DEVELOPMENT AND PERFORMANCE EVALUATION OF TUMOR TARGETING POTENTIAL OF FOLATE SPACER FUNCTIONALIZED SOLID LIPID NANOPARTICLES

Methods: The synthesized nanoconjugates (SLNFA, SLN1FA, and SLN4FA) were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, and transmission electron microscopic studies. Nanoconjugates were evaluated for entrapment, in vitro drug release (under various pH conditions) and hemolytic studies. Cell uptake and cytotoxicity studies were performed on human malignant cell lines (MCF-7) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: This study explored the effect of PEG spacer length on the targeting potential of folate-conjugated SLN. PTX entrapment and in vitro drug release from nanoconjugates augmented, and hemolytic toxicity of nanoconjugates slashed with the molecular weight of PEGs. Further, nanoconjugates with PEG 4000 displayed highest tumor-targeting potential as compared to other spacer conjugated nanoconjugates due to optimized steric hindrance and receptor mediated endocytosis among other PEGs. Conclusion: Engineering of the dendritic surface with targeting ligand such as FA can enhance the site-speciﬁc anticancer drug delivery. PEGylation of SLN can improve the circulation time of SLN in the body. This is a debut study reporting FA conjugation to the surface through four PEGs as spacer and optimized the spacer chain length for effective cancer targeting through SLN. This report as a whole is believed to shed new light on the role of spacer chain length in targeting potential of folate-anchored SLN. Hemolytic toxicity, Cytotoxicity, Cancer, Polyethylene glycols as spacer. Objective : The objective of the present investigation was to assess the tumor-targeting potential of ligand-spacer engineered solid lipid nanoparticles (SLN) as nanoscale drug delivery units for site-specific delivery of a model anticancer agent, paclitaxel (PTX). SLNs were engineered by direct and indirect conjugation of folic acid (FA) through different types of polyethylene glycols (PEGs) (MW: 4000) as spacers.


INTRODUCTION
According to the global cancer report issued by the 48 World Health Organization (WHO), cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries, and deaths from cancer worldwide are projected to continue rising, with an estimated 12 million deaths in 2030 [1]. Although there has been potential progress in the prevention, detection, and treatment of cancer over the past 50 years adequate therapy remains elusive due to late stage diagnosis, inadequate strategies for addressing aggressive metastasis, and lack of clinical procedures for overcoming multidrug-resistant (MDR) cancer [2,3]. MDR can be intrinsic (inherent) or acquired through chemotherapy exposure [4]. The development of MDR contributes to the persistence of the disease in spite of high dose and combination chemotherapy, which is often the last treatment option [5]. However, this often leads to toxic side effects and poor clinical outcomes. The majority of clinically approved chemo 68 motherapeutic agents target cell growth patterns and are not selective for cancer cells [6]. Nanotechnology offers an unprecedented opportunity in rational delivery of drugs and genes to solid tumors [7] following systemic administration due to their unique accumulation behavior at the tumor site [8,9]. Examples of nanotechnology applied in pharmaceutical product development include nanoparticles, dendrimers, and liposomes [10]. Targeted anticancer drug delivery includes drug delivery by avoiding reticuloendothelial system 80 (RES) [11], utilizing the enhanced permeability and retention effect and tumor specific targeting [12]. Nanoparticles made from solid lipids are attained major attraction as novel drug carrier for intravenous and nasal application as they have been proposed as an alternative particulate carrier system [13]. The solid lipid nanoparticles (SLNs) are sub-micron colloidal carriers (50-1000 nm), which are composed of physiological lipid dispersed in water or in an aqueous surfactant solution [14]. SLNs as colloidal carriers for drugs combine the advantage of polymeric nanoparticles, fat emulsions and liposomes simultaneously and avoiding some of their disadvantages [15]. They have been widely used for controlled drug delivery through intravenous, transdermal, ocular, and oral administration routes. The first study on SLNs for paclitaxel (PTX) delivery was reported by Miglietta et al. in 2000 [16]. Developed tripalmitin-based SLNs stabilized by soya phosphatidylcholine, with average diameter below 500 nm, with proven stability in isotonic glycerol solution. Encapsulation or conjugation of bioactive molecules in these functional solid lipid nanoparticles may reduce their toxicity, enhance their solubility, and prolong blood clearance. PEGylation is effective approach for drug delivery because of the escape from the recognition by the RES. Different types of PEGylated solid lipid nanoparticles using polyvinyl chloride have already been reported. Recently, conjugation of targeting ligand through polyethylene glycols (PEG) as spacer considered as most potential approach to improve the targeting properties of SLNs was synthesized folate PEGmodified SLNs nanoconjugates as a drug delivery system. The role of PEG chains as spacer show important potential with targeting ligand employed for the functionalization of surface groups [17]. Thus, PEG enhances targeting in addition to enhancing solubilization of bioactive and reducing immunogenicity and toxicity of the carrier, which is a property of critical importance for drug delivery systems [18]. Furthermore, the introduction of a folate group at the end of PEG chains conjugated to solid lipid nanoparticles induces tumor-targeting potential to the carrier due to its receptor mediated endocytosis. Folate as ligand for tumor targeting has already applied with liposomes, nanoparticles, and dendrimers 137 Folate-based cancer targeting is an established

Singh et al.
concept and solid lipid nanoparticles have been widely explored for targeting employing folate as ligand [19]. In this line, different reports on usage of various PEGs as spacer are available but effect of spacer chain length on targeting potential is lacking. Furthermore, the importance of spacer in cancer targeting has been well accepted. Steric hindrance as well as ligand presentation is believed to be a pre-requisite to achieve ligand receptor-based cancer targeting [19]. The purpose of the present investigation was to compare the tumor-targeting potential of ligand and ligand-spacer-engineered solid lipid nanoparticles nanoconju1gates as nanoscale drug delivery units for site-specific delivery of a model anticancer agent, Paclitaxel 1 (PTX). In the present study, SLN-spacerfolate nanoconjugates were synthesized with PEGs (Mw: 1000, 4000) which in addition to the protection exercised by the PEGs chains was also functionalized by a folate targeting ligand at the end of the one PEG chain with each nanoconjugates. In this manner, the folate group, due to its location at the distal end of PEG chain, is accessible to interact with complementary receptor. In addition, a folate-SLN nanoconjugate has also been synthesized to be used as a control to compare the tumortargeting potential of synthesized nanoconjugates [20]. This work is expected to throw new light on the role of spacer chain length in targeting potential of folate-anchored SLNs [21].

Materials
PTX was received gift sample from Sun Pharmaceutical Industries Ltd, Gujarat, (India). Soya Phosphatidyl Choline was purchased from HiMedia. Reagents and solvents analytical grade were purchased from local suppliers unless stated otherwise. Deionized water was used throughout the study.

Preparation of SLNs
Preparation of plain SLNs was carried out employing solvent injection method, which involves the rapid diffusion of solvent across the solventlipid phase into the aqueous phase. Tristearin, PC, stearylamine, and drug were dissolved in ethanol, maintained at an elevated temperature of 70°C with continuous stirring. This solution was injected into an aqueous solution of Tween 80 maintained at the same temperature given above with continuous stirring and sonicated under probe sonicator. The prepared nanoparticulate formulation was optimized for various parameters such as lipid lecithin ratio, drug-lipid ratio, surfactant ratio, stirring time, stirring speed, and sonication time to obtain nanosized SLNs with maximum drug entrapment. Lipid employed in the production of SLNs was first subjected to optimization by varying the ratio of tristearin: PC from 1:0.5 to 1:2, keeping tristearin quantity as constant [22,23] Tables 1 and 2. (CDI). This debut study is described in detail through Scheme 1 and 2 (a and b). All synthesized nanoconjugates with conjugating agents. The conjugation of FA on SLN was accomplished using two methods; by direct conjugation on periphery and through different PEGs as spacer effect of conjugation on the absorption maxima of SLNs is described in Alshubaily et al. [20].

Determination of drug content and entrapment efficiency
Drug entrapment of the PTX in SLNs was determined by dispersing the known molar concentration of PTX loaded SLNs in cellulose dialysis bag (MWCO 1000 Da, Sigma, Germany). This solution was dialyzed with the help of magnetic stirring (50 rpm; Remi, Mumbai, India) in cellulose dialysis bag against PBS (pH 7.4) under sink condition for 10 min to remove any unentrapped drug from the formulation. One milliliter aliquot was withdrawn and diluted 10 times in a volumetric flask with 30:70 methanols: PBS (pH 7.4). Absorbance was measured spectrophotometrically (Shimadzu, 1800 Japan) at 237 nm to indirectly estimate the amount of drug entrapped within the system. The dialyzed formulation was then lyophilized and further characterized. The amount of drug entrapped in folate coupled SLNs was also determined by employing the similar procedure as reported for PTX loaded SLNs [24].

Particle size determination
The average particle size and size distribution of the SLNs were determined by photon correlation spectroscopy using a Zetasizer DTS ver. 4.10 (Malvern Instrument, UK). The samples of SLN dispersions were diluted to 1:9 v/v with deionized water. The particles size and size distribution were represented by average (diameter) of the Gaussian distribution function in the logarithmic axis mode [25] (Table 3).     (Fig. 1).

Particle morphology (transmission electron microscope [TEM])
TEM was used as a visualizing aid for particle morphology. The sample (10 µL) was placed on the grids and allowed to stand at room temperature for 90 s. Excess fluid was removed by touching the edge with filter paper. All samples were examined under a TEM (Philips Morgagni 268, Eindhoven, Netherlands) at an acceleration voltage of 100 kV, and photomicrograph [27].

Microscopic study
Microscopic study of synthesized nanoconjugates SLNFA, SLNP1FA, and SLNP4FA was performed to characterize their size. TEM of the prepared nanoconjugates was done to characterize the systems after drying on 3 mm form an (0.5% plastic powder in amyl acetate) coated copper grid (300 mesh) at 60 KV (Philips Morgani, 268D; TEM [26], Holland) after staining negatively using uranyl acetate (4%) and photomicrographs were taken at suitable magnifications at Electron Microscopy Section of AIMS, Delhi (Fig. 3).

Nuclear magnetic resonance ( 1 H NMR) spectroscopy
The synthesized nanoconjugates SLNFA, SLNP1FA, and SLNP4FA were analyzed by 1 H NMR spectroscopy at NIPER, Hyderabad. The nanoconjugates were solubilized in D 2 O and analyzed at 300 MHz by 1 H NMR Spectrometer model Avance-II (Bruker, Germany). Various shifts and peaks were observed, which were interpreted for different groups (Fig. 2).

In vitro drug release studies
In vitro drug release profile of entrapped PTX from SLNs, SLNFA, SLNP1FA, and SLNP4FA was studied using dialysis tube. The SLN formulations were first separated from free drug by passing through sephadex G-50 column. The separated SLN formulation (5 ml) was taken in to dialysis tube (molecular weight cutoff 10,000 Da) and placed in a beaker containing 100 ml of phosphate buffer (pH 7.4). The beaker was placed over a magnetic stirrer and the temperature of the assembly was maintained at 37±1°C throughout the study. Samples were withdrawn at definite time intervals with replacement by same volume of phosphate buffer. The withdrawn samples were analyzed for   drug content by measuring absorbance at 237 nm against blank [28] ( Fig. 4, Table 4).

Drug content and entrapment efficiency
Drug entrapment was determined by dialysis using a dialysis membrane and was found to be 79.3±0.5%. Keeping all optimization parameters under consideration, entrapment was found to be optimum in the selected formulation. A comparison was made between drug entrapment, surface morphology, particle size, and polydispersity index (PDI). Particle size (201.1±3.7 nm) of PTX loaded plain SLNs formulation was smaller than PTX loaded folate coupled SLNs and it was found to be 249.4±2.6 nm this may be due to formation of extra layer over the SLN surface due to the coating of folate on the surface of SLNs. PDI of plain formulations was also lower (PDI=0.234) than that of coupled formulation (PDI 0.283) this may be due to coupling of the SLN with the folate which disturbed the surface of the SLN. The drug entrapment efficiency of plain SLN was 79.3±0.5% which is more as compared to coupled SLN which having drug entrapment efficiency 71.7±0.5%, this may be because of the leaching out the drug form the SLN during incubation period (Table 3).

FT-IR spectrum
In FT-IR spectrum of SLNP1FA and SLNP4FA, C-O stretch (ether linkage) strong, and sharp peak was obtained at 1114.71 cm -1 1107.21 cm -1 due to polyether backbone of PEG, CH-NH-C(=O) amides bending peaks at 1462.46, 1415.88 cm -1 , and 1458.03 cm -1 due to amide bond formation between hydroxy group of PEG 1000 and 4000 and amine groups of SLN through CDI conjugation mechanism, Esters C-O stretching peaks at 1228.75, 1192.07 cm -1 and 1298.69, 1250.31 cm -1 confirmed the ester bond formation between the free hydroxy group of PEG 1000 and 4000 to carboxylic group of FA through DCC/DMAP conjugation protocol, and remaining peaks of aromatic compounds were indicated presence of FA (Fig. 1).

H NMR spectroscopy
The 1 H NMR spectrum and shifts of SLNFA as compared to that of simple SLNs furnish the proof of conjugation. In SLNFA newer peak of R-(C=O)-NH-CH 2 -CH 3 of FA linkage appeared at 1.0-2.0 ppm. Similarly, SLNP1FA and SLNP4FA nanoconjugates exhibit characteristic peak of amide bond carbonyl proton and amide bond nitrogen proton appeared at 7.1-7.9 ppm and 3.0-3.4 ppm, and characteristic peak of ester bond formation between PEG and FA appeared at 3.7-4.1 ppm in contrast to SLN 1 H NMR spectrum [29] (Fig. 2).

Microscopic study
The electron microscopic analysis of nanoconjugates proves them to be as nanometric sized vesicles, which was evident by TEM photograph. The data revealed the increase in particle size on folate conjugation, directly and through PEGs spacer (Fig. 3). The size of nanoconjugates slightly increased with molecular weight of PEGs and the order of size of nanoconjugate were found to be SLNP4FA> SLNP1FA> SLNFA> SLN.

Drug release study
The release of drug PTX from SLN nanoformulations was monitored by a dialysis method. The dialysis was carried out at 37°C using dialysis membranes with molecular weight cutoff of 10 kDa and phosphatebuffered saline (pH 7.4) as the sink solution. The concentration of drug was analyzed at various time points during the dialysis process to obtain the in vitro drug release profile. In vitro drug release data of PTX associated with nanoconjugates is presented in Fig. 4. Moreover, the release patterns of SLNP1FA and SLNP4FA were sustained with increasing chain length of PEGs (Mw: 1000, 4000) in both release conditions. This may be attributed to the surface engineering of folate and folate PEGs as it led to more sealing at the nanometric periphery and hydrophobic interactions, which delayed the drug release. The rate of drug release of nanoconjugates was observed to be low due to close structure and steric hindrances due to large chain length of PEGs as spacer. Hence, folate attachment through PEG's spacer on SLNs can be proposed for better sustained and controlled release drug delivery system (Table 4).

Stability testing
The storage stability testing indicated that plain SLN and coupled SLN formulations stored at 4±1°C were more stable than those stored at room temperature. Average particle size of nanoparticles was found to increases on storage, which can be due to the aggregation of particles at different storage conditions. This effect was least in the case of formulations stored at 4±1°C, which indicates that the aggregation can be regulated by temperature and ideal storage condition being at 4±1°C.
The different SLN formulations were stored at 4±1°C and room temperature, and the percent of residual drug content was calculated after 30, 60, and 90 days by assuming the initial drug content to be 100%. Percent residual drug content of formulations was found to be 95.2±1.3% at 4±1°C and 90.3±2.4% at room temperature after 180 days in formulation plain SLN and 96.8±2.0% at 4±1°C and 93.4±2.1% at room temperature after 90 days in formulation coupled SLN . The residual drug content of formulations stored at room temperature was found to be lower in comparison to formulations stored at 4±1°C, which indicated that the formulations tend to degrade more at higher temper (Table 5).
SLNP4FA was responsible for higher uptake and target ability due to receptor specific targeting of SLNs due to surface conjugation of FA through PEG (Mw: 4000) as spacer, folate enhanced the receptor mediated endocytosis of nanoformulations and PEGs used as a spacer to couple ligand (FA) moieties to surfaces of nanometric carrier thus potentially provide both "bioinert" and "bioactive" functions to nanoformulations. Based on obtained IC 50 value of synthesized nanoconjugates arranged for target ability: SLNP4FA>SLNP1FA>SLNFA.
Cell topographic study of PTX and PTX loaded nanoformulations (SLN, SLNFA, SLNP1FA, and SLNP4FA) were performed with a view to assess the ability of different drug loaded formulations to target human mammary carcinoma cells, MCF-7 ( Fig. 9.2 to 9.8). Similar to the results of the percent cell growth inhibition assay, cell topographic studies also displayed higher uptake of folate-spacer conjugated nanoformulations contrast with plain PTX and SLNFA (Fig. 6). The higher uptake was possibly due to the conjugation of folate through PEGs as spacer on the surface of the SLN as compared to direct conjugation of folate on the surface of the SLN. This might be due to higher drug loading, resulting in a higher release. Among SLNP1FA and SLNP4FA, the highest uptake was observed with the SLNP4FA because PEG (Mw: 4000) exhibits more tumor accumulation. The results support the strategy that optimized spacer chain length for effective tumor targeting can provide higher uptake of anticancer bioactive to the cancer cells [30].
This study is in agreement that the SLNFA and folate-spacer (SLNP1FA and SLNP4FA) nanometric nanoformulations entered into the cancer cell by receptor mediated endocytosis (folate receptors are overexpressed in MCF-7 cell line). Singh et al. previously reported the direct and indirect conjugation of folate through one spacer (PEG Mw: 4000) on the surface of nanocarriers. This is the 1 st time conjugation of folate directly and indirectly using two spacer (PEGs Mw: 1000 and 4000) with the SLN was performed and assessed the tumor targeting potential against MCF-7 cell lines. In vitro MTT and cell uptake assay concluded that SLNP4FA bears significant tumor targeting potential as compared to free PTX and PTX loaded nanoformulations (SLNFA and SLNP1FA). Based on obtained results tumor targeting potential can be ranked as follows [31]: SLNP4FA>SLNP1FA>SLNFA. Singh et al.

CONCLUSION
Engineering of the dendritic surface with targeting ligand such as FA can enhance the site specific anticancer drug delivery. PEGylation of SLN can improve the circulation time of SLN in the body. In the present study, combination of both of these approaches displayed promising results in terms of tumor-targeting potential. The present study was aimed to developed surface-engineered SLN by conjugation of folate directly or indirectly through different types of PEGs (Mw: 1000, and 4000) as spacers with more biocompatibility and less toxicity, but at the same time, the ability to deliver drug at the desired site of action. The reduced toxicity and enhanced tumor-targeting potential with SLN4FA can be used most suitably as a controlled and targeted drug delivery system for the delivery of anticancer drugs like PTX. The nanoformulations can be given through the intravenous route as a long-term circulatory nanoparticulate system. This is a debut study reporting FA conjugation to the surface through four PEGs as spacer and optimized the spacer chain length for effective cancer targeting through SLN. This report as a whole is believed to shed new light on the role of spacer chain length in targeting potential of folate-anchored SLN.