Synthesis, characterization and radiolabeling of folic acid modified nanostructured lipid carriers as a contrast agent and drug delivery system

Author’s Accepted Manuscript Synthesis, characterization and radiolabeling of folic acid modified nanostructured lipid carriers as a contrast agent and drug delivery system Eser Ucar, Serap Teksoz, Cigdem Ichedef, Ayfer Yurt Kilcar, E. Ilker Medine, Kadir Ari, Yasemin Parlak, B. Elvan Sayit Bilgin, Perihan Unak www.elsevier.com/locate/apradiso

PII: DOI: Reference:

S0969-8043(16)30896-X http://dx.doi.org/10.1016/j.apradiso.2016.11.002 ARI7641

To appear in: Applied Radiation and Isotopes Received date: 17 March 2016 Revised date: 7 September 2016 Accepted date: 1 November 2016 Cite this article as: Eser Ucar, Serap Teksoz, Cigdem Ichedef, Ayfer Yurt Kilcar, E. Ilker Medine, Kadir Ari, Yasemin Parlak, B. Elvan Sayit Bilgin and Perihan Unak, Synthesis, characterization and radiolabeling of folic acid modified nanostructured lipid carriers as a contrast agent and drug delivery system, Applied Radiation and Isotopes, http://dx.doi.org/10.1016/j.apradiso.2016.11.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, characterization and radiolabeling of folic acid modified nanostructured lipid carriers as a contrast agent and drug delivery system Eser Ucar1, Serap Teksoz1*, Cigdem Ichedef1, Ayfer Yurt Kilcar1, E.Ilker Medine1, Kadir Ari1, Yasemin Parlak2, B. Elvan Sayit Bilgin2, Perihan Unak1 1

Department of Nuclear Applications, Institute of Nuclear Sciences Ege University, Bornova, 35100 İzmir, Turkey 2

Department of Nuclear Medicine, School of Medicine, Celal Bayar University, Manisa

*

Corresponding author. tel: +90-232-3113466, fax: +90-232-3886466. e-mail: [email protected]

ABSTRACT Nanostructured lipid carriers (NLCs) are the new generation of solid lipid drug delivery systems. Their suitability as contrast agents for gamma scintigraphy is an attracting major attention. The aim of current study was to prepare surface modified nanostructured lipid carrier system for paclitaxel (PTX) with active targeting and imaging functions. In accordance with the purpose of study, PTX loaded nanostructured lipid carriers (NLCs) prepared, modified with a folate derivative and radiolabeled with technetium-99m tricarbonyl complex (99mTc(CO)3+). Cellular incorporation ratios of radiolabeled nanoparticles (99mTc(CO)3-PTXNLC) were investigated in vitro on three cancer cell lines. Additionally in vivo animal studies conducted to evaluate biological behavior of 99mTc(CO)3-PTX-NLC on female Wistar Albino rats. Biodistribution results showed that the folate derivative modified 99mTc(CO)3-PTX-NLC had considerably higher uptake in folate receptor positive organs. The data obtained from present study could be useful in the design of biodegradable drug carriers of PTX and folate receptor based tumor imaging agents.

Keywords: Radiolabeling; paclitaxel; nanostructured lipid carriers; technetium-99m; cell culture; biodistribution

1. Introduction Cytotoxic drugs form an important part of chemotherapy in treatment of cancer. Poor pharmacokinetic profiles and low specificity of these drugs affect their therapeutic efficacy negatively (Wong et al., 2007). Designed nano-scale biomaterials as drug delivery systems are promising to minimize the side effects of cytotoxic drugs and to enhance their selective distribution. Major nanoparticle formulations reported as drug carriers are dendrimers, vesicles, micelles, carbon nanotubes (Goldberg et al., 2007). In recent years, lipid based nanoparticles have been described as an alternative carriers for poorly water-soluble and lipophilic drugs. They have low toxicity due to their physiological lipid compositions. Among lipid based nanoparticles, nanostructured lipid carriers (NLC) are categorized as a second generation lipid nanoparticles. NLCs have the advantages of active compound protection, high bioavailability and the controlled release of the low soluble drugs (Ng et al., 2015). NLCs have imperfect matrix due to their solid and liquid lipid mixture composition. Thus prepared system provides improved drug loading capacity and prevents drug expulsion during storage (Hu et al., 2005). A drug delivery system with multiple features can be more effective and specialized system (Mussi and Torchilin, 2013). For this purpose, active targeting function can be achieved by addition of ligands to the system such as transferrin, arginine-glycine-aspartic acid (RGD) and folic acid. In many cases these ligand-receptor interactions end up with efficient uptake of carrier system by receptor-mediated endocytosis (Pirollo and Chang, 2008). Folate receptor (FR) has been studied widely as a targeting moiety which has very high affinity for folic acid. Because folic acid is internalized by FR-mediated endocytosis, it has

been utilized as a high specific targeting agent for the transport of attached therapeutic agents and carrier systems that do not normally enter tumor tissues. Folate-linked drug carriers bypass normal permeability barriers by FR-mediated endocytosis. Alpha isoform of folate receptor is overexpressed at high levels on human cancers. Among normal tissues, significant levels of FR alpha are found in proximal tubules of kidney. Function of FR in proximal tubules is to prevent loss of folic acid in the urine by transcellular reabsorption (Low and Kularatne, 2009). FR in kidney could be used for targeting drugs to proximal tubular cells (Dolman et al., 2010). Different folate conjugates have been attached to drug carrier systems via a PEG spacer by other research groups. Prolonged circulation times and effective target binding advantages were indicated for these systems (Lee and Low, 1995; Guo et al., 2000; Xiang et al., 2008; Gao et al., 2013). Nanoparticles can be designed to use in molecular imaging to prepare a multifunctional system. Optical properties (e.g., quantum dots) or magnetic properties (e.g., iron oxide nanoparticles) of nanoparticles can be useful for imaging.

Nevertheless lipid based

nanoparticles do not produce signals by themselves (Ferro-Flores et al., 2014). Radiolabeling of lipid nanoparticles has been studied for various purposes such as determination of pharmacokinetics, transfer of imaging or therapy agents. In diagnostic nuclear medicine, technetium-99m (99mTc) is an ideal radionuclide due to its appropriate half-life (6 h.) and gamma energy (140 keV). Tiwari and Pathak radiolabeled two lipid based nanoparticle formulations with

99m

Tc and compared them according to their pharmacokinetics,

biodistribution and drug release characteristics. They stated that nanostructured lipid carriers (NLC) have more advanced features over solid lipid nanoparticles (SLN) and oleic acid is the major factor to improve characteristics, pharmacokinetics and biodistribution of nanoparticles (Tiwari and Pathak, 2011).

Paclitaxel

(PTX),

(2α,4α,5β,7β,10β,13α)-4,10-Bis(acetyloxy)-13-{[(2R,3S)-3-

(benzoylamino)- 2-hydroxy-3 -phenylpropanoyl] oxy}-1,7 di hydroxy -9-oxo-5,20-epoxytax11-en-2-yl benzoate, is a typical and commonly used anticancer agent. It is used for the treatment of breast and ovarian cancer. Injectable form of PTX is composed of a 1:1 mixture of ethanol and Cremophor EL (polyethoxylated castor oil) which has reported serious side effects. In the past two decades, many alternative drug carrier systems have been investigated to prepare a water-soluble formulation and to avoid these adverse effects. Nevertheless, an active targeted delivery system for PTX is still needed (Feng and Mumper, 2013; Kollipara et al., 2010). The goal of this work was to construct a surface interacting with FR overexpressed tissues for PTX loaded NLCs. For this purpose, folate-polyethylene glycol-cholesterol hemisuccinate (Fol-PEG-CHEMS) was synthesized. Then, paclitaxel loaded folate modified nanostructured lipid carriers (PTX-NLC) prepared and radiolabeled with

99m

Tc(CO)3+. Targeting efficacy of

PTX-NLC evaluated in cell culture by fluorescence microscopy and incorporation assays. Furthermore, tissue distribution of nanostructured lipid carriers encapsulating paclitaxel was evaluated in vivo by biodistribution and gamma camera imaging studies. 2. Materials and Methods The human breast tumor cell line (MCF7) and human epithelial cervix adenocarcinoma cell line (HeLa) were obtained from the American Type Culture Collection (Rockville, MD, USA). Adenocarsinoma human alveolar epithelial cell line (A-549) was obtained from Bioengineering Department of Ege University (İzmir, Turkey). Female Wistar Albino rats were purchased from Kobay D.H.L.A.Ş. (Ankara, Turkey). Chemicals were generally reagent grade from Sigma-Aldrich Chemical Co. Water was purified by filtration through a WatersMillipore purification system (Milli-Q Gradient A-10; Milli-Q; Millipore S.A., Molsheim, France) with conductivity of 18 MX cm. Na99mTcO4 was obtained from a

99

Mo/99mTc

generator (Celal Bayar University, Faculty of Medicine, Manisa, Turkey) using 0.9% saline. Radioactivity was detected using Cd(Te) detector equipped with a RAD 501 (Turkey) singlechannel analyzer. Bioscan AR-2000 radioanalyzer was used to scan plastic-backed cellulose thin layer radiochromatography (TLRC) strips (Merck-105565) for quality controls. High performance liquid chromatography (HPLC) system consists of a Shimadzu quaternary gradient pump (Model LC-10Atvp Shimadzu), an automated syringe injector (20 µL loop) on a 5 µm reverse phase coloumn (ODS-2 HYPERSIL Dim.(mm)250x4.6). Nuclear Magnetic Resonance (NMR) data were taken in Faculty of Science, Ege University using 400 MHz liquid MERCURYplus-AS 400 model NMR. Zeta potential and particle size measured by Malvern Nano ZS zetasizer for characterization of PTX-NLCs. Morphology of PTX-NLCs was investigated using FEI Tecnai G2 Spirit Bio(TWIN) model high contrast transmission electron microscope (TEM) operated at 120 kV. Cell images were taken using Olympus BX 53 Fluorescence microscopy. Thermo Scientific Varioskan Flash multimode reader was used for cell viability study. Sanyo incubator was used for in vitro study. The static imaging was performed using a gamma camera (Diacan Instruments) in Celal Bayar University, School of Medicine, Department of Nuclear Medicine. 2.1 Synthesis of Fol-PEG-CHEMS Folate-polyethylene glycol-cholesterol hemisuccinate (Fol-PEG-CHEMS) was synthesized by previously described method (Xiang et al., 2008). Tetrahydrofuran (THF) was dried for CHEMS-NHS synthesis. 109 mg cholesteryl hemisuccinate (CHEMS) was reacted with 52 mg N-hydroxysuccinimide (NHS) and 135 mg dicyclohexylcarbodiimide (DCC) in dry THF overnight at room temperature. At the end of the reaction dicyclohexyl urea was formed. The mixture was filtreted and the solid was washed with dry THF. THF was evaporated and the solid product was dried under vacuum. CHEMS-NHS was purified by recrystalization in 100% ethyl acetate. For the synthesis of folate-PEG-bisamine, 53 mg folic acid, 17 mg NHS

and 20 mg DCC were dissolved in a mixture of THF and dimethyl sulfoxide (DMSO). 335 mg PEG-bisamine and 0.25mmol (35µL) triethylamine (TEA) were added. The reaction mixture was allowed to proceed overnight at room temperature. THF was evaporated in vacuum line. The product was purified by Sephadex G-25 gel-filtration chromatography. 1 mL samples in each vial were lyophilized. 65 mg Fol-PEG-bisamine and 14 mg CHEMSNHS were dissolved in 15 mL CHCl3, and reacted overnight at room temperature for synthesis of Fol-PEG-CHEMS. The solvent was then removed and the residue was hydrated in 50 mM Na2CO3 to form Fol-PEG-CHEMS micelles. The micelles were then dialyzed against deionized water using a Cellu Sep dialysis membrane with a molecular weight cut-off (MWCO) of 6-8 kDa to remove low molecular weight by-products. The product Fol-PEGCHEMS was then dried by lyophilization. 1H NMR analyses were performed in DMSO to confirm the identity of the final product. 2.2 Preparation of paclitaxel loaded nanostructured lipid carriers (PTX-NLCs) NLCs are prepared by solvent diffusion method in aqueous system. 0.16 mmol stearic acid(SA) (47 mg), 0.02 mmol oleic acid(OA) (6 mg/7 µL), 0.001 mmol PTX (1.2 mg/0.2 mL) were dissolved in 2 mL ethanol and 2 mL acetone by ultrasonicator at 70 ºC. Firstly, 6 mg Fol-PEG-CHEMS, then SA-OA-PTX mixture was dispersed into 40 mL distilled water under agitation at 400 rpm in 70 ºC for 30 min. The resultant pre-emulsion cooled to room temperature. pH value was adjusted to 1.2 with 0.1 M HCl. After centrifugation at 10.000 rpm for 90 min, obtained precipitate re-dispersed into 3 mL distilled water and Poloxamer 188. The resultant dispersion was fast frozen by liquid nitrogen and stored under -85 ºC. Then the sample was lyophilized for 72 h. Zeta potential and particle size measured by dynamic light scattering (DLS) for characterization of PTX-NLCs. Morphology of PTX-NLCs was investigated using TEM operated at 120 kV.

Determination of drug content in NLCs. The mobile phase consisted of acetonitrile and distilled water (50:50, v/v) and flow rate was 1 mL/min. The detection wavelength was 210 nm. The drug loading (DL) and the entrapment efficiency (EE) were calculated from following equations (Yang et al., 2013). EE (%) = [(WT – WF ) / WT] x 100 DL(%) = [(WT – WF ) / WL] x 100 WT is weight of PTX added in system, WF is weight of free PTX in supernatant and W L is weight of lipids added in system. 2.3 Radiolabeling of Paclitaxel Loaded NLCs Synthesis of 99mTc(CO)3+. The method described by Alberto et al. was modified to prepare Tc(I)-tricarbonyl complex (Alberto et al., 1998). Synthesis of

99m

Tc(CO)3+ was performed directly from Na99mTcO4 in

physiological serum under 1 atm pressure of carbonmonoxide using sodium borohydride as reducing agent. Radiochemical purity of compound was determined by thin layer radiochromatography (TLRC) and high performance liquid radiochromatography (HPLRC) methods. Aluminum-backed silica gel coated strips were used as stationary phase and acetonitrile was used as developing media for TLRC method. Solvents used for HPLC method were consisted of 0.1% trifluoroacetic acid (TFA) (solvent A) and methanol (solvent B). The elution was induced by 100% A (3 min). Time program was used for elution of solvents. The eluent switched at 3 min to 75% A and 25% B and at 9 min to 66% A and 34% B, followed by a linear gradient from 66% A and 34% B to 100% B during the period from 9 to 20 min, which was followed by 100% B for 8 min before switching back to 100% A. The flow rate was 1 mL/min. 99m

Tc(CO)3 Radiolabeling of PTX-NLCs.

Radiolabeling parameters, incubation time, pH and PTX-NLC amount were optimized to obtain maximum labeling efficiency. Data regarding optimization study were given as supplementary material. 148 MBq of

99m

Tc(CO)3+ was mixed with PTX-NLC solution (210

µg / 210 µL) and incubated at pH 6, 80°C for 60 min. Quality control of radiolabeled system was determined by TLRC and HPLRC methods. Plastic-backed cellulose strips used as stationary phase and pyridine-acetic acid-water (3:5: 1.5, v/v) mixture was used as mobile phase for TLRC method. The same HPLC conditions were used as quality control of 99m

Tc(CO)3+. In vitro stability of PTX-NLC system was determined by incubating 500 µL of

99m

Tc(CO)3-PTX-NLC in saline at room temperature. Radiolabeling stability was analyzed by

TLRC at time intervals of 0, 60, 120, 180, 240 and 1440 minutes. 2.4 In Vitro Studies MCF7, HeLa and A549 cells which have different folate receptor expressions were used for cell culture studies. MCF7 and HeLa cells were grown in Eagle’s minimum essential medium supplemented with 2 mM glutamine, 1.5 g/L sodium bicarbonate, 10% fetal bovine serum (FBS), 0.1 mM nonessential amino acid, and 1 mM sodium pyruvate. A549 cells were cultured in Dulbecco’s minimum essential medium supplemented with 2 mM glutamine, 1.5 g/L sodium bicarbonate, 10% FBS, and 1 mM sodium pyruvate. In all experiments, cells were grown at 37 ºC in a humidified incubator equilibrated with 5 % CO2. The cells were maintained in exponential growth by sub culturing with trypsin- EDTA (0.25 % by w/v in Hanks’ balanced salt solution). Cells were then pelleted and re-suspended in cell medium. Cytotoxicity studies The 50% growth inhibition (IC50) values of studied cells for PTX and PTX-NLC was investigated by WST-8 assay procedure. In a 96-well plate MCF7, HeLa and A549 cells were seeded at a density of 105 cells per well in a volume of 0.1 mL and cultured at 37°C until

cells get confluent. Growth medium containing different concentrations of samples were prepared and added to the cells. Medium without cell and reagent was used as negative control. After 24 h incubation time with samples, % cell viability was determined spectrophotometrically by a microplate reader using 10 µL WST dye solution per well at 450 versus 690 nm. The tests were performed triplet and each experiment repeated six times. Negative control was regarded as zero absorbance. The results were calculated by using the following equation: [1- (measured absorbance value / absorbance value of negative control) x 100]. Time depended in vitro incorporation studies To investigate time dependent incorporation of radiolabeled PTX-NLCs, cells were incubated for 30, 60, 120 and 240 minutes with mediums including [99mTc(CO)3]+ and 99mTc(CO)3-PTXNLC (50 μCi / 500 μL per well). The cells were washed with 3 x 0.5 mL phosphate-buffered saline (PBS) and 500 μL RIPA lyse buffer solution was added. A portion of the lysed cell suspension (25 μL) was used to determine protein content via bicinchoninic acid assay. Consequently, binding efficiency percentage per cell was calculated by using protein values. Fluorescence imaging studies At first, for the FITC labeling of SLN-PTX, 1 mg SLN-PTX solution was prepared in 1 mL 0.1 M sodium carbonate buffer (pH: 9). Then 1 mg FITC was dissolved in 1 mL anhydrous DMSO. 50 µL of FITC solution was added to the SLN-PTX solution very slowly in 5 µL portions with continuous stirring. Obtained solution was incubated 8 hours at 4 ºC in the dark. 50 mM NH4Cl was added to the solution and incubated 2 more hours at 4 ºC. Unbound FITC was separated from the conjugate by gel filtration using Sephadex G-25 column. MCF7, Hela and A549 cell lines were cultured on chamber slide for fluorescence imaging studies. Cells were washed with PBS and medium with FITC labelled PTX-NLC was added

on the cells. Experiments were performed under dark conditions. 4,6-diamino-2-phenylindol (DAPI) staining was performed to image the cell’s nucleus. After 4 h incubation time cells were washed with PBS(x2) and images were taken using Fluorescence microscopy with green filter (520 nm). 2.5 Biodistribution Studies All animal experiments for

99m

Tc(CO)3 radiolabeled paclitaxel loaded folic acid derivative

(Fol-PEG-CHEMS) conjugated lipid nanoparticles were carried out according to the relevant instructions set by the Institutional Animal Review Committee of Celal Bayar University (No:20 / 06.03.2013). Biodistribution studies were performed on 6 weeks old 220-280g female Wistar Albino rats (n=3 for 30., 60., 120. and 240. minutes). Receptor blocking was done for 12 of rats by the intravenous injection of folic acid solution (50µg/0.1 mL) 10 minutes before the injection of 99mTc(CO)3-PTX-NLC. Then 0.2 mL of the 99mTc(CO)3-PTXNLC solution containing 60 µg of paclitaxel loaded lipid nanoparticles in water with an activity of 15-18 MBq was administered by intravenous injection. Rats were sacrificed by intraperitoneal sodium pentabarbital administration at intended time points. Selected organs were removed, weighed and counted by a Cd(Te) detector in order to determine the percentage of injected dose per gram of organ weight (%ID/g organ). 2.6 Gamma-imaging Studies Imaging studies performed on female Wistar Albino rats in two groups as normal (N) and receptor blocked (RB). Then 0.3 mL of the 99mTc(CO)3-PTX-NLC solution containing 60 µg of paclitaxel loaded lipid nanoparticles in water with an activity of 18.5 MBq was administered by intravenous injection Receptor blocking study was done by intravenous injection of folic acid solution (50µg/0.1 mL), 10 minutes before the

99m

Tc(CO)3-PTX-NLC

injection. The rats were anaesthetized intraperitoneally using a cocktail of ketamine (50

mg/kg) / xylazine (10 mg/kg). Images were taken at 30, 60, 90, 120, 180, 240 and 1440 minutes after the administration of 99mTc(CO)3-PTX-NLC. 2.7 Statistical Analysis Statistical significance was assessed with one-way analysis of variance and non-linear regression by the GraphPad program for cell culture studies. Differences in the mean values of the measured activities were evaluated statistically by the SPSS 13 program (Univariate Variance Analyses and Pearson Correlation). Probability values < 0.05 were considered significant. Pearson correlation was carried out among different organs for

99m

Tc(CO)3-PTX-

NLC. 3. Results and Discussion 3.1 Synthesis of Fol-PEG-CHEMS Fol-PEG-CHEMS was prepared as targeting molecule according to the literature (Xiang et al., 2008). 1H NMR analyses performed to confirm the identity of the product (DMSO, δ ppm): related peaks for the folate moiety 8.65(d), 7.63(d), 6.63(d), 4.49(d), 4.26(m), the PEG moiety 3.77(m), and the CHEMS moiety 5.31(bd), 2.26(m). Results of the 1H NMR analyses were compatible with the data in previously reported study (Xiang et al., 2008). 3.2 Preparation and characterization (Size, Morphology, Charge) of PTX-loaded NLCs As described before, oleic acid content is an important factor for the particle size and morphology of nanoparticles. A modified solvent diffusion method carried on and 30% oleic acid content was used to prepare NLCs (Hu et al., 2005). Particle size and polydispersity indices of prepared NLCs were bigger and decreased after homogenization by using sonicator. Intensity average hydrodynamic diameter and polydispersity indices of nanoparticles dispersed in water were obtained as 237.1 nm and 0.27 respectively (Figure 1a). Repulsive forces must be dominant for colloidal stability of colloidal drug delivery systems and zeta

potential value is desired to be above ±30 mV range (Tiwari and Pathak, 2011). In the present study negatively charged particles with -33.9 mV zeta potential proved colloidal stability of the system (Figure 1b). EE and DL results were 49% and 0.5% respectively. The transmission electron microscopy (TEM) images are displayed in (Figure 2). TEM images revealed round shape of nanoparticles and their size below 100 nm. Recently reported TEM studies also confirmed that nanostructured lipid carrier systems have a spherical shape and a size below 130 nm (Alam et al., 2014; Banerjee et al., 2014). 3.3 Radiochemical purity of 99mTc(CO)3+ and radiolabeling of PTX-NLCs. 99m

Greater than 90% radiolabeling efficiency was achieved for

Tc(CO)3-PTX-NLC. Both

99m

Tc(CO)3+ and radiolabeled NLCs were maintained at top portion of TLRC strips with

different Rf values 0.93 and 0.76 respectively. Specific activity of 99mTc(CO)3-PTX-NLC was calculated as 0.63-0.70 MBq/µg. The intact percentages of

99m

Tc(CO)3-PTX-NLC incubated

in saline were 98.9 ± 0.1 %, 98.4 ± 0.4 %, 97.9 ± 0.2 %, 97.5 ± 0.4 % and 96.5 ± 0.3 % at 1, 2, 3, 4 and 24 h, respectively. The graph of stability data was given as supplementary material.

99m

Tc(CO)3-PTX-NLC was displayed good in vitro stability according to

radiolabeling efficiency data (>90%) during 24 hours (n=3). HPLC radiochromatograms in figure 3 exhibits 99mTc(CO)3+ and 99mTc(CO)3-PTX-NLC peaks with different retention times 7.18 min. and 4.00 min. respectively. According to sufficient radiolabeling yield achieved in our study, PTX-NLC system is suitable for radiolabeling with 99mTc(CO)3+. 3.4 In Vitro Studies (Toxicity, Time Dependent Incorporation, Fluorescent Imaging) of PTXNLCs Paclitaxel loaded NLCs were examined on three cell lines. HeLa and MCF7 cell lines have high and relatively low folate receptor expressions respectively. A549 cells were used as folate receptor negative control.

Cytotoxicity studies were performed on cell lines with different concentrations of PTX-NLC. Cell viabilities (%) determined on A549, HeLa and MCF7 cells for 24 hours are given in figure 4. The IC50 values of PTX and NLC-PTX for A549, HeLa and MCF7 cells are presented in Table 1. There was statistical significance in the IC50 values of NLC-PTX compared to PTX (p>0.5). In their review Doktorovova et al. had been compared lipid nanoparticles toxicity by using IC50 values obtained in various studies. Correlatively with our results they reported that IC50 values are found mostly in the range of 0.1-1.0 mg/mL particles in dispersion (Doktorovova et al., 2014). According to time depended in vitro incorporation results given in figure 5., NLC-PTX showed higher uptake in every cell line than control group

99m

Tc(CO)3-

99m

Tc(CO)3+. The

highest incorporation ratio was observed on HeLa cell line and the lowest was on A549 cell line. Incorporation ratios on cell lines were consistent with the relative levels of folate receptor expressions in the cells. Considering time dependent incorporation study results there is statistically difference between uptake values of NLC-PTX and [99mTc(CO)3]+ (p<0.05). Cellular fluorescence for MCF7, HeLa and A549 cell lines treated with FITC labeled PTXNLC is shown in figure 6. As seen in images there is significant fluorescence intensity on three cell lines. Previous results obtained by other research groups are also consistent with obtained data in our study. (Zhao et al., 2012; Guo et al., 2000). 3.5 Biodistribution of PTX-NLCs Organ distribution of 99mTc(CO)3-PTX-NLC was determined using receptor blocked (RB) and normal (N) female rats in different time intervals (30. min., 60. min., 120. min., 240. min.). Radiolabeled nanoparticles were distributed rapidly after intravenous injection in both normal

and receptor blocked group. Biodistribution data is presented as percent injected dose per gram of organ (ID/g %) in Table 2. ID/g % of selected organs in normal and receptor blocked groups are given in Fig. 7. After 30 minutes

99m

Tc(CO)3-PTX-NLC exhibited high distribution in uterus, ovary, breast, kidney,

stomach and lung of N rats as compared to RB rats (p < 0.05). FR-α expression in the germinal epithelium of ovary and surface epithelium of uterus was reported by Wu and coworkers (Wu et al., 1999). There are supporting literature reports as to normal lung and stomach tissues have folate receptor expressions in low levels (Sudimack and Lee, 2000; Weitman et al., 1992; Konda et al., 2002). It is also known that alpha and beta isoforms of folate receptors are seen on the apical membrane of the proximal tubules of kidney (Lu and Low, 2012; Salazar and Ratnam, 2007). The main role of FRs in kidney is transcellular reabsorption of folate in the urine. 99mTc(CO)3-PTX-NLC uptake in renal tissue is an expected result of FR expression in proximal tubule cells (Razjouyan et al., 2015; Kim et al., 2015). Correlatively with recently reported studies, after intravenous injection of

99m

Tc(CO)3-PTX-

NLC, radioactivity levels in kidney displayed difference between N and RB rats (p < 0.05) (Guo et al., 2011; Zhao et al., 2011). Similarly, Trump et al performed biodistribution study with 99mTc(CO)3-DTPA-folate and reported high renal uptake 4h after intravenous injection of radiolabeled system (Trump et al., 2002). Zhao et al prepared folate modified nanostructured lipid carriers for docetaxel delivery (FA-DTX-NLC) and compared its biodistribution with non-targeted formulation (DTX-NLC). They reported high concentration of FA-DTX-NLC in tumor and kidney in comparison with DTX-NLC. It seems that the retention of folate targeted 99m

Tc(CO)3-PTX-NLC in the kidney is related with folate receptors expressed on the proximal

tubules of the kidney (Mathias et al., 1998; Konda et al., 2002). In the light of these informations our findings on healthy animal models may be related to receptor expressions in stated organs. Another probability about lung localization of nanoparticles after intravenous

injection is their sequestration by lung capillary as a result of aggregation (Rossin et al., 2005). 3.6 Gamma-imaging Studies To evaluate the localization of

99m

Tc(CO)3-PTX-NLC, gamma-imaging study was performed

after intravenous injection. Figure 8 shows the static scintigrams of normal and receptor blocked female rats in different time intervals. Although there is no significant differences between N and RB rat gamma images as seen in figure 8., there is low liver uptake after 24 hours in N rat when compared with RB rat. 4. Conclusion In this study, the drug delivery system composed of folic acid modified lipid core was prepared for targeted delivery of PTX. Nanostructured lipid carrier system was formulated to form an imperfect matrix and entrap more drug content. Desirable particle size and zeta potential values were obtained. Nanoparticles successfully radiolabeled with

99m

Tc(CO)3+.

Biological behavior and possible folate receptor targeting property of prepared system was investigated by in vivo and in vitro studies. Our results indicate that

99m

Tc(CO)3-PTX-NLC

system may be beneficial to detect folate receptor overexpressed tissues in animal models. Development of nanotechnology-related biodegradable drug delivery systems is promising to make theranostic agents tumor-specific and to avoid their side effects. We believe that, the performed study could contribute to further studies on radiolabeled nanoparticles for the design of targeted imaging agent. Disclosure Statement This study was funded by The Scientific Technological Research Council of Turkey (TUBITAK, Project no: 113S369) and Ege University Science and Technology Centre (EBILTEM, Project no: 2014BIL035).

Acknowledgements The authors acknowledge Prof. António Rocha Paulo and Dr. Célia Fernandes, Centro de Ciências e Tecnologias Nucleares, for their guidance during synthesis process. Eser Ucar would like to thank PhD student Özge Kozguş for helping cytotoxicity studies.

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Fig. 1a. Zeta potential distribution of PTX-NLCs.1b. Size distribution of PTX-NLCs dispersed in water by intensity. Fig. 2. TEM images of PTX-NLCs Fig. 3. HPLC radiochromatograms of

99m

Tc(CO)3 and 99mTc(CO)3-PTX-NLC

Fig. 4. The effect of PTX-NLC amount on viability of HeLa, MCF-7 and A549 cell lines at 24 h. Fig. 5. In vitro incorporation of radiolabeled PTX-NLC at different times Fig. 6. Fluorescence microscopy images of HeLa, MCF-7 and A549 cells treated with PTXNLC. Fig. 7. Injected dose per gram (%ID/g) ratios of folate receptor(FR) positive organs for normal (N) and receptor blocked (RB) rats. Fig. 8. Static images of 99mTc(CO)3-PTX-NLC for normal(N) and receptor blocked(RB) rats at 30, 60, 90, 120, 180, 240 and 1440 minutes after injection.

Table 1. IC50 values of A549, HeLa and MCF-7 cells incubated with NLC-PTX and PTX for 24 h.

NLC-PTX (µg/ml) PTX (µg/ml)

A549 125.5 0.031

Cell Line HeLa 77.91 0.014

MCF-7 71.31 0.012

Table 2. The biodistribution (%ID/g organ-Bg) of

99m

Tc(CO)3-PTX-NLC for selected organs

in receptor blocked (RB) and normal (N) rats. 30 min. %ID/g organ Bg

RB rats

60 min. N rats

RB rats

120 min. N rats

RB rats

240 min. N rats

RB rats

N rats

Heart

1.10±0.12

4.44 ± 2.15

2.59±1.18

3.97 ± 1.82

2.90±1.31

3.93 ± 1.21

1.72±1.05

1.24 ± 0.46

Lung

1.75±1.33

9.90 ± 1.93

2.77±2.89

8.41 ± 0.20

6.78±2.31

12.11 ± 4.03

4.35±2.63

3.35 ± 0.52

Liver

0.72±0.69

6.02 ± 1.35

4.76±4.05

6.88 ± 4.15

9.81±4.49

6.25 ± 0.85

5.01±1.41

4.64 ± 1.63

Kidney

1.43±1.25

10.39 ± 3.11

8.95±6.14

10.96 ± 5.78

15.42±7.97

15.64 ± 5.02

11.85±5.53

10.21 ± 2.05

Small Intestine

0.86±0.19

5.07 ± 2.24

3.65±2.30

8.03 ± 5.71

16.13±1.00

11.31 ± 3.65

4.61±0.76

2.51 ± 0.76

Large Intestine

0.74±0.63

4.38 ± 1.75

5.21±2.18

8.11 ± 6.10

18.68±5.41

13.68 ± 6.04

13.65±7.56

11.23 ± 2.86

Stomach/5

3.34±0.85

16.84 ± 2.59

4.22±1.06

20.64 ± 5.98

13.62±5.41

31.83 ± 1.90

9.56±4.13

10.95 ± 4.95

Spleen

0.79±0.55

6.66 ± 3.03

2.43±0.00

4.19 ± 2.04

3.32±1.10

4.17 ± 1.78

2.44±0.84

1.08 ± 0.67

Pancreas

1.08±0.79

3.77 ± 0.94

3.81±1.71

4.74 ± 0.95

4.25±0.52

4.82 ± 1.24

1.72±0.89

2.12 ± 1.29

Muscle

0.87±0.21

1.29 ± 0.59

1.42±0.63

1.36 ± 0.76

1.93±0.43

1.04 ± 0.06

0.61±0.40

0.25 ± 0.11

Uterus

1.06±0.75

4.06 ± 0.53

2.42±1.19

6.62 ± 1.93

6.08±1.62

5.37 ± 1.49

3.58±0.06

1.81 ± 1.06

Ovary

1.80±0.68

5.00 ± 2.43

3.14±1.85

9.96 ± 2.01

7.38±0.18

7.98 ± 1.18

2.41±2.20

1.67 ± 0.06

Breast

1.15±0.22

7.36 ± 0.15

4.29±2.91

6.41 ± 0.75

5.67±2.73

7.33 ± 2.07

5.43±0.80

2.34 ± 0.60

Fat

0.57±0.47

2.21 ± 0.79

3.23±2.13

2.11 ± 1.26

4.66±2.64

1.92 ± 0.62

1.12±0.56

0.31 ± 0.22

Bladder

5.58±0.38

22.38 ± 18.88

7.35±5.40

62.67 ± 16.67

31.22±7.85

26.46 ± 11.08

5.83±1.95

3.28 ± 0.20

Head

0.13±0.11

1.42 ± 0.53

0.24±0.17

0.83 ± 0.11

0.68±0.34

1.20 ± 0.50

0.33±0.16

0.36 ± 0.17

Blood

6.10±1.65

13.34 ± 0.31

3.50±2.71

8.75 ± 2.25

6.30±4.40

13.40 ± 1.40

6.34±4.30

5.12 ± 1.70

Highlights  Proposal of 99mTc carbonyl-labeled and active targeted nanostructured lipid carriers as a multifunctional system.  Cytotoxicity and fluorescence imaging studies for nanoparticles were evaluated on lung, cervical and breast cancer cell lines.  In vitro cell binding studies were performed for radiolabeled carrier system.  Tissue distribution of 99mTc carbonyl-labeled nanoparticles was investigated on female Wistar Albino rats by Biodistribution and gamma imaging studies.

Graphical abstract