of DOX. Free DOX, SLN-D and G-SLN-D did not exhibit any significant inhibitory effect on the A549 cell lines when concentration of DOX was below 0.001 ␮g/mL. Further the cytotoxic response amplified when the concentration of DOX either in free form or within the SLNs was increased. Nearly the entire cells died when the concentration of DOX was 100 ␮M/mL, indicating internalization of satisfactory quantity of DOX inside the nuclei during the incubation time of 72 h (Fig. 3b). Cytotoxic response was in the order G-SLN-D > SLND > galactose + G-SLN-D > plain DOX in the concentration range of 1–100 ␮M/mL, The A549 cells were observed to be more sensitive to doxorubicin in G-SLN-D and the cytotoxicity increased on increasing the concentration, as shown in Fig. 3b(ii). It may possibly be due to receptor–ligand interaction involving lectin receptors and galactose which probably promoted better internalization. SLN-D were less cytotoxic as compared to G-SLN-D but more cytotoxic than free DOX. This may be accredited to the absence of ligand–receptor activity and the formulation enters the cell only by passive diffusion. Augmented internalization, accumulation of nanoparticles accompanied with sustained release of DOX loaded in the nanoparticles within the cells could be responsible for the augmented cytotoxic effect. Additionally, when the cells were incubated with galactose (1 mg/mL) + G-SLN-D the percent cell inhibition was less as compared to G-SLN-D. This may be owing to the ability of free galactose to selectively bind with the lectin receptors on the surface of tumor cells thereby saturating them. Thus, a strong sensitization was achieved with G-SLN-D, which appears to diminish cell resistance to the drug. Further, galactosylated SLNs enter the cells just via passive diffusion/non-specific endocytosis could be attributed to efficient cytotoxic action. The conclusions of cell cytotoxicity assay were further supported by the quantitative cell uptake studies, which revealed ∼1.5-fold

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Fig. 3. (a) Comparative hemolytic toxicity study (n = 6; p ≤ 0.05*,a ). *Significant difference between free DOX Vs SLN-D and M-SLN-D, a significant difference between S-D and G-S-D. (b) Cell cytotoxicity of G-SLN-D, SLN-D, galactose + G-SLN-D, plain DOX (A) after 24 h, (B) after 48 h and (C) after 72 h. Each data point represented as mean ± SD (n = 4).

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Fig. 4. (i) Cellular uptake efficiency of plain FITC, SLN-F, G-SLN-F and galactose + G-SLN-F in A549 cells. Each data point represented as mean ± SD (n = 4). (ii) Fluorescent photomicrographs of FITC uptake in the A549 cells from (A) G-SLN-D, (B) SLN-D, (C) galactose + G-SLN-D after 6 h of incubation.

higher uptake of DOX in G-SLN-D in comparison with SLN-D and free DOX upon incubation with A549 cells.

3.7. Cellular uptake study Cellular uptake of DOX from SLNs formulations as the function of galactose conjugation and incubation time was evaluated on A549 tumor cell lines using flow cytometer. The A549 cells were incubated with plain FITC, FITC labeled plain SLNs (SLN-F), FITC labeled galactosylated SLNs (G-SLN-F) and galactose + G-SLN-F. The rapid cellular localization of fluorescent SLN could be correlated with their nano size and composition; as they are chiefly constituted of bio-acceptable and biodegradable entity, solid lipids and lecithin [23]. The outcomes of the study indicated 22 ± 1.4%, 40 ± 1.3%, 63 ± 1.3% and 37 ± 1.2% cellular internalization after 2 h of incubation with plain FITC, SLN-F, G-SLN-F and galactose + G-SLN-F, respectively, where it was marked 30 ± 1.6%, 74 ± 1.3%, 99 ± 0.8% and 70 ± 1.4% respectively after 6 h of incubation. Cell uptake study shows the time-dependent cellular internalization of SLN. Cells associated with galactosylated SLNs depict the highest fluorescent intensity. This may be owing to receptor-mediated adhesion followed by phagocytic uptake of the complex [54,55]. In case of SLN-F, the internalization might be owing to fluid phase endocytosis/phagocytosis mediated nonspecific interlocalization. FITC alone might nonspecifically adsorbed on to the surface of the cells and thus depicting some fluorescence [56] [Fig. 4(i)]. On the contrary, incubation of free galactose with G-SLN-F leads to preferential

binding of galactose with lectin receptors thereby saturating them and ruling out the entry of galactosylated SLNs via lectin receptormediated endocytosis. Reduced cellular entry upon addition of galactose and also in case of uncoated SLNs clearly evidenced that the amplified uptake and cytotoxic action is facilitated by lectin receptors mediated uptake of galactosylated SLNs. Analogous to the results of quantitative cellular uptake studies, the fluorescent photomicrographs showed higher uptake of G-SLN-F in tumor cells, followed by SLN-F > galactose + G-SLN-F at equivalent concentrations of FITC [Fig. 4 (ii)].

3.8. In vivo pharmacokinetic and biodistribution studies Pharmacokinetic studies were executed to explore the effect of the presence of lipoidal matrix on the circulation life time of SLNs formulation and release profile of drug in systemic circulation (Table 2b; Fig. 5a). The data (Fig. 5a) revealed the plasma profiles of various DOX formulations after single i.v. injection in albino rats. The resulting pharmacokinetic parameters were determined and are revealed in Table 2b. The outcomes of the study revealed that the both of the SLNs formulation possessed slightly lower but prolonged Cmax compared to intravenous administration of free drug. However, DOX was detected in the serum until the end of 24 and 48 h after administration of SLN-D and G-SLN-D, respectively, and only for 8 h in case of free DOX solution and in very trace quantities, and thereafter no DOX was found. The MRT (mean residence time) of the G-SLN-D and SLN-D formulation was found to

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Fig. 5. (a) Serum concentration of DOX attained at various intervals (n = 6; p ≤ 0.05*,a ). *Significant difference between free DOX Vs S-D and M-S-D, a significant difference between SLN-D and G-SLN-D. (b) Biodistribution of free drug and formulations attained at various time intervals in different tissues (n = 6; p ≤ 0.05*,a ). *Significant difference between free DOX Vs S-D and M-S-D, significant difference between S-D and M-S-D.

be increased by about 8.2 and 3.25 folds respectively in comparison to free DOX. This designated the sustained release temperament of the DOX from the lipidic nanostructure. The outcomes of the study also manifested an increased residence time of G-SLN-D (Fig. 5a). Furthermore, the serum AUC0–24 , AUC0–inf , AUMC0–t and AUMC0–inf of G-SLN-D was 1.66, 1.76, 3.60 and 4.44 folds higher than SLN-D and 5.25, 5.55, 37.58 and 45.73 folds higher than free DOX, respectively. The results distinctly indicate long circulation property of SLNs with ligand anchored formulation having the more prolonged retention in the systemic circulation. The plasma level of DOX was sustained to a greater extent in case of G-SLN-D than SLN-D possibly due to double encirclement effect to drug diffusion upon galactosylation [26]. Furthermore, the high Clt and Kel of plain DOX as compared to both the SLNs formulation demonstrated their slow clearance from the body, which is supported by its extended halflife. These results of the pharmacokinetic studies presented to this point manifest that the bio-macromolecular drug delivery vehicles had markedly better bioavailability and prolonged retention in the systemic circulation. It is accredited that intensely porous vasculature like tumor tissue, has elongated capillary permeability and can accrue galactosylated SLNs via the EPR effect [57]. This will augment the confinement of nano lipid particles into tumor mass. The organ distribution studies were convened to enumerate the possible effectiveness of G-SLN-D to deliver DOX to tumor site and at the same time bypass non-target tissues (spleen, heart, kidney, and liver). The amount of free DOX was found to have greatest access to kidney and secondarily to tumors and liver (Fig. 5b). However, the free drug concentration was observed to decrease rapidly

and no free DOX was detectable in kidney and tumors at the end of 24 h. On the contrary the concentration of DOX in the SLNs formulation was found to be higher in tumors and liver (Fig. 5a). The data propound the greatest access of free DOX to the kidney, which is its major clearance organ. The concentrations of DOX observed in tumor tissues with SLNs formulations were higher as compared to free drug (Fig. 5b). This may be illustrated on the basis of enhanced permeation and retention (EPR) effect, which clamped the admission of the formulation in normal tissues while encouraged selective entry in tumors. G-SLN-D was found to have scored much better against DOX (Fig. 5b). No drug was found in tumor tissues after 24 h when free drug was administered. However sizeable quantities of the drug could be enucleated in the case of SLNs formulation. The DOX level in tumor tissues was marked the most with time in case of galactosylated SLNs. This may be illustrated on the basis of the impendence of lectin receptors on tumor tissues that favored selective entry of G-SLN-D in tumors. The concentration of DOX loaded in G-SLN-D also found in significant amount in liver possibly because of promoted macrophagic uptake due to the presence of lectin receptors on macrophages. However, the degree of increment in liver was comparatively lower than tumor tissues, because galactose conjugation shielded the hydrophobic nature of SLN and thus precluded specific uptake by reticulo-endothelial system (RES). This discloses the localized action, and transcendent retention potential of the formulation at target sites, thereby enhancing the therapeutic efficacy by providing the opportunity to minimize the dose of drug as well as SLNs required for its delivery. The above effect can be

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attributed to the modified vasculature at the tumor sites. G-SLND significantly diminished the delivery of drug to heart indicating their prospective in minimizing the cardiotoxicity associated with DOX therapy. Thus the present formulation utilizes EPR effect provided by the biomacromolecule by prolonged retention and greater accessibility to these leaky vasculatures. The outcome of the experiment designates that the galactosylated SLNs extended the circulation time and facilitated the tumor specific delivery of the bioactive. The developed formulation stated EPR as well as extravasation effects providing synergistic action, thereby enhancing the therapeutic potential. 4. Conclusions Since the developed galactosylated SLN formulation is intended to deliver the drugs in the nuclear region of cancer cells, it was essential to examine its intracellular fate especially in the nucleus of A549 cells. To validate the hypothesis of increased cytotoxicity because of galactosylation, cell uptake and intracellular localization of G-SLN-F was evaluated in A549 cells. The results obtained from the studies concluded that an unloaded SLN formulation, obtained with lipid matrices, is not toxic to cell-lines. SLN are rapidly entrapped into cell-lines, as shown with the fluorescent SLN and galactosylated fluorescent SLN. This behavior confirms that SLN are easily internalized. The cytotoxicity of DOX incorporated in SLN is consistently higher than that of the drug solutions on cell-lines. This effect is particularly marked on A549 with doxorubicin-loaded galactosylated SLN. However, the increase of doxorubicin cytotoxicity compared to the solution has previously been observed with polymeric micelles, polymeric nanoparticles and liposomes carrying doxorubicin. Kabanov and coworkers elucidated the augmented intracellular uptake and enhanced cytotoxicity of doxorubicin conferred in Pluronic copolymer micelles [58]. Couvreur and coworkers delineated that doxorubicin-loaded polyalkylcyanoacrylate nanoparticles were found more cytotoxic than doxorubicin solution against P388 leukemia cells. Present work illustrated the higher sensitivity of the cells to the drugs incorporated into galactosylated SLN than to the drugs loaded in SLN and in solution may be related to the marked uptake and accumulation of drug-loaded in galactosylated SLN in the cells, where the loaded galactosylated-SLN should release the drugs, so enhancing their action. Conflict of interest The authors report no conflicts of interest in this work. Acknowledgements The authors are thankful to the Department of Pharmaceutical Sciences, Dr. H.S. Gour University, Sagar (M.P.) for providing the necessary infrastructure and facilities. The authors are grateful for the fellowship and grant provided by the Council of Scientific & Industrial Research, Human Resource Development Group (CSIR HRDG), New Delhi, India. The authors are grateful for the fellowship provided by the AICTE, India. The authors also thank Sun Pharma, Vadodara for providing doxorubicin HCl as gift sample and Bose Institute, Kolkata for cell line study. References [1] K. Jain, P. Kesharwani, U. Gupta, N.K. Jain, A review of glycosylated carriers for drug delivery, Biomaterials 33 (2012) 4166–4186. [2] C. Bies, C.-M. Lehr, J.F. Woodley, Lectin-mediated drug targeting: history and applications, Adv. Drug Deliv. Rev. 56 (2004) 425–435. [3] P. Kesharwani, R.K. Tekade, N.K. Jain, Dendrimer generational nomenclature: the need to harmonize, Drug Discov. Today 20 (2015) 497–499.

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