European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 449–458

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Research paper

Development and characterization of sulfasalazine loaded fucosylated PPI dendrimer for the treatment of cytokine-induced liver damage Richa Gupta, Neelesh Kumar Mehra, Narendra Kumar Jain ⇑ Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar, India

a r t i c l e

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Article history: Received 7 June 2013 Accepted in revised form 28 October 2013 Available online 1 November 2013 Keywords: Fucose PPI dendrimers Kupffer cells Sulfasalazine Targeting Macrophages Pharmacokinetic

a b s t r a c t The present investigation was aimed at exploring the targeting potential of sulfasalazine (NF-jB inhibitor drug) loaded fucose tethered poly (propylene imine) (PPI) dendritic nanoarchitecture (SSZ–FUCO-PPID) to Kupffer cells for effective management of cytokine-induced liver damage. The SSZ–FUCO-PPID formulation was characterized for entrapment efficiency, in vitro release, stability, toxicological investigations, macrophage uptake, NF-jB inhibition, and in vivo studies. In cell uptake assay the uptake of SSZ–FUCOPPID was found to be higher and preferentially by J774 macrophage cell line. Cytokine assay suggested that the SSZ–FUCO-PPID potentially inhibited the IL-12 p40 production in LPS activated macrophages. Western blot analysis clearly suggested that SSZ–FUCO-PPID inhibited the activation of NF-jB as indicated by the absence of p-IjB band. Pharmacokinetic study revealed improved bioavailability, half-life and mean residence time of SSZ upon fucosylation of dendrimers. The biodistribution pattern clearly established the higher amount of SSZ–FUCO-PPID in liver. Hematological data suggest that the fucosylated formulations are less immunogenic as compared to unconjugated formulations. The results suggest that the SSZ–FUCO-PPID formulation holds targeting potential to Kupffer cells for the treatment of cytokine-induced liver damage. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Currently the researchers have been continuously exploring the numerous nanosized carrier systems including the liposomes, nanoparticles (gold nanoparticles, solid lipid nanoparticles, and nanostructured lipid carriers), carbon nanotubes and dendrimers for wide variety of biomedical applications [1–10]. The hyperbranched dendrimers have emerged as one of the captivated nano-particulate carrier system to the scientific community in the form of controlled and targeted drug delivery. Dendrimers possess unique physicochemical properties such as highly branched, three-dimensional, mono-dispersed macromolecules with multivalency, large number of end terminal peripheral groups and interior cavities, and host–guest interactions properties [1,3,5,9–16]. The United States Food and Drug Administration (FDA) have approved Vivagel™ (SPL 7013 Gel) for the prevention of HIV/HSV. Vivagel™ is a water based vaginal product of 3% w/w/ SPL7013 mixed in carbopolÒ gel buffered to pH physiologically compatible with the normal human vagina, available in the market [17]. Dendrimers have been reported as a carrier for targeted drug delivery, ⇑ Corresponding author. Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar 470 003, India. Tel./fax: +91 7582 265055. E-mail addresses: [email protected] (N.K. Mehra), [email protected], [email protected] (N.K. Jain). 0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.10.018

gene/DNA delivery, solubility enhancement and diagnosis. The numerous reports are available of surface modified PPI dendrimers with carbohydrates (mannose, and galactose) in targeted drug delivery of theragnostics [13,18–20]. Fucose had been shown to be promising among various targeting ligands investigated so far for macrophage targeting because the macrophages possess a large number of receptors that can recognize carbohydrate units. Therefore, we hypothesized that fucose anchored PPI dendrimers could be a better alternative for the targeting of macrophages of liver. Higuchi et al. investigated potential role of fucose anchored cationic liposome/NF-jB decoy complexes in the treatment of cytokine-related liver disease [21]. The livermacrophages (Kupffer cells) play an imperative role in inflammatory liver signaling. Exposure to pathogens like bacteria and viruses, leads to activation of the transcription factor i.e. nuclear factor-jB (NF-jB). NF-jB was first discovered in 1986 as a nuclear transcription factor required for immunoglobulin kappa light chain transcription in B-cells. It is expressed in all cell types and holds a central role as a regulator of the response to cellular stress. The term NF-jB does not refer to a single molecule but rather to a family of dimeric transcription factors consisting of the five Rel subunits, namely p50, p52, RelA (known as p65), RelB and c-Rel, expressed in the cytoplasm of Kupffer cells [22]. NF-jB plays pivotal role in inflammatory liver signaling because it is one of the most important regulators of pro-inflammatory gene expression.

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Activation of the NF-kB/Rel transcription family by nuclear translocation of cytoplasmic complexes plays a central role in inflammation of pro-inflammatory genes. NF-jB can be activated within minutes by numerous stimuli, including inflammatory cytokines such as TNF- and interleukin-1, T-cell activation signals, growth factors and stress inducers [23–26]. Since activated NF-jB mediates the production of inflammatory cytokines, it brings the need for selective targeting of NF-jB in Kupffer cells for the treatment of cytokine-induced liver damage [27]. Hence the delivery of an NF-jB inhibitor drug to Kupffer cells would be an attractive option for the treatment of the disease. Sulfasalazine (SSZ) is a known anti-inflammatory and potent NF-jB agent that inhibits NF-jB dependent transcription in microto milli-molar concentrations; conversely 5-ASA or sulfapyridine SP do not block NF-jB activation at all doses. SSZ inhibits phosphorylation of IjB thus the nuclear localization of NF-jB is subdued and as a result no cytokine production takes place [28–31]. The purpose of the present study was to investigate targeting potential and NF-jB inhibition activity of SSZ loaded fucosylated PPI dendrimers (SSZ–FUCO-PPID) as compared to free drug and un-conjugated dendrimers. To best of our knowledge the present study is a debut report that explores fucosylated dendrimers for Kupffer cells targeting. Various formulations of dendrimers were synthesized and characterized for entrapment efficiency, in vitro drug release, stability and hemolytic toxicity. Ex vivo cytotoxicity and cellular uptake and NF-jB inhibition were studied against macrophage cell line, J774. Finally, in vivo pharmacokinetic, biodistribution and hematological parameters were also evaluated for free SSZ and dendrimer formulations. 2. Materials and methods 2.1. Materials SSZ was a benevolent gift from M/s IPCA Laboratories (Mumbai), India. L-fucose and dialysis membrane (MWCO, 12–14 kDa) were purchased from HiMedia Laboratories Pvt. Ltd. Mumbai, India. Ethylene diamine (EDA) and acrylonitrile were purchased from Central Drug House (CDH), New Delhi, India. Raney Nickel was purchased from Merck (India). All other reagents and solvents were used as received.

2.3. Fucose conjugation to 5.0 G PPI dendrimers (FUCO-PPID) Fucose conjugation to 5.0 G PPI dendrimers was carried out following the method of mannose conjugation previously reported from our laboratory [7,11,18,33], with slight modification (Fig. 1). Briefly, L-fucose (8 mM) was dissolved in 0.1 M sodium acetate buffer (pH 4) and subsequently added to lyophilize PPI dendrimers (1 mM). The mixture was agitated at ambient temperature for 72 h to ensure completion of reaction. Resulting solution was concentrated under vacuum at 60 °C (Jyoti Scientific Industries, Gwalior, India). FUCO-PPID was purified by dialyzing against distilled water in a dialysis tubing (MWCO 6 kDa, Sigma) for 24 h to remove unconjugated fucose molecules along with other impurities, which diffuse out into water; and finally FUCO-PPID was retained inside and lyophilized (Hetro Dry Winner, Germany). The synthesized PPI and FUCO-PPID dendrimers were characterized. The Fourier transform-infrared spectroscopy was carried out using KBr pellet method in an IR spectroscope Perkin-Elmer FT-IR Spectroscope, USA. The 1H NMR spectroscopy of dendrimer was carried out at 300 MHz, after dissolving in CDCl3 (Bruker DPX, USA). The Transmission Electron Microscopy (TEM) was performed to characterize dendrimers in terms of their size. TEM was performed after drying dendrimer solution on carbon grid and staining negatively by 1% phosphotungstic acid (PTA) by metal shadowing techniques at Electron Microscopy Division (Morgani, 268D, Holland). The zeta potential of plain 5.0 G PPI and SSZ/FUCO-PPID dendrimeric formulations was determined by photon correlation spectroscopy in a Malvern Zetasizer nano ZS90 (Malvern Instruments, Ltd., Malvern, UK) at room temperature (RT). 2.4. Drug loading in dendrimer formulations The drug (SSZ) was loaded in PPID and FUCO-PPID following the equilibrium dialysis method, as reported earlier from our laboratory, with slight modification [3,11,14]. Briefly, the known molar concentrations of PPID and FUCO-PPID were mixed separately with SSZ in 1:1 ratio in PBS (pH 7.4) and incubated for 24 h with continuous slow magnetic stirring at 50 rpm (Remi, Mumbai, India) using Teflon bars. The content was then dialyzed in a dialysis tubing (MWCO 6 kDa, Sigma, USA) against PBS (pH 7.4) under sink conditions for 30 min to remove free SSZ from the dendrimeric formulations. The amount of free drug was estimated

2.2. Synthesis and characterization of 5.0 generation PPI (5.0 G PPI) dendrimers The fifth generation (5.0 G) PPI dendrimers were synthesized by divergent approach using Double Michael addition reaction as reported previously from our laboratory [3,11,14,18,20]. Briefly, double Michael addition reaction was used to synthesize half generation (0.5 G; ACN terminated PPI dendrimers) by adding acrylonitrile to the initiator core ethylenediamine (EDA), the exothermic reaction caused the temperature to rise to about 38 °C, the reaction mixture was heated at 80 °C for 1 h to complete the addition reaction. This was filtered by removal of excess acrylonitrile (ACN) as a water azeotrope by vacuum distillation (16 mbar, bath temperature 40 °C) to obtain 0.5 G PPI [EDA-dendr-(CN)4] as crystalline solid. The 0.5 G PPI dendrimers were subjected to heterogeneous hydrogenation by mean of Raney Nickel as catalyst to produce full 1.0 G (ANH2) dendrimers. To the hydrogenation vessel filled with Raney Nickel catalyst, EDA-dendr-(CN)4 dissolved in methanol was added and the resultant mixture was hydrogenated at 40 atm hydrogen pressure at 70 °C for 1 h, filtered and solvent was evaporated at reduced pressure. PPI dendrimers up to 5.0 G were synthesized by repetition of the reaction sequence [3,11,14,32].

Fig. 1. FT-IR spectra of (A) 5.0 G PPI, and (B) FUCO-PPID dendrimers formulation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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spectrophotometrically at kmax 359 nm (UV-1601, Shimadzu, Japan) and the amount of SSZ loaded in dendrimeric formulations (Scheme 1) was calculated. The dialyzed formulation was lyophilized and used for further characterization. The percentage entrapment efficiency of PPID and FUCO-PPID dendrimeric formulations was calculated using the following equation:

EE% ¼

Weight of entrapped drug  100 Weight of entrapped drug þ free drug

2.5. In vitro drug release The in vitro release from the known amounts of SSZ loaded plain (SSZ-PPID) and fucosylated PPI dendrimers (SSZ–FUCO-PPID) was studied in PBS (pH 7.4) and acetate buffer (pH 4.0) in a modified dissolution method [11,14,34]. The dialysis bags (MWCO 6 kDa, Sigma, USA) were filled with SSZ-PPID and SSZ–FUCO-PPID separately at 37 ± 2 °C with slow magnetic stirring under sink conditions. Samples were withdrawn at predetermined time points and replenished with the equivalent volume of fresh medium into the recipient compartment maintaining the strict sink conditions. The concentration of SSZ was determined in triplicate after appropriate dilution of aliquots spectrophotometrically (kmax 359 nm; UV-1601, Shimadzu, Japan) against the similar blank medium. 2.6. Stability studies The SSZ loaded dendrimer formulations (SSZ-PPID and SSZ– FUCO-PPID) were stored under dark (amber color vials) and light (colorless vials) at 4 ± 0.5 °C, 25 ± 0.5 °C and 50 ± 0.5 °C in stability chambers (Remi CHM-6S, India) for a period of 5 weeks. The samples were analyzed initially and weekly up to 5 weeks for any sign

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of precipitation, turbidity, crystallization, change in color, consistency and drug leakage. The obtained data were used for the analysis of any physical or chemical degradation at storage conditions. Drug leakage was determined by monitoring the release of drug from the formulations after storage at different conditions (4, 25, 50 ± 0.5 °C). The formulation samples (2 mL) were dialyzed in a dialysis tubing (MWCO 6 kDa, Sigma, USA) against external medium (PBS pH 7.4) and analyzed for SSZ content spectrophotometrically at kmax 359 nm and repeated every week up to 5 weeks. The percentage increase in drug release from the formulations was used to analyze the effect of accelerated condition of storage on the formulation [7]. 2.7. Comparison of hemolytic toxicity The red blood corpuscles (RBCs) suspension for hemolytic study was prepared following the reported procedure, with minor modifications [3,11,32,34]. Briefly, the human venous blood was collected in Hi-Anticlot blood collection vials (HiMedia Labs, Mumbai, India). The RBCs were separated from the whole blood by centrifugation (Remi, Mumbai, India) at 3000 rpm for 5 min. Supernatant was collected and suspended in normal saline to get 10% hematocrit value. 0.5 mL of suitably diluted free SSZ, PPID, SSZ-PPID and SSZ–FUCO-PPID was added separately to 4.5 mL of normal saline and incubated for 1 h with RBCs suspension. After centrifugation, supernatants were diluted with an equal volume of normal saline and absorbance was measured at 540 nm. RBCs suspension was added to 5 mL of 0.9% w/v NaCl solution (normal saline) and 5 mL distilled water, respectively to obtain 0% and 100% hemolysis. The degree of hemolysis was determined using the following equation:

Hemolysis ð%Þ ¼

Abs  Abs0  100 Abs100  Abs0

where Abs, Abs100 and Abs0 are the absorbance of sample, a solution of 100% and 0% hemolysis, respectively. 2.8. Cell culture In the present study J774 macrophages cell line was used for evaluation of SSZ-PPID and SSZ/FUUCO-PPID dendrimeric formulations. The 100 lL aliquots containing macrophage cell lines J774 with more than 95% viability were suspended in 0.9 mL of fresh supplemented RPMI 1640 culture medium supplemented with 10 U/mL penicillin, 10% fetal bovine serum (FBS), 100 lg/mL streptomycin, 1 mM sodium pyruvate and 10 mM HEPES medium and transferred into a tissue culture plate with 96 well tissue culture plates and incubated for 48 h at 37 °C with 5% CO2 and 95% humidified atmosphere to acquire more than 75% confluency [7,33,35].

Scheme 1. Schematic representation of SSZ encapsulated fucosylated loaded PPI dendrimer (SSZ–FUCO-PPID). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.8.1. Cytotoxicity studies (SRB assay) Cytotoxicity of developed dendrimeric formulations was assessed via sulforhodamine (SRB) assay. This assay relies on the uptake of the anionic pink amino-xanthine dye, ‘SRB’ by basic amino acids where it forms an electrostatic complex with the basic amino acid residues of proteins under moderately acidic conditions. The greater the number of cells, the greater amount of dye is taken up, and after fixing, when the cells are lysed, the released dye will give a more intense color and greater absorbance. One hundred micro-liters of samples (PPID, SSZ-PPID and SSZ–FUCO-PPID) to be tested in culture medium was added to the 96-wells plate containing J774 cells in concentration range of 1–1000 lM and incubated for 24 h. The cells were fixed with icecold trichloro acetic acid (TCA) for 1 h at 4 °C. Culture plates were washed with distilled water five times and allowed to dry in the air, 50 lL sulforhodamine B (SRB) solution was added to each well

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of the dry 96-well tissue culture plates and allowed for staining at room temperature (RT) for 30 min. SRB solution was removed by washing the plates quickly with 1% v/v acetic acid, five times, to remove unbound dye. Washed plates were dried in the air. The bound SRB was solubilized by the addition of 100 lL unbuffered Tris Base (10 mM; pH 10.5) to each well and shaken for 5 min on a shaker platform. Plates were read in a 96-well plate reader at 492 nm [36,37]. Unbound/blank cells were used as control. The experiment was repeated three times for each sample. Cell viability of each group was compared with control and was expressed as % cell viability using following equation:

Cell viability ð%Þ ¼

½Atest  100 ½Acontrol

where Atreated is the absorbance of treated cells with various samples at 492 nm and Acontrol is the absorbance of control at 492 nm. 2.8.2. Macrophage uptake assay The macrophages uptake efficiency as a function of conjugation of dendrimeric macromolecules was determined by incubating the macrophage cells J774 with the fluorescein iso-thiocyanate (FITC) dye loaded dendrimers formulations at 1 and 6 h time interval. The FITC was loaded in dendrimers formulations following the standard protocol according to the earlier reported method [7,38]. SSZ-PPID and SSZ–FUCO-PPID were loaded with FITC by incubating dendrimers (0.1 lM) formulations with 5 mL of 0.03% FITC solution for 24 h, with intermittent shaking. Ten lL of various FITC loaded formulations SSZ-PPID and SSZ–FUCO-PPID was suspended in the wells containing J774 macrophage cells. After incubation for 1 and 6 h, cell lines were detached by pipetting and collected by centrifugation at 5000 rpm for 2 min to remove nano-carriers adhering to cell surface. Cells were washed 5 times with PBS. Cell associated fluorescence was measured by fluorescence activated cell sorter (FACS) instrument (‘‘BD’’ Bioscience, FACS Aria, Germany) using 480 nm excitation, 520 nm emission and the appropriate sensitivity settings [39] and macrophage uptake efficiency was measured in terms of observed intensity of fluorescence. 2.9. Nuclear factor-kB(NF-jB) inhibition study 2.9.1. Cytokine assay Ten lg/mL of lipo-polysaccharides (LPs) was introduced in 96 well tissue culture plates containing macrophage cells J774 in the absence (control) and presence of samples to be tested (free SSZ, SSZ-PPID and SSZ–FUCO-PPID). Cells were incubated for 48 h to ensure stimulation in case of control (stimulated cells produce higher amount of IL-12, which is the indication of NF-jB activation). Cultured supernatant was harvested and cytokine (IL12p40) concentrations in the supernatants were determined by enzyme linked immune-sorbent assay (ELISA) [40]. The results were represented as % inhibition in IL-12 (IL-12 p40) concentration. 2.9.2. Western blot analysis Ten lg/mL of LPS was introduced in 96 wells plate containing macrophage cells in the absence (control) and presence of samples to be tested (free SSZ, SSZ-PPID and SSZ–FUCO-PPID) and incubated for 48 h. Cells were washed with PBS and subsequently lysed in 25 mM Tris HCl (pH 7.5), 1 mM EDTA, 0.25 mM dithioerythritol, 0.1 M KCl, 1% Nonidet P40 (NP40), ‘‘complete’’ protease inhibitor cocktail (Boehringer, Mannheim) and phosphatase inhibitor. The lysate cells were transferred in to Eppendorf tubes, ultra-centrifuged (Z36HK, HERMLE LaborTchnik GmbH, Germany) at 15,000g and the supernatant was recovered. Cell extracts were analyzed according to the standard protocols. Western blots were pretreated

in blocking buffer (5% of BSA) and subsequently incubated with anti p-IjB antibody. Bands were detected using the western blotting detection reagent [41]. 2.10. In vivo studies The present in vivo studies were carried out on 8 weeks old male albino rats (Sprague-Dawley, 150 ± 20 g). The in vivo experimental protocol was duly approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) of Dr. Hari Singh Gour University, Sagar, India (letter number Animal Eths. Comm.2011/12/439). For in vivo studies drug concentration in blood samples and organ homogenates was determined by high performance liquid chromatography (HPLC) method using mobile phase methanol:acetonitrile:water: 35:35:30 v/v) with 1 mL/min flow rate using C18-RP column [42]. 2.10.1. Blood level studies The animals were divided into five groups each comprising of three rats and marked adequately. All animals were fasted overnight before administration of dose but allowed water ad libitum. The developed dendrimeric formulations (SSZ-PPID and SSZ– FUCO-PPID) and free SSZ solubilized in PBS (pH 7.4) (suspended in case of free SSZ) in the dose of 20 mg/kg body weight were administered intravenously to each animal of the first, second, third, fourth and fifth groups, respectively through caudal vein. One rat from each group was sequentially taken and 0.2 mL of blood was withdrawn from retro-orbital plexus in 0.9% saline after 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, 18.0, 24.0, 30.0 and 36.0 h, respectively from each group. In each case blood was centrifuged at 3000 rpm (Remi, Mumbai, India) for 10 min. The upper supernatant (plasma) was collected separately with the help of micropipette. One hundred lL of acetonitrile was added to the 100 lL of sample. The contents were vortexed (Superfit vortexer, India) for 1 min and 5 mL methanol was added to it. The mixture was extracted for 10 min and centrifuged at 3000 rpm for 10 min. The supernatant was decanted into another vial and evaporated to dryness at 60 °C. The dried residue was reconstituted with 1 mL methanol and centrifuged at 15,000 rpm for 10 min. Clear supernatant was collected and amount of SSZ was determined by HPLC method [7,14,42] and various pharmacokinetic parameters including maximum plasma concentration (Cmax), area under the plasma concentration curve (AUC), area under the mean plasma concentration curve (AUMC), and mean residence time (MRT) were calculated. 2.10.2. Tissue biodistribution studies Tissue biodistribution studies were carried out for quantitative measurement of SSZ in different organs including liver, spleen, kidney and lungs. Animals were divided into three groups, each comprising three animals and fasted overnight prior to administration. To the first group plain SSZ solution (1 mg/kg) in PBS (pH 7.4); to the second and third groups SSZ-PPID and SSZ–FUCO-PPID, respectively were administered through the caudal vein. At specific time points (1, 6 and 24 h) animals were sacrificed by decapitation method and organs were excised and homogenized. The homogenates were deproteinized with acetonitrile and the homogenates centrifuged, filtered and estimated for the drug content by HPLC method [3,7,11,14,20,42]. The results were expressed as percentage dose recovered from each organ. 2.10.3. Hematological studies Hematological parameters for red blood corpuscles (RBCs), white blood corpuscles (WBCs), and differential counts were determined according to the earlier reported method [20,43]. Animals were divided into four groups, including one group as control and all groups comprised of three animals each (n = 3). Animals

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of group second, third and fourth were administered with free drug (SSZ), SSZ-PPID and SSZ–FUCO-PPID formulation, respectively in a dose of 20 mg/kg body weight intravenously. Animals were maintained on same regular diet up to 7 days. After 7 days blood samples were collected by retro-orbital route from the animals and analyzed for RBCs, WBCs and differential count (monocytes, lymphocytes and neutrophils, % Hb, MCH and HCT). 2.10.4. Statistical analysis The results were expressed as mean ± standard deviation (n = 3) and statistical analysis was performed with Graph Pad Instat Software (Version 3.00, Graph Pad Software, San Diego, California, USA) using one-way analysis of variance (ANOVA) followed by Tukey– Kramer test for multiple comparisons. A probability p < 0.05 was considered statistically significant. The pharmacokinetic parameters were calculated using the Kinetica 5.0 PK/PD analysis software, Thermo Fischer Scientific, USA. 3. Results and discussion In the current scenario hyperbranched dendrimer is attracting great attention in the treatment of cancer, diabetes, malaria etc in biomedical applications [2,3,11,16,20]. The 5.0 generation PPI dendrimers were synthesized using ethylene diamine (EDA) as core using the previously reported method from our laboratory [3,11,14,32,43]. The FTIR spectrum of 5.0 G PPI dendrimers is shown in Fig. 1(A). Fucose conjugation to 5.0 G PPI dendrimers (FUCO-PPID) was carried out by ring opening reaction followed by reaction of aldehyde groups of fucose in 0.1 M sodium acetate buffer (pH 4.0) with the amino groups present on the end terminal surface of the dendrimers [7,9,44]. This reaction leads to the formation of Schiff’s base i.e. AN@CHA, which may possibly get reduced into the secondary amine (ANHACH2) and remain in equilibrium with Schiff’s base. The un-conjugated fucose and other impurities in fucose conjugated dendrimers were removed by dialysis method [44]. The characteristic peaks of FUCO-PPID showed the peak of NH deformation of secondary amine at 1653.14 cm1, attributed to conjugation between the aldehyde groups of fucose with the amine group of 5.0 G PPI dendrimer, confirming the formation of Schiff base (Fig. 1B) while characteristic peak at 3422.07 cm1 showed the free terminal amine groups of dendrimer. The synthesis of fifth generation PPI dendrimer was confirmed by 1H NMR spectroscopy (Bruker DRX, USA), the spectra are shown in Fig. 2(A and B). The NMR spectrum showed the multiplets (m) between 0.6–1.25 ppm and 1.3–1.9 ppm for alkane and primary alkane, respectively. The obtained NMR peaks between 2.3 and 2.9 ppm correspond to methylene (ACH2). The peak at 7.933 ppm confirmed the presence of primary amines. The results matched with the reported synthesis of PPI dendrimers [3,11,43,45]. The fucose conjugation was confirmed by NMR spectrum, the shift at the d value 7.735 ppm in NMR spectrum showed the presence of ACONH group of the conjugated system (Fig. 2B). The electron microscopy was performed to ascertain the nanometric size range of dendrimers. The TEM studies suggested that the dendrimers and its conjugates were in the nanometric size range (Fig. 3A and B). Recently Jain and co-workers reported the average particle size of plain 5.0 G PPI dendrimer to be 6 ± 0.05 nm with a polydispersity index (PDI) of 0.83 ± 0.035 nm. The narrow PDI value (0.83 ± 0.035 nm) and average particle size clearly suggest the narrow particle size distribution [3]. Our results are in accordance with the earlier published reports [3,11,14,20,32,43–46]. The synthesized 5.0 G PPI dendrimers had honey like consistency with dark brownish color having high viscosity in a physical state [43]. The surface charge of the plain 5.0 G PPI dendrimers and SSZ/FUCO-PPID was determined and

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found to be 16.00 ± 0.85 and +5.0 ± 0.07, respectively. The reduction in the zeta potential of SSZ/FUCO-PPID conjugated may be ascribed due to fucosylation of dendrimers. The SSZ was loaded in the PPID and FUCO-PPID dendrimers through equilibrium dialysis method reported earlier, with slight modifications [3,11,14]. The percent drug loading was calculated indirectly by estimating the un-entrapped drug spectrophotometrically during equilibrium dialysis method. The percent drug loading of PPID and FUCO-PPID dendrimeric formulation was found to be 28.87 ± 0.23% and 40.27 ± 0.82%, respectively. The percentage SSZ loading of FUCO-PPID was significantly increased compared to PPID, possibly due to the fact that with increase in generations the structure became more compact and peripherally closed for higher generations of dendrimers. Fucosylation further increased complexation and provided steric hindrance, preventing the drug leakage from open structure. The in vitro drug release studies suggested that both SSZ-PPID and SSZ–FUCO-PPID showed burst release of SSZ at pH 4.0 (Fig. 4). The drug release was found to be 97.60 ± 0.68% and 76.07 ± 0.006% for SSZ-PPID and SSZ–FUCO-PPID, respectively at 24th h at pH 7.4 while SSZ–FUCO-PPID formulation released about 83.75 ± 0.56% SSZ at same pH. At the acidic microenvironment SSZ–FUCO-PPID dendrimer formulation released 94.76 ± 0.33% SSZ at 48th h. The attachment of the hydrophilic fucose molecules on the surface of the 5.0 G EDA–PPI dendrimers shrunk the hydrophobic interior cavities of the dendrimer at pH 7.4 and allowed the SSZ to stay for a longer duration of time. On the other hand higher drug release rate at pH 4.0 (simulated to pH of phagolysosomes as reported by Mor & co-workers [47]) may possibly be attributed to the protonation of terminal amine groups of PPI dendrimers, causing them to repel each other and exposing the drug to the medium (Fig. 4). The results of in vitro studies, including drug loading studies, indicate higher loading of SSZ and sustained release manner upon surface modification (SSZ–FUCO-PPID). The stability of SSZ-PPID and SSZ–FUCO-PPID formulation was determined at different conditions under dark (amber color vials) and light (colorless vials) at 4 ± 0.5 °C, 25 ± 0.5 °C and 50 ± 0.5 °C in a stability chamber (Remi, CHM-6S, India) for a period of 5 weeks. The formulations were found to be most stable in dark condition at 4 ± 0.5 °C. The stability data are in conformity with the earlier published reports [3,11,14,43]. The drug leakage was found to be lower at room temperature as compared to that at 4 ± 0.5 °C which may be due to the shrinking of the dendrimeric hyperbranched nano-architecture leading to decrease in cavity enclosing drug molecules. The release of the SSZ was found to be greater at the extended temperature possibly due to shielding of inner channels or cavities of PPI dendrimers. At higher temperature fucose molecules gain higher kinetic energy, and as a result, the ring is wide open and the fucose molecules shield the dendrimer cavities, spread in the medium. It was also found that the drug leakage was more in formulations stored in light than those kept in dark. This may be attributed to structure cleavage at higher temperature and light leading to bond breakage due to higher reaction kinetics. Thus it can be concluded that the SSZ–FUCO-PPID is more stable in dark and at 4 than at 25, 50 ± 0.5 °C [48]. The hemolytic toxicity of the PPI dendrimers remains a major limitation in the use of such poly-cationic hyperbranched dendrimer mediated targeted drug delivery systems. The surface modification of the PPI dendrimers by attachment of the numerous targeting ligand including mannose, galactose, folic acid and sialic acid etc. was found to decrease the erythrocytes toxicity, may be due to shielding or coating of the charged quaternary ammonium ion, which is generally formed on the NH2 terminated end functional groups of full generation of PPI dendrimers responsible for hemolysis [11,14,32,43,48]. The hemolytic toxicity of free SSZ, PPI, SSZ-PPID and SSZ–FUCO-PPID was found to be 13.01 ± 0.13%,

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Fig. 2. 1H NMR spectra of (A) 5.0 G PPI dendrimer, and (B) SSZ–FUCO-PPID formulations.

Fig. 3. Transmission electron microscopic nanographs of (A) 5.0 G PPI, and (B) SSZ– FUCO-PPID formulations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Cumulative percent SSZ released

SSZ-PPID (pH 4.0) SSZ-PPID (pH 7.4)

SSZ-FUCO-PPID (pH 4.0) SSZ-FUCO-PPID (pH 7.4)

100 90 80 70 60 50 40 30 20 10 0 0

10

20

30

40

50

60

70

80

Time (hr)

14.76 ± 0.72%, 17.19 ± 0.44% and 4.45 ± 0.26%, respectively. However, the hemolytic toxicity of SSZ–FUCO-PPID was found to be decreased by 4.45 ± 0.26% (p 6 0.05) as compared to SSZ-PPID, PPI and free SSZ. The fucose attachment considerably reduced the hemolysis of RBCs possibly due to shielding of the end terminal ANH2 (amine) functional groups responsible for hemolysis of the RBCs. In this way contact of cell membranes of RBCs with cationic end terminal amine functional groups of PPI dendrimers is prevented and hence less hemolysis occurs [20]. Recently, Jain and co-workers reported that mannosylation and sialylation of the 4.0 G PPI dendrimers showed significantly reduced hemolysis of

Fig. 4. The cumulative SSZ released at pH (4.0 and 7.4) from SSZ-PPID and SSZ– FUCO-PPID formulations (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the RBCs to 1.7 ± 0.46%, 2.6 ± 0.37%, 3.3 ± 0.59%, and 3.8 ± 0.47% at 0.1%, 0.2%, 0.3% and 0.4% w/v concentrations owing to the inhibition of interaction of RBCs with the charged quaternary ammonium ions [11]. Our results are in agreement with earlier published reports on surface engineered PPI dendrimers [3,11,14,32,45].

R. Gupta et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 449–458

The in vitro cytotoxicity assay was carried out on macrophages J774 cells via SRB assay by incubating different formulations for 24 h. Unloaded plain 5.0 G PPI dendrimers did not show any significant cytotoxicity after 24 h incubation even at higher concentration (100 lM). The free SSZ exerted the cytotoxicity in a dose-dependent manner and showed negligible cytotoxicity up to 100 lM, however above 100 lM concentration it exerts significant cytotoxicity (% cell viability < 40). The higher cytotoxicity of SSZ-PPID than free SSZ might be due to the higher interaction of positively charged PPI dendrimers with negatively charged cell membranes. SSZ–FUCO-PPID showed more cytotoxicity than PPID, SSZ-PPID and free SSZ. The fucosylation of dendrimer might have contributed to higher cytotoxicity of SSZ–FUCO-PPID due to more accumulation of SSZ inside the cell via receptor-mediated endocytosis (Fig. 5). However, our aim was not only to kill macrophages but also to rule out that the dose selected for the treatment of disease is not cytotoxic to the cells. The available data revealed that the dose selected was non-cytotoxic to macrophages. The macrophage uptake assay clearly revealed that with the increase in time mean FITC measured intensities were found to be increased in macrophage cell line (J774), which may be due to more cellular uptake of FITC loaded fucosylated dendrimers than unconjugated dendrimers formulations. The SSZ–FUCO-PPID dendrimer may possibly enter into the cells through receptor-mediated endocytosis mechanism (Fig. 6). Results of cytokine assay showed that the incubation of J774 macrophages cells with LPS (10 lg/mL) for 48 h caused the appearance of IL-12 p40 in the culture supernatants. However, treatment of the cells with sulfasalazine (SSZ) and various dendrimers formulations suppressed IL-12 p40 production in a dose-dependent manner [40]. On comparing dendrimers formulations more significant % inhibitions were observed with SSZ–FUCO-PPID as compared to SSZ-PPID and free SSZ. This may be ascribed to the higher cellular uptake due to the receptor-mediated internalization of SSZ–FUCOPPID (Fig. 7). Phosphorylation of IjB, an important step in NF-jB activation, is mediated by IKK kinase and the presence of phosphorylatedIjB (p-IjB) in cell lysates of J774 cells is the indication of NF-jB activation. Western blot analysis showed a clear band (between 50 and 37 kDa) of p-IjB with control (J774 cells incubated with LPS only). Chen et al. also reported the band of p-IjB between 46 and 30 kDa in Western blot analysis [49]. However band intensities were found to be decreased when cells were treated with SSZ and SSZ loaded dendrimers and MWCNTs. The decreased band intensities are possibly due to the inhibition of NF-jB activation. There was less or no production of p-IjB, thus no band was observed, when cells were treated with SSZ–FUCO-PPID. Disappearance of

Fig. 5. % Cell viability of free SSZ, PPID, SSZ-PPID and SSZ–FUCO-PPID dendrimers formulations. Values represented as mean ± SD (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

455

Fig. 6. Fluorescence uptake of the dendrimers formulations (n = 3) p 6 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

band suggested the complete inhibition of NF-jB activation (Fig. 8). The blood level study was carried out following i.v. administration of developed formulations to study the effect of presence of macromolecules on the release profile and retention of dendrimer formulations in systemic circulation. The concentration of drug was determined in blood plasma samples at various time intervals (Fig. 9). The release of SSZ from the dendrimers formulations was found to be sustained in vivo also. The pharmacokinetic data show that the formulation had a slightly lower Cmax (143 ± 0.53 lg/mL) in case of SSZ–FUCO-PPID as compared to free SSZ (168.01 ± 0.93 lg/mL) (Table 1). The elimination rate constant (kel) of the SSZ loaded fucosylated dendrimers was determined to be 0.08 h1, which was lower than non-fucosylated PPI dendrimers (0.12 h1) and free SSZ formulation (0.14 h1). The lowering of kel of SSZ delivered through the fucosylated dendrimeric formulation as compared to free SSZ clearly suggests its slow elimination from the body. These results are in agreement with other pharmacokinetic parameters obtained in this study. The area under the first moment curve (AUMC0–1) was calculated and found to be 7337.83, 10161.99 and 18398.92 lg h2/mL for free SSZ, SSZ-PPID and SSZ–FUCO-PPID dendrimers formulations, respectively. The AUMC(0–1) of SSZ–FUCO-PPID dendrimers formulation was nearly two-fold that of SSZ-PPID. The mean residence time (MRT) of the SSZ–FUCO-PPID (12.15 h) was found to be increased significantly in comparison with SSZ-PPID (8.64 h) and free SSZ (7.30 h). The increased MRT suggested the sustained release profile of SSZ from the fucosylated PPI dendrimers as compared to free SSZ-PPID and free SSZ. Previously our laboratory has reported the MRT, AUC(0–t), AUMC(0–t) and Kel of the doxorubicin loaded dextran conjugated PPI dendrimers (PAD-PPI-DOX) to be 7.59 h, 35.530 lg h/mL and 269.438 lg h2/mL, respectively [46]. It indicates that the metabolism (and hence excretion) has been

Fig. 7. % Inhibition of IL-12 (IL-12p40) level in macrophage cell line J774 by dendrimers formulations and free SSZ. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 8. Determination of phosphorylated-IjB (p-IjBa) protein in LPS activated macrophage cells by Western blot analysis at 48 h incubation time.

Fig. 9. Comparative plasma drug concentration profile of free SSZ, SSZ-PPID, and SSZ–FUCO-PPID formulations. Values represented as mean ± SD (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

escaped in significant manner by entrapping SSZ in the SSZ–FUCOPPID system as compared to the free SSZ. The plasma concentration vs time curve also reveals an increased residence time of SSZ-PPID (8.64 h) and the reason for this may be the same as for the SSZ–FUCO-PPID. Biodistribution study was performed to evaluate targeting efficiency of SSZ from different formulations. The concentration of SSZ in different organs was estimated via HPLC [42] at 1st, 6th and 24th h. The results indicated that the free SSZ accumulated progressively in liver, where up to 30.25 ± 0.56% of dose was localized after 1 h of administration but after 24 h only 4.54 ± 0.12% and 2.70 ± 0.19% of SSZ were found in liver and spleen, respectively. The amount of SZZ in body depends upon its distribution, metabolism and excretion. Initially, the free amount of SSZ in liver was highest but declined consequently in case of free SSZ, might be due to the rapid elimination from liver (prime site of its action). However in case of SSZ-PPID dendrimeric formulation the SSZ was found to be primarily accumulated in liver and spleen and reduced from 24.10 ± 0.63% at 1 h to 10.90 ± 0.29% at 24 h in liver

Fig. 10. SSZ levels attained in different organs after administration of free SSZ, SSZPPID, and SSZ–FUCO-PPID formulations. Values represented as mean ± SD (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(Fig. 10). Thus higher uptake of formulations in macrophage rich organs must be ascribed to the RES uptake. But in case of SSZ– FUCO-PPID, the drug was primarily found to be accumulated in liver and spleen that may be due to the receptor-mediated endocytotic uptake of fucosylated dendrimer formulations by macrophages. In this case, the drug in liver at 1st and 24th h was found to be 45.28 ± 0.78% and 35.73 ± 0.86%, respectively of initial dose. In spleen, the drug at 1st h was found to be 16.90 ± 0.44% and 18.89 ± 0.51% of initial dose, which was increased up to 22.78 ± 0.35% for SSZ–FUCO-PPID. At 24th h the drug in spleen was found to be 4.08 ± 0.12% and 7.94 ± 0.23% of initial dose. So, when this data were compared with the data obtained from free drug solution and SSZ-PPID it was inferred that fucosylated dendrimer was highly accumulated in macrophage rich organs and provided a sustained release of drug. Yeeprae et al. reported similar results of accumulation of mannosylated system in macrophage rich organs [50]. The biodistribution studies clearly establish the superiority of the SSZ–FUCO-PPID, when compared against the plain drug toward increasing the accumulation of SSZ in the liver. The data can be well correlated for dendrimers with the study where glycodendrimeric primaquine entrapped nanoparticles were used by Bhadra et al. for disposition of drug to liver only [51,52]. The blood parameters like RBCs, WBCs and differential lymphocytes count were evaluated to assess the relative effect of different drug loaded formulations as compared to the free SSZ (Table 2). The RBCs count was found to be decreased below normal values more in case of SSZ-PPID than that of SSZ–FUCO-PPID. This result is correlated to more hemolyatic toxicity of SSZ-PPID as compared to SSZ–FUCO-PPID. The WBCs count of SSZ-PPID was found to increase significantly as compared to normal values. However, for SSZ–FUCO-PPID, the increase was less than that of unconjugated formulation and also less than the normal count in controlled group. Similarly, a relative increase in lymphocyte and neutrophils count was observed with SSZ-PPID, however in case

Table 1 Pharmacokinetic parameters of free SSZ, SSZ-PPID, and SSZ–FUCO-PPID formulations. Formulations

Free SSZ SSZ-PPID SSZ–FUCO-PPID

Parameters Cmax (lg/mL)

AUC(0–1) (lg h/mL)

AUMC(0–1) (lg h2/mL)

t1/2 (h)

Kel (h1)

MRT (h)

168.01 160.70 143.8

1004.55 1176.77 1514.24

7337.83 10161.99 18398.92

5.06 5.98 8.42

0.14 0.12 0.08

7.30 8.64 12.15

Probability p < 0.01; standard deviation < 5%. Values represented as mean ± SD (n = 3). MRT = mean residence time; Cmax = maximum plasma concentration; t1/2 = half life; Kel = elimination constant; AUC = area under curve.

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R. Gupta et al. / European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 449–458 Table 2 Hematological parameters of albino rats treated with SSZ and SSZ loaded dendrimers formulations. Formulations

Hematological parameters RBCs count (106/lL)

Control Drug (SSZ) SSZ-PPID SSZ–FUCO-PPID

9.1 ± 0.10 7.70 ± 0.19 6.49 ± 0.50 8.96 ± 0.59

WBC count (103/lL)

10.30 ± 0.44 11.66 ± 0.42 13.66 ± 0.64 10.45 ± 0.83

Differential counts (103/lL) Monocytes

Lymphocytes

Neutrophils

1.69 ± 0.12 1.57 ± 0.06 1.43 ± 0.12 1.30 ± 0.22

7.71 ± 0.55 8.73 ± 0.15 11.50 ± 0.50 8.70 ± 0.44

1.47 ± 0.15 1.77 ± 0.05 2.03 ± 0.25 2.53 ± 0.47

Hb (g/dL)

MCH (pg)

HCT %

13.09 ± 0.69 11.28 ± 0.39 9.53 ± 0.61 12.87 ± 0.76

18.41 ± 0.42 16.23 ± 0.29 14.16 ± 1.05 17.53 ± 0.50

43.59 ± 0.66 36.03±.80 30.30 ± 0.82 43.16 ± 0.76

#Values represented as mean ± SD. (n = 3).

of SSZ–FUCO-PPID, the increase was lesser. This could be attributed to the fact that the cell cytotoxicity of SSZ-PPID was increased due to its presence of poly-cationic charge, which could lead to increased level of lymphocytes as well as neutrophils. Levels of Hemoglobin, MCH and %HCT were found to decrease more in case of unconjugated than that of conjugated (fucosylated) formulations. Data obtained from this study can be well correlated with earlier reports [19,20]. 4. Conclusion PPI dendrimers as emerging nano-vectors for sustained, controlled and targeted theragnostics delivery are yet under investigation for their clinical efficacy. The present study concludes that the surface modified PPI dendrimer presents biocompatible framework, exhibits reduced toxicity, and provides a sustained drug release behavior in vitro as well as in vivo. SRB assay, macrophage uptake assay, fluorescence microscopy, pharmacokinetic and biodistribution data demonstrated that fucosylated dendritic nanoconjugate has potential to deliver significantly higher amount of drug (SSZ) to liver for higher therapeutic outcomes. The result of hemolytic toxicity and hematological studies revealed that fucose conjugation can be utilized to reduce the toxicity associated with unconjugated dendrimers. Cytokine assay also suggested that SSZ–FUCO-PPID exhibits significantly higher inhibition of IL-12 p40 secretion. Western blot assay suggested that SSZ–FUCO-PPID could significantly inhibit NF-jB activation, which has become one of the major targets in product development. The activation of NF-kB is emerging as one of the major mechanism of tumor cell resistance to cytokines and chemotherapeutics agents in the treatment of cytokine-induced liver damage or in numerous cancers. Overall we can conclude that sulfasalazine loaded fucose conjugated PPI dendrimers (SSZ–FUCO-PPID) hold enormous potential as site-specific, targeted, safe, therapeutically more effective drug delivery system for Kupffer cell targeting for the treatment of cytokine-induced liver damage. Kupffer cells play an important role in inflammatory liver signaling. Use of specific inhibitors that can block NF-jB activation should be beneficial in improving the cancer therapy/cytokine-induced liver damage. Acknowledgment The authors Richa Gupta and Neelesh Kumar Mehra would like to acknowledge the University Grants Commission (UGC), New Delhi, India for financial assistance in the form of Junior Research Fellowship (JRF). References [1] N.K. Mehra, V. Mishra, N.K. Jain, Receptor based therapeutic targeting, Ther. Deliv. 4 (3) (2013) 1–26. [2] S. Mignani, S.E. Kazzouli, M. Bousmina, J.P. Majoral, Expand classical drug administration ways by emerging routes using dendrimer drug delivery systems: a concise review, Adv. Drug Deliv. Rev. (2013), http://dx.doi.org/ 10.1016/j.addr.2013.01.001.

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Development and characterization of sulfasalazine loaded fucosylated PPI dendrimer for the treatment of cytokine-induced liver damage.

The present investigation was aimed at exploring the targeting potential of sulfasalazine (NF-κB inhibitor drug) loaded fucose tethered poly (propylen...
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