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Imaging, biodistribution and in vitro study of smart 99mTc-PAMAM G4 dendrimer as novel nano-complex
Accepted Manuscript Title: Imaging, biodistribution and in vitro study of smart 99m Tc-PAMAM G4 dendrimer as novel nano-complex Authors: Asghar Narmani, Kamal Yavari, Javad Mohammadnejad PII: DOI: Reference:
S0927-7765(17)30511-8 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.07.089 COLSUB 8752
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
14-3-2017 31-7-2017 31-7-2017
Please cite this article as: Asghar Narmani, Kamal Yavari, Javad Mohammadnejad, Imaging, biodistribution and in vitro study of smart 99mTcPAMAM G4 dendrimer as novel nano-complex, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.07.089 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 proof before it is published in its final 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.
Imaging, biodistribution and in vitro study of smart 99mTc-PAMAM G4 dendrimer as novel nano-complex Asghar Narmania, Kamal Yavari*b, Javad Mohammadnejada a
Department of life science engineering, Faculty of New Science and Technology, University of Tehran, 1439957131, Tehran, Iran b
Department of Nuclear Science and Technology Research Institute, 11365-8486, Tehran, Iran
Graphical abstract
Highlights: 1. Conjugate the 99mTc to folate-PAMAM G4 can be useful as a nuclear imaging agent for tumors overexpressing FRs. 2. Smart PAMAM G4 illustrated excellent potency in targeting of the cancer cell lines in both in vitro and in vivo studies. 3. The in vitro inhibitory effects of PAMAM G4 nano-complexes enhanced by 5-FU. 4. The smart nano-complex indicated active targeting efficiency for the tumor site in tumor bearing BALB/c mice. 5. Folate-modified PAMAM dendrimers showed significantly high accumulation in the tumor, liver and spleen tissues.
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Abstract Overexpression of folic acid receptor in various human tumors cells makes it as good candidate for targeting delivery of chemotherapeutic and radiopharmaceutical agents. In this research, FA used for functionalization of PEG modified PAMAM G4 dendrimer as a smart delivery of 5-FU and 99mTc for the breast carcinoma in order to chemotherapeutic and imaging goals. One aim of this research was assess the FA-mediated cell viability assay of PEG-PAMAM G4-FA-5FU99m Tc and in vitro uptake of PEG-PAMAM G4-FA-99mTc as the novel nano-complex determined on C2Cl2 (normal cell) and MCF-7 (breast cancer cell) cell lines. Other main goals were studied. Morover, an investigation in to in vivo imaging and biodistribution was carried out via a novel radio tracer by which tumor accumulation and site were obviously detected. The targeted tumor images taken by tail intravenous injection demonstrated that nano-complex can be smartly used in imaging study of the clinical practices. Also, the biodistribution of this nano-complex was investigated and the organ predestination of 99mTc labeled nano-complex (%ID/g) was ascertained. Keywords: Breast cancer targeting; Folic acid and 5-FU; 99mTc; Cellular uptake and viability; Biodistribution; Imaging 1.Introduction Nanotechnology offers promising perspectives for the diagnosis and treatment of many diseases related to the lymphatic system, such as cancer, infectious disease or autoimmune diseases [1]. In the past few years, several nanometric carriers have been developed to help diagnostic agents and drugs to reach the lymphatic system [2, 3]. The diagnosis of breast cancer due to its morbidity and high incidence, is the most important issue in cancer therapy [4]. Recently, many academics are particularly interested in molecular imaging techniques in order to monitor breast cancer in human and animal models. In general, molecular imaging is defined as the visualization, characterization and measurement of biomedical processes at the molecular and cellular levels in living systems [5, 6]. On the other hand, new nanoparticle based radiopharmaceuticals with excellent specificity and affinity needs to be developed to increase imaging quality. Currently, non-targeted methods in chemotherapy and imaging are found to be ineffective, because they are unable to differentiate between the cancerous and normal cells [7, 8]. The folate receptor (FR), a glycosylphosphatidylinositol-anchored membrane glycoprotein, due to overexpression (10 times with respect to normal cells) in various human carcinomas such as breast, ovary, kidney, lung etc; is highly restricted in most normal human tissues [9-11]. FR has high affinity and is promising for folic acid (FA) moiety, as FA moieties can be covalently bind to FR and provoke endocytosis process [12]. On the other hand, PEG as an amphiphilic polymer, can favorably modify the surface hydrophilicity/hydrophobicity of particles and sterically stabilize them, and consequently, suppress the binding of serum proteins, and can overcome the cytotoxic and hemolytic effects of nanocarriers such as positive charge PAMAM G4 dendrimer, decrease the aggregation of nanocarrier and enhance the tumor accumulation of nanocarrier by facilitating the enhanced permeability and retention (EPR) [13-15].
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The use of bi-functional chelating agent such as HYNIC results in an increase in the efficiency of radiolabeling with respect to direct radiolabeling [16-18]. HYNIC which is a nicotinic acid analogue that can bind to amine nanocarrier via amide bonds and on the other hand, can form complex with Technetium-99 m (99mTc) is one of the main radiopharmaceutical agents in molecular imaging [9, 19-21]. 99mTc (half-life, 6.02 h; γ ray ¼ 142 keV) practically has a wide application in imaging due to its widespread accessibility, low cost and low absorbeddose burden to the patient [21]. Drug delivery systems (DDSs) also have efficient potentials in therapeutic applications such as 5-fluorouracil (5-FU), doxorubicin (DOX) etc.; as a chemotherapeutic agent, it is one of the broad-spectrum antitumor drugs [22]. Polymer systems can be used to trap chemotherapeutic agents, increase their half-life in serum, increase sustained and controlled drug release in the body, can help site specific targeting of agents and so on [23-25]. In the present context, amine terminated generation 4 PAMAM dendrimer was used for drug delivery goals due to its specific characteristics such as high drug loading, surface functional groups, good biocompatibility, excellent pharmacokinetics qualities, etc [26-28]. After PEGylation of positive charge PAMAM G4 surface to omit or reduce the positive charge induced cytotoxicity, folic acid (FA) as a site specific targeting molecule was used to engineer the nanocarrier. Then, 5-FU chemotherapeutic drug for inhibition of the tumor cell growth was loaded and incorporated into nanocarrier, and nano-complex was labeled via 99mTc as an imaging radiopharmaceutical agent. Thereafter, stability and cellular uptake, assessment of cell viability on MCF-7 (breast cancer) and C2C12 (normal cell) cell lines, were tested and biodistribution and imaging were investigated in this research. 2. Materials and Methods 2.1. Materials and apparatus The PAMAM G4 dendrimer in methanolic solution, Folic acid (FA), 5-Fluorouracil (>99.0 % HPLC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), Suc-HYNIC and TFA (Trifluoroacetic acid), N-tris (hydroxymethyl) methylglycine (tricine), MTT salt (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), penicillin, streptomycin, and fetal bovine serum (FBS) and cellulose dialysis membranes were purchased from Sigma-Aldrich. MAL-PEG-NHS ester 7500 was purchased from Jenkem. 99mTc was obtained from a 99Mo/99mTc generator. The C2C12, 4T1 and MCF-7 cell lines were purchased from Pasteur Institute of Iran. Spectroscopic analyses of samples were performed using a Fourier transform infrared (FT-IR) (Perkin–Elmer 843), proton nuclear magnetic resonance (1H NMR) (Bruker Avance III 400MHz, Germany) spectrometer. Transmission electron microscope (TEM) images were taken by means of Philips EM 208 S (Netherlands). DLS and zeta potential analyses of all syntheses were determined by NanoBrook 90 Plus (Brookhaven, USA). Imaging was performed using single head γ-camera (Siemens-Germany) and γ-counter (EG & G ORTECH 4001 M). 2.2. Dendrimer surface modification In order to enclose the positive charge of PAMAM G4 dendrimer, the required values of both compounds were calculated and the reaction was implemented according the reports of Peng et al [29]. Practically, 38.6 mg of MAL-PEG-NHS (MW=7500 Da) (5.14 × 10-6 mol, 10 folds of 3
molar ratio to PAMAM G4) and 36.58 mg of PAMAM G4 (5.15 × 10-7 mol) were dissolved in 5 and 10 ml of dimethylsulfoxide (DMSO, 5 ml), respectively, then PEG containing solution was added to PAMAM G4 dissolved solution drop by drop and at 300 rpm for 3 overnights for 3 day, in the wake of this process, the reaction mixture was vigorously dialyzed (cut-off of 12000 Da) against PBS buffer (3 times, 4 L) and DI water (3 times, 4 L) for 3 days, respectively, to separate the excess reactants. PEGylation PAMAM G4 (P-P) and all subsequent syntheses were characterized by spectroscopy devices. 2.3. Dendrimer surface engineering and drug loading For active targeting of the nano-complex, FA was conjugated to the previous synthetic sample. To achieve this conjugation, briefly 8.48 mg of FA was dissolved in 6 ml of DMSO and 18 ml of DMF (with 1:3 ratio), and 64.49 mg of EDC.HCl (14 folds of molar ratio to FA) as a FA activator was added to FA containing solution at light protected and nitrogen atmosphere condition. Thereafter, activated FA was added to P-P solution dropwise overnight in the mentioned condition again and PEG-PAMAM G4-FA (P-P-FA) dialyzed with PBS and DI water was prepared just as the last [30]. In the second part, 2.96 × 10-3 mg of 5-FU (50 folds of molar ratio to PAMAM G4) was dissolved in the final synthetic nano-complex and stirred at low rpm overnight [31]. 2.4. Linker conjugation and radiolabeling of dendrimer For this conjugation, 0.104 g (0.78 mmol) of Suc-HYNIC-Boc as a bi-functional chelating agent was mixed and stirred with 1 ml of TFA (20 equivalent) at room temperature to achieve the SucHYNIC-TFA. Then, 4.5 mg of lyophilized Suc-HYNIC-TFA was reacted with 9 mg of P-P-FA dendrimers in DMSO as a solvent at light protected condition [32, 33]. After dialysis of synthesized PEG-PAMAM G4-FA-Suc-HYNIC (P-P-FA-Suc), 1 mg of both P-P-FA-Suc and PP-FA-5FU-Suc were incubated with 200 μl of 99mTcO4– solution (72-195.0 MBq) in the presence of tricine solution (7 mg of tricine in 33 μL of 0.1 M HCl, 550 μL of water, 7 mL of 0.9% NaCl and 33 μL Sn(II) solution (6.3 mg SnCl2.2H2O in 170 μL of 0.1 M HCl) as co-ligand. To ascertain the radiochemical purity of the 99mTcO4– labeled nano-complexes (P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc), ITLC strips eluted with saline buffer were used [32]. 2.5. Radiolabeling stability Stability studies of P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc nano-complexes were evaluated in serum and PBS by the usage of ITLC strips. Subsequently, 150μl (200μCi) of the PP-FA-Suc-99mTc and P-P-F-A-5FU-Suc-99mTc nano-complexes were added to the fresh human serum and PBS and incubated at 37 °C. Thereafter, at different time intervals, the same values of the P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc nano-complexes were added to 1 cm at the bottom point of Whatman No. 1 filter paper (ITLC) and each of the stripes was placed in the solvent (1 mM NaCl) from the drop point region was counted by means of a γ-counter [32, 33]. 2.6. Cell culture
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Human breast cancer MCF-7, mouse breast cancer and myoblast normal C2C12 cell lines were purchased from the Pasture Institute of Iran and maintained in RPMI and DMEM (Gibco, UK) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 μg/ml streptomycin and 100 IU/ml penicillin. 2.7. Cell internalization Internalization studies of P-P-FA-99mTc were conducted using PBS 1X and 0.1 M NaOH. C2C12, 4T1 and MCF-7 cells were harvested at 5 × 104cells/300 μl in medium and cultured in 24 well plates overnight. Then, 500 nM (175 μCi), 250 nM (90 μCi) and 125 nM (50 μCi) of PP-FA-99mTc were added to each well and incubated. At various time intervals (1, 3 and 24 h), RPMI and DMEM medium of wells were removed and medium activity, as a non-uptake drug conjugates, was read by γ-counter. In addition, the cultured cells in any of the wells were washed with filtered PBS 1X, incubated for 15 min and 400 μl of 0.1 M NaOH to separate cells and cell activity was counted using γ-counter as uptake and receptor conjugated carriers. Differences between the medium and cell activities were considered as an internalized P-P-FA-Suc-99mTc [32]. 2.8. MTT assay The growth inhibitory effects of PAMAM G4, P-P, P-P-FA, P-P-FA-5FU, P-P-FA-Suc-99mTc and P-P-FA-5FU-suc-99mTc with 1000, 500, 250, 125 and 62.5 nM concentrations on MCF-7 and C2C12 cells were determined using the MTT assay with 9 repeat (at 24, 48 and 72 hour after treatment). Briefly, the cells were cultured on 96-well microtiter plates and were allowed to attach overnight. Subsequently, cells were treated with different type of drugs. After the posttreatment times, MTT solution (5 mg/ml in PBS) was added to each well and incubated for another 4 h. Consequently, the liquid suspension was removed and cells were resuspended in DMSO. The optical density (OD) of these DMSO solutions was read at 570 nm, and OD differences of treated and non-treated cells revealed the cell viability. 2.9. Mouse model with breast tumor Five- to-six week-old female BALB/c mice (avg. body weight 16.0 ±1.5 g) were used for the in vivo studies. The tumor was established by subcutaneous injection of 4T1 tumor cell line in the right side of the post neck region. The mouse breast carcinoma 4T1 cell line was grown continuously as a monolayer at 37 °C and 5% CO2. The 4T1 cells were prepared for the injection in the female BALB/c mice. A 2 ml of 0.25% trypsin/1 mM EDTA solution was added to the plate and swirled to cover the plate and incubated at room temperature for 2 min, followed by addition of 5-6 ml serum-free media to harvest the trypsinized cells from the plate and transfer to a conical tube. These cells were centrifuged, the supernatant were discarded and the pellet was resuspended in serum-free medium. The cells were diluted with a serum-free medium to the desired concentration (1 × 106 cells/0.2 ml). These 4T1 tumor cells in serum-free medium suspension were inoculated subcutaneously. Then, the mice were monitored daily for tumor onset by palpating the injection area with the index finger and thumb for the presence of tumor. After 12 to 15 days of inoculation, the mice injected with 4T1 cells developed palpable solid tumors (619.2 ± 98.5 mm3 in volume). 5
2.10. In vivo imaging Imaging was performed on female, 7–8 weeks old tumor bearing BALB/c mice (n=3). The planar γ-camera imaging was performed by means of an in vivo single head γ-camera. The mice were treated with 5.4 MBq (200μCi) of 99mTc labeled dendrimer (P-P-FA-Suc-99mTc) through the tail intravenous (IV) injection. The mice were studied for imaging evaluation of radiolabeled smart nano-complex at 1, 3 and 24 h of post-injection time. 2.11. Biodistribution study Normal and tumor bearing BALB/c mice were divided into two groups (n=12). Practically, 200 μCi of the radiolabeled dendrimer (P-P-FA-Suc-99mTc) in PBS was injected, intravenously. After 1, 3, and 24 h post-injection interval times, the mice were euthanized and the main organs including the heart, lung, adrenal, stomach, intestine thyroid, liver, spleen, kidney, muscle, bone, bladder, pancreas, blood, skin, tail, whole body and tumor were removed and weighted (stomach and intestine were washed with PBS, firstly). The radioactivity of P-P-FA-Suc-99mTc accumulated organs were detected by a γ-counter and the results were expressed as percentages of the injected dose per gram (% ID/g) organs. 2.12. Statistical analysis Comparisons between the groups were expressed as mean ± standard error of the mean. Statistical differences were evaluated using one way ANOVA and Tukey’s HSD with the level of statistical significance at p < 0.05. 3. Results and Discussion 3.1. Characterization of synthesized nano-complexes FT-IR and 1H NMR spectroscopies were applied to confirm the synthetic nano-complexes. In FT-IR analysis (Fig. 1), the pure PAMAM G4 characteristic absorption peaks were obtained at 3306 cm-1 (N–H stretch of amines); 3180 cm-1 (antisymmetric N–H stretch of substituted primary amine) and 1252 cm-1 reflecting the C–O stretching vibration; 1112 cm-1 (C–C bending) [34, 35]. Absorption peaks at 1643 cm-1 (–C=O stretch); 2887 cm-1 (C–H stretch); 3408 cm-1 (N– H stretch of amide) and 1734 cm-1 (C=O stretch of carbonyl group) were revealed for P-P nanocomplex [36, 37]. There was an upshift of C=O stretch of acid group of carboxymethyl mPEG of amide linkages. For P-P-FA nano-complexes, important peaks of folic acid were at 3545, 3415 cm-1 (N–H stretch of primary amine and amide); 3328 cm-1 (alkyl C–H and C=C stretch); 1695 cm-1 (aromatic C=C bending and stretching); 1492 cm-1 (CH–NH–C=O amides bending); 837 cm-1 (aromatic C–H bending and benzene 1,4-disubstitution); and for the NHS conjugated folic acid, 3690 cm-1 (amide N–H and C=O stretching); 2993 cm-1 (carboxylic acid C=O and O–H stretching unconjugated); 1712 cm-1 (ketones C=O unconjugated stretch), which confirmed the synthesis of NHS ester of folic acid [35, 38]. The 5-FU loaded P-P-FA nano-complex FT-IR analysis revealed absorption peaks at 3238 cm-1 (N–H stretching vibration); 3068 cm-1 (C–H stretching vibration); 2929 and 2829 cm-1 (–CH2– symmetric and asymmetric stretching vibration). Also, other peaks such as 1725, 1648 and 1249 cm-1 that are related to the carbonyl 6
stretching vibrations were determined; 1232 cm-1 (C–F stretching vibration); 991 cm-1 (N–H and C–H wagging vibration); 811 cm-1 (C–F stretching vibration) [39]. The peaks of conjugated linker were achieved at 3038 cm-1 (CH2 stretching vibrations); 1701 cm-1 (COO– stretching vibration); 3693 cm-1 (amide N–H stretching); 835 cm-1 (aromatic C–H bending), which indicated the HYNIC conjugation to our nano-carrier. Absorption peak at 935, 920, 892 and 898 cm-1 were related to TcO4– in P-P-FA-99mTc and P-P-FA-5FU-Suc-99mTc [40]. 1 H NMR spectra of the synthetic nano-complexes were also implemented (Fig. 2). The P-P-FA nano-complex aromatic folate proton peaks were obtained at δ 6.71, δ 7.60, and δ 8.69 ppm, and PEG proton peak was revealed at δ 3.70 ppm [34, 35]. The multiple proton peaks of PAMAM were found at δ 1.29, δ 1.34 ppm and and δ 2.45 to δ 2.68 ppm [35, 38]. In addition, 5FU proton peak was ascertained at δ 7.58 ppm. Proton peaks in the range of δ 0.83 to δ 2.06 ppm and δ 3.29 ppm were assigned to aliphatic protons of CH2NHC=O and amide bond formation. Size diameter determination of synthetic nano-complexes were ascertained by means of TEM (Fig. 3), and final nano-complexes size were determined between 30 to 80 nm in diameter. The surface charges of the samples were also ascertained and -2.10 ± 1.11 and -1.69 ± 0.51 mV were obtained for P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc nano-complexes, respectively (Table 1). The values of 99mTc were also measured by the Energy Dispersive X-ray Spectroscopy (EDS or EDX). The EDS is a chemical microanalysis technique used in conjunction with scanning electron microscopy (SEM) to provide elemental identification and quantitative compositional information. The weight and atomic percentage of 99mTc, F and other elements were determined in P-P-FA-5FU-99mTc and P-P-FA-99mTc samples via EDS. The weight percentage of carbon and 99mTc were ascertained as 93.66 and 6.34% for P-P-FA-Suc-99mTc, respectively. Also, the weight percentage for carbon, F and 99mTc elements were determined as 64.76, 25.37 and 9.87% in P-P-FA-5FU-Suc-99mTc, respectively (Fig. 3). 3.2. PBS and serum stability Radiochemical stability of P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc was evaluated by using the ITLC at different time intervals (1, 2, 4 and 24 h) (Fig. 4 A and B). After incubation of 200 μCi of P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc (radiolabeling yield >95%) in PBS and serum at 37 °C for 24 h, more than 76 % conservation of total radioactivity was revealed. The P-P-FA-Suc-99mTc stabilities were determined as 85.28±1.99 % and 86.67±2.11 % in PBS and serum after 1 h, respectively. It was the same for P-P-FA-5FU-Suc-99mTc nano-complex in the same time (85.32±1.15 % in PBS and 83.75±0.88 % in serum). Also, it was 77.2±0.76% and 77.98±0.74 % in the PBS and 76.27±1.02 % and 78.35±1.47 % in the serum samples after 24 h for P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc, respectively. In comparison with antibody labeling efficiency, PAMAM G4 nanoparticle could be considered as good candidate for radiolabeling and imaging [41]. In general, stability decreases after 24 h which could be attributed to protein interactions such as albumin in the serum [42].
3.3. Cell internalization Cellular uptake of FA targeted nano-carrier was evaluated on normal and cancer cell lines to investigate FR-mediated endocytosis (Fig. 4 C and D). Higher internalization of P-P-FA-Suc7
99m
Tc was observed in MCF-7 cell line with respect to the normal cells. The results indicated that 64.11±0.88 % of nano-complex (500 nM) was internalized by MCF-7 cancer cell line after 24 h, while in the same condition, 45.74±0.48 % for C2Cl2 as a control cell line was determined. A comparison of the first and last uptake values demonstrated a considerable uptake difference between the normal and cancer cell. For example, MCF-7 cells internalized 41.11% of the smart nano-complex after 24 h at 500 nM; however, it was 12.52 % for normal cell lines in the same condition which crucially revealed the importance of FA targeting [11, 21]. Generally, the cellular uptake of P-P-FA-Suc-99mTc was depicted as time and concentration dependent behavior in a direct relation. 3.4. Cell viability assay Cell viability was implemented by MTT assay on human breast cancer (MCF-7) and normal (C2Cl2) cell lines for all synthetic nano-complexes (Fig. 5). Synthesis of the novel nanocomplexes triggered our interest to evaluate the cytotoxic effect of surface modified and engineered PAMAM G4 dendrimer as a carrier system for the delivery of chemotherapeutic radiolabeled agent on cell lines. All compounds included pure PAMAM G4, P-P, P-P-FA, P-PFA-5FU, P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc in different concentrations (62.5, 125, 250, 500 and 1000 nM). It is worth mentioning that PEGylation of PAMAM G4 demdrimer can considerably decrease the cytotoxic effects of positive charge PAMAM G4 and effectively increase the biocompatibility and stability of polymer. For instance, in concentration of 1000 nM the PEG reduced the cytotoxicity of PAMAM G4 from 43.75±0.55 % to 79.86±1.56 % of cell viability in MCF-7 cell line (p<0.01) [13, 15]. Also, at up to 500 nM concentration, the P-P-FA strengthens the percentage of the cell viability which may be due to augmentation of PEGylated PAMAM G4 and also usage of cell from FA as a nutritional compound (p<0.05). The cell viability was enhanced from 53.95±0.61% to 66.68±0.73% after PEGylation at 250 nM (after 24 h in MCF-7) which increased to 84.07±0.54 % after FA addition to P-P [9, 10]. A comparison of the C2Cl2 normal cell line with MCF-7 regarding the effects of various types of nano-complex types showed that pure PAMAM G4 had cytotoxic effects on normal cell in comparison with cancer cell lines and PEGylation mainly reduced the side effects of PAMAM G4 on the normal cell lines. The P-P-FA nano-complex in both cell lines demonstrated nearly the same effects, but mainly different from those of concentration. Drug loaded nano-complex (P-P-FA-5FU) has been extremely cytotoxic in comparison with others. It showed concentration dependent cell viability assay, as 23.82±0.56% and 14.69±0.84% cell viability were obtained at 62.5 and 1000 nM 1000 nM concentration for MCF-7, and 27.51±0.65% and 23.42±0.46% of cell viability were obtained for C2Cl2 at the same concentration after 24 h. These revealed that drug loaded nano-complex has more effective cytotoxicity on cancer cell in contrast with normal cell lines. Radiolabeled samples were tested on cell viability through both P-P-FA-Suc-99mTc and P-PFA-5FU-Suc-99mTc nano-complexes. Due to the low half-life of 99mTc, radiolabeled complexes were also assessed at 3 and 24 h post treatment. Synergistic effects of drug and 99mTc were observed; however, major cell inhibition effect was related to 5FU. Apparently, high cell inhibition effect of radioactive agent was obtained at high dose of 99mTc. Values of 69.80±1.98 % and 86.19±0.51 % of cell viability were obtained at 1000 nM of P-P-FA-Suc-99mTc after 3 and 24 h of treatment, while 13.10±1.4 % and 87.34±2.35 % of the cell viability were observed for P8
P-FA-5FU-Suc-99mTc at 3 and 24 h for 1000 nM, respectively (Fig. 5). Normal cell as a control was also the same for 99mTc radiolabeled nano-complexes. 3.5. In vivo imaging Fig. 6 A to C shows the γ-camera taken images of female BALB/c mice bearing breast carcinoma tumor at 1, 3 and 24 h interval times as a molecular imaging technique in clinical and basic research fields. P-P-FA-Suc-99mTc nano-complex as novel radiotracer was completely localized at site specific area of tumor after IV injection as indicated in the images. 3.6. Biodistribution study For biodistribution study, we attempted to label functionalized PAMAM G4, targeting highly metastatic cells with 99mTc, for potential imaging of tumors and metastases. Fig. 6 D and E demonstrates the biodistribution profile of P-P-FA-Suc-99mTc radiotracer in the mice with breast tumor at 1, 3 and 24 h after intravenous administration and was expressed as percentage of injected dose per gram (%ID/g) of organs. Biodistribution studies at 1, 3 and 24 h of postinjection revealed the specific localization of nano-complex at the tumor site. The liver, spleen and tumor were indicated as a substantial accumulation of radioactive nano-complex, respectively. Tumor uptake of P-P-FA-Suc-99mTc radiotracer showed time-dependent manner against the liver and spleen uptake especially at the last time. The tumor uptake (%ID/g) was 4.66±0.64 % at 1 h post-injection which increased to 13.76 ± 1.39 % after 24 h revealing excellent tumor targeting. As the tumor uptake of nano-complex was interestingly higher than that of other organs except the liver and spleen, we could conclude that these organs did not actively uptake P-P-FA-Suc-99mTc given that they did not over-express folate receptor that leads to a high retention of radioactivity in the tumor. Also, high plasma concentration of the nanocomplex indicated good circulation of radiolabeled dendrimer as the site specific targeting delivery systems, and with respect to peptide and antibody based radiotracers, late clearance from plasma were shown [9, 11, 43]. The in vivo biodistribution profiles demonstrate that the radioactivity in the kidney was beyond the normal levels 1 h after nano-complex IV administration, illustrating that P-P-FA-Suc-99mTc was mainly excreted through the kidney. These data were in line with the results of Chen et al [41, 44, 45]. In this study, we evaluated smart PAMAM G4 labeled with a radionuclide as a tumor-targeting imaging agent for the first time. P-P-FA-Suc-99mTc biodistribution and γ-camera imaging demonstrated its ability to target highly metastatic tumors. The uptake values of the other organs of the normal and tumor bearing BALB/c mice are presented in Table 2. 4. Conclusion Herein, we report PEG modified and FA functionalized PAMAM G4 as a smart low toxic nano-carrier asset to be used in therapeutic, imaging and biodistribution domains. Our results demonstrate that a novel nano-complex has excellent potentials for the delivery of 5-FU chemotherapeutic agents for breast cancer cell lines and tumor accumulation with best internalization to cancer cells. This nano-complex was investigated for quantitative in vivo biodistribution according to the accumulation into the tumor, liver and spleen following intravenous administration in the breast tumor bearing mice. The tumor accumulation indicated 9
the targeting capacity of the smart nano-complex. Moreover, imaging study of the site specific targeting radiotracer was ascertained and the results confirm the excellent power of nanocomplex in the tumor bearing mice model. In conclusion, the novel smart synthetic nanocomplex is applicable for the treatment, tracing and imaging of tumors at clinical levels. Acknowledgments This paper has been extracted from an MSc thesis. The authors would like to acknowledge all the supports of University of Tehran. Tehran, Iran (Grant no. 28701/06/09).
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13
Fig 1. FT-IR spectra of PAMAM G4 nano-complexes. The main locations of any compounds and linkage formation in conjugates were illustrate in text via their specific absorption peaks.
Fig 2. 1H NMR spectra of P-P-FA-Suc-99mTc (up) and P-P-FA-5FU-Suc-99mTc (down) nanocomplexes. The folate proton peaks were obtained at δ 6.71, δ 7.60, and δ 8.69 ppm, and PEG proton peak was revealed at δ 3.70 ppm. The 5FU proton peak was ascertained at δ 7.58 ppm.
14
15
Fig 3. TEM image (up) and EDS (down) graph of P-P-FA-Suc-99mTc (left) and P-P-FA-5FUSuc-99mTc (right) nano-complexes. The size of both nano-complex was determined between 30 to 80 nm in diameter.
95
A
P-P-FA-Suc-99mTc
Serum
90
Stability (%)
PBS
85
80
75
70 1
2
4
24
Time (h)
95
B
P-P-FA-5FU-Suc-99mTc
Serum
90
Stability (%)
PBS
85
80
75
70 1
2
4
Time (h)
16
24
70
125 nM
C
250 nM
Uptake (%)
60
500 nM
50 40 30 20 1
3
24
Time (h) 70
125 nM
D
250 nM
Uptake (%)
60
500 nM
50 40 30 20 1
3
24
Time (h)
Fig 4. The PBS and serum stability and cell uptakes. PBS and serum stability of 99mTc radiolabeled nano-complexes at various time intervals (A and B). Cellular uptake of C2Cl2 and MCF-7 cell in 125 nM, 250 nM and 500 nM concentrations after 1, 3 and 24 h post treatment time intervals (C and D).
17
120
P
MCF-7 (24 h)
Cell viability (%)
100
P-P P-P-FA
80
P-P-FA-5FU P-P-FA-Tc
60
P-P-FA-5FU-Tc 40 20 0 0
62.5
125
250
500
1000
Concentration (nM) 120
P
C2Cl2 (24 h)
Cell viability (%)
100
P-P P-P-FA
80
P-P-FA-5FU 60
P-P-FA-Tc P-P-FA-5FU-Tc
40 20 0 0
62.5
125
250
Concentration (nM)
18
500
1000
120
P
MCF-7 (48 h)
Cell viability (%)
100
P-P P-P-FA
80
P-P-FA-5FU 60 40 20 0 0
62.5
125
250
500
1000
Concentration (nM) 120
P
C2Cl2 (48 h)
P-P
Cell viability (%)
100
P-P-FA 80
P-P-FA-5FU
60 40 20 0 0
62.5
125
250
Concentration (nM)
19
500
1000
120
MCF-7 (72 h)
P
Cell viability (%)
100
P-P P-P-FA
80
P-P-FA-5FU 60 40 20 0 0
62.5
125
250
500
1000
Concentration (nM)
120
C2Cl2 (72 h)
P
Cell viability (%)
100
P-P P-P-FA
80
P-P-FA-5FU 60 40 20 0 0
62.5
125
250
Concentration (nM)
20
500
1000
120
MCF-7 (3 h)
P-P-FA-Tc
Cell viability (%)
100
P-P-FA-5FU-Tc
80 60 40 20 0 0
62.5
125
250
500
1000
Concentration (nM) 120
C2Cl2 (3 h)
P-P-FA-Tc
Cell viability (%)
100
P-P-FA-5FU-Tc
80 60 40 20 0 0
62.5
125
250
500
1000
Concentration (nM)
Fig 5. Cytotoxicity assessments by MTT assay in MCF-7 and C2Cl2 cell lines after 3, 24, 48 and 72 h of post-treatment with P, P-P, P-P-FA, P-P-FA-5FU, P-P-FA-Suc-99mTc and P-P-FA-5FUSuc-99mTc nano-complexes.
21
90
D 80 70
ID/g (%)
60
1h
50
3h
40
24h
30 20 10 0
22
90
E 80 70
1h
ID/g (%)
60
3h
50
24h 40 30 20 10 0
Fig 6. The imaging study and biodistribution of synthetic nano-complex in mice body. γ-Camera image of tumor bearing BALB/c mice after 1 h (A), 3 h (B) and 24 h (C) of P-P-FA-Suc-99mTc tail IV injection. Evaluation of P-P-FA-Suc-99mTc biodistribution in normal (A) and tumor bearing (B) BALB/c mice (%ID/g) (D and E).
23
Table 1. Particle diameter and their surface charges determined via DLS for dendrimer-modified nanoparticles PAMAM modification
Dispersed in
Diameter/nm
Surface charge (mV)
PAMAM PAMAM-PEG PAMAM-PEG-FA PAMAM-PEG-FA-5FU PEG-PAMAM G4-FA-5FU PEG-PAMAM G4-FA-5FU-Suc PEG-PAMAM G4-FA-Suc PEG-PAMAM G4-FA-Suc-Tc PEG-PAMAM G4-FA-5FU-Suc-Tc
H2O H2O H2O H2O H2O H2O H2O H2O H2O
4.49 17.28 58.27 58.45 58.45 52.09 59.33 69.88 76.58
25.38 ± 2.8 -24.38 ± 5.16 -25.63 ± 1.62 -21.71 ± 0.71 -21.71 ± 0.71 -2.58 ± 0.60 -7.89 ± 2.96 -2.10 ± 1.11 -1.69 ± 0.51
Table 2. In vivo biodistribution values of P-P-FA-Suc-99mTc nano-complex in various organs (%ID/g). Organs
Blood Heart lung Adrenal Stomach intestine Thyroid Liver Spleen Kidney Muscle Bone Bladder Tail Pancreas Skin Whole body
Normal mice
Tumorous mice
1h
3h
24 h
1h
8/21±1.68 1/86±0.17 7/46±1.01 2/65±0.43 15/31±4.14 1/66±0.27 4/45±0.98 82/08±5.63 26/42±1.56 6/48±0.8 0/79±0.26 0/94±0.38 0/75±0.34 2/37±0.09 3/27±0.78 1/67±0.02 0/98±0.25
7/06±1.59 1/62±0.03 5/7±0.2 0/64±0.05 6/71±0.34 1/69±0.06 3/51±0.84 72/21±1.98 25±0.98 5/24±0.73 0/43±0.31 0/76±0.23 0/82±0.28 3/03±0.31 1/26±0.06 1/12±0.1 1/25±0.41
4/21±0.49 0/77±0.06 3/6±0.22 7/21±0.37 0/83±0.06 0/69±0.01 0/37±0.16 46/74±2.07 16/73±0.45 4/38±0.71 0/26±0.19 0/46±0.19 0/44±0.22 2/36±0.9 0/79±0.12 0/61±0.07 3/67±0.69
9/89±1.21 2/49±0.15 6/51±0.53 2/69±0.05 7/18±0.85 2/01±0.36 2/44±0.65 83/21±3.72 13/31±1.1 6/16±0.25 1±0.04 0/79±0.07 1/41±0.03 4/39±0.25 2/07±0.21 2/75±0.27 1/57±0.29
7/68±0.89 1/72±0.02 3/68±0.5 0/87±0.27 6/32±0.59 1/99±0.25 1/19±0.14 69/34±1.36 14/43±0.79 4/09±0.2 1/51±0.03 0/5±0.03 0/71±0.02 2/55±0.16 0/72±0.07 1/24±0.03 1/57±0.35
5/46±0.94 4/21±0.8 1/54±0.07 1/35±0.65 2/97±0.67 1/96±0.29 3/59±0.46 13/62±1.29 3/28±0.28 4/45±0.43 2/13±0.91 1/12±0.13 0/84±0.17 1/79±0.61 2/25±0.1 3/01±0.23
4/66±0.64
10/61±0.48
13/76±1.39
Tumor
24
3h
24 h
6/02±0.78
S0927-7765(17)30511-8 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.07.089 COLSUB 8752
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
14-3-2017 31-7-2017 31-7-2017
Please cite this article as: Asghar Narmani, Kamal Yavari, Javad Mohammadnejad, Imaging, biodistribution and in vitro study of smart 99mTcPAMAM G4 dendrimer as novel nano-complex, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.07.089 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 proof before it is published in its final 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.
Imaging, biodistribution and in vitro study of smart 99mTc-PAMAM G4 dendrimer as novel nano-complex Asghar Narmania, Kamal Yavari*b, Javad Mohammadnejada a
Department of life science engineering, Faculty of New Science and Technology, University of Tehran, 1439957131, Tehran, Iran b
Department of Nuclear Science and Technology Research Institute, 11365-8486, Tehran, Iran
Graphical abstract
Highlights: 1. Conjugate the 99mTc to folate-PAMAM G4 can be useful as a nuclear imaging agent for tumors overexpressing FRs. 2. Smart PAMAM G4 illustrated excellent potency in targeting of the cancer cell lines in both in vitro and in vivo studies. 3. The in vitro inhibitory effects of PAMAM G4 nano-complexes enhanced by 5-FU. 4. The smart nano-complex indicated active targeting efficiency for the tumor site in tumor bearing BALB/c mice. 5. Folate-modified PAMAM dendrimers showed significantly high accumulation in the tumor, liver and spleen tissues.
1
Abstract Overexpression of folic acid receptor in various human tumors cells makes it as good candidate for targeting delivery of chemotherapeutic and radiopharmaceutical agents. In this research, FA used for functionalization of PEG modified PAMAM G4 dendrimer as a smart delivery of 5-FU and 99mTc for the breast carcinoma in order to chemotherapeutic and imaging goals. One aim of this research was assess the FA-mediated cell viability assay of PEG-PAMAM G4-FA-5FU99m Tc and in vitro uptake of PEG-PAMAM G4-FA-99mTc as the novel nano-complex determined on C2Cl2 (normal cell) and MCF-7 (breast cancer cell) cell lines. Other main goals were studied. Morover, an investigation in to in vivo imaging and biodistribution was carried out via a novel radio tracer by which tumor accumulation and site were obviously detected. The targeted tumor images taken by tail intravenous injection demonstrated that nano-complex can be smartly used in imaging study of the clinical practices. Also, the biodistribution of this nano-complex was investigated and the organ predestination of 99mTc labeled nano-complex (%ID/g) was ascertained. Keywords: Breast cancer targeting; Folic acid and 5-FU; 99mTc; Cellular uptake and viability; Biodistribution; Imaging 1.Introduction Nanotechnology offers promising perspectives for the diagnosis and treatment of many diseases related to the lymphatic system, such as cancer, infectious disease or autoimmune diseases [1]. In the past few years, several nanometric carriers have been developed to help diagnostic agents and drugs to reach the lymphatic system [2, 3]. The diagnosis of breast cancer due to its morbidity and high incidence, is the most important issue in cancer therapy [4]. Recently, many academics are particularly interested in molecular imaging techniques in order to monitor breast cancer in human and animal models. In general, molecular imaging is defined as the visualization, characterization and measurement of biomedical processes at the molecular and cellular levels in living systems [5, 6]. On the other hand, new nanoparticle based radiopharmaceuticals with excellent specificity and affinity needs to be developed to increase imaging quality. Currently, non-targeted methods in chemotherapy and imaging are found to be ineffective, because they are unable to differentiate between the cancerous and normal cells [7, 8]. The folate receptor (FR), a glycosylphosphatidylinositol-anchored membrane glycoprotein, due to overexpression (10 times with respect to normal cells) in various human carcinomas such as breast, ovary, kidney, lung etc; is highly restricted in most normal human tissues [9-11]. FR has high affinity and is promising for folic acid (FA) moiety, as FA moieties can be covalently bind to FR and provoke endocytosis process [12]. On the other hand, PEG as an amphiphilic polymer, can favorably modify the surface hydrophilicity/hydrophobicity of particles and sterically stabilize them, and consequently, suppress the binding of serum proteins, and can overcome the cytotoxic and hemolytic effects of nanocarriers such as positive charge PAMAM G4 dendrimer, decrease the aggregation of nanocarrier and enhance the tumor accumulation of nanocarrier by facilitating the enhanced permeability and retention (EPR) [13-15].
2
The use of bi-functional chelating agent such as HYNIC results in an increase in the efficiency of radiolabeling with respect to direct radiolabeling [16-18]. HYNIC which is a nicotinic acid analogue that can bind to amine nanocarrier via amide bonds and on the other hand, can form complex with Technetium-99 m (99mTc) is one of the main radiopharmaceutical agents in molecular imaging [9, 19-21]. 99mTc (half-life, 6.02 h; γ ray ¼ 142 keV) practically has a wide application in imaging due to its widespread accessibility, low cost and low absorbeddose burden to the patient [21]. Drug delivery systems (DDSs) also have efficient potentials in therapeutic applications such as 5-fluorouracil (5-FU), doxorubicin (DOX) etc.; as a chemotherapeutic agent, it is one of the broad-spectrum antitumor drugs [22]. Polymer systems can be used to trap chemotherapeutic agents, increase their half-life in serum, increase sustained and controlled drug release in the body, can help site specific targeting of agents and so on [23-25]. In the present context, amine terminated generation 4 PAMAM dendrimer was used for drug delivery goals due to its specific characteristics such as high drug loading, surface functional groups, good biocompatibility, excellent pharmacokinetics qualities, etc [26-28]. After PEGylation of positive charge PAMAM G4 surface to omit or reduce the positive charge induced cytotoxicity, folic acid (FA) as a site specific targeting molecule was used to engineer the nanocarrier. Then, 5-FU chemotherapeutic drug for inhibition of the tumor cell growth was loaded and incorporated into nanocarrier, and nano-complex was labeled via 99mTc as an imaging radiopharmaceutical agent. Thereafter, stability and cellular uptake, assessment of cell viability on MCF-7 (breast cancer) and C2C12 (normal cell) cell lines, were tested and biodistribution and imaging were investigated in this research. 2. Materials and Methods 2.1. Materials and apparatus The PAMAM G4 dendrimer in methanolic solution, Folic acid (FA), 5-Fluorouracil (>99.0 % HPLC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), Suc-HYNIC and TFA (Trifluoroacetic acid), N-tris (hydroxymethyl) methylglycine (tricine), MTT salt (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), penicillin, streptomycin, and fetal bovine serum (FBS) and cellulose dialysis membranes were purchased from Sigma-Aldrich. MAL-PEG-NHS ester 7500 was purchased from Jenkem. 99mTc was obtained from a 99Mo/99mTc generator. The C2C12, 4T1 and MCF-7 cell lines were purchased from Pasteur Institute of Iran. Spectroscopic analyses of samples were performed using a Fourier transform infrared (FT-IR) (Perkin–Elmer 843), proton nuclear magnetic resonance (1H NMR) (Bruker Avance III 400MHz, Germany) spectrometer. Transmission electron microscope (TEM) images were taken by means of Philips EM 208 S (Netherlands). DLS and zeta potential analyses of all syntheses were determined by NanoBrook 90 Plus (Brookhaven, USA). Imaging was performed using single head γ-camera (Siemens-Germany) and γ-counter (EG & G ORTECH 4001 M). 2.2. Dendrimer surface modification In order to enclose the positive charge of PAMAM G4 dendrimer, the required values of both compounds were calculated and the reaction was implemented according the reports of Peng et al [29]. Practically, 38.6 mg of MAL-PEG-NHS (MW=7500 Da) (5.14 × 10-6 mol, 10 folds of 3
molar ratio to PAMAM G4) and 36.58 mg of PAMAM G4 (5.15 × 10-7 mol) were dissolved in 5 and 10 ml of dimethylsulfoxide (DMSO, 5 ml), respectively, then PEG containing solution was added to PAMAM G4 dissolved solution drop by drop and at 300 rpm for 3 overnights for 3 day, in the wake of this process, the reaction mixture was vigorously dialyzed (cut-off of 12000 Da) against PBS buffer (3 times, 4 L) and DI water (3 times, 4 L) for 3 days, respectively, to separate the excess reactants. PEGylation PAMAM G4 (P-P) and all subsequent syntheses were characterized by spectroscopy devices. 2.3. Dendrimer surface engineering and drug loading For active targeting of the nano-complex, FA was conjugated to the previous synthetic sample. To achieve this conjugation, briefly 8.48 mg of FA was dissolved in 6 ml of DMSO and 18 ml of DMF (with 1:3 ratio), and 64.49 mg of EDC.HCl (14 folds of molar ratio to FA) as a FA activator was added to FA containing solution at light protected and nitrogen atmosphere condition. Thereafter, activated FA was added to P-P solution dropwise overnight in the mentioned condition again and PEG-PAMAM G4-FA (P-P-FA) dialyzed with PBS and DI water was prepared just as the last [30]. In the second part, 2.96 × 10-3 mg of 5-FU (50 folds of molar ratio to PAMAM G4) was dissolved in the final synthetic nano-complex and stirred at low rpm overnight [31]. 2.4. Linker conjugation and radiolabeling of dendrimer For this conjugation, 0.104 g (0.78 mmol) of Suc-HYNIC-Boc as a bi-functional chelating agent was mixed and stirred with 1 ml of TFA (20 equivalent) at room temperature to achieve the SucHYNIC-TFA. Then, 4.5 mg of lyophilized Suc-HYNIC-TFA was reacted with 9 mg of P-P-FA dendrimers in DMSO as a solvent at light protected condition [32, 33]. After dialysis of synthesized PEG-PAMAM G4-FA-Suc-HYNIC (P-P-FA-Suc), 1 mg of both P-P-FA-Suc and PP-FA-5FU-Suc were incubated with 200 μl of 99mTcO4– solution (72-195.0 MBq) in the presence of tricine solution (7 mg of tricine in 33 μL of 0.1 M HCl, 550 μL of water, 7 mL of 0.9% NaCl and 33 μL Sn(II) solution (6.3 mg SnCl2.2H2O in 170 μL of 0.1 M HCl) as co-ligand. To ascertain the radiochemical purity of the 99mTcO4– labeled nano-complexes (P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc), ITLC strips eluted with saline buffer were used [32]. 2.5. Radiolabeling stability Stability studies of P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc nano-complexes were evaluated in serum and PBS by the usage of ITLC strips. Subsequently, 150μl (200μCi) of the PP-FA-Suc-99mTc and P-P-F-A-5FU-Suc-99mTc nano-complexes were added to the fresh human serum and PBS and incubated at 37 °C. Thereafter, at different time intervals, the same values of the P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc nano-complexes were added to 1 cm at the bottom point of Whatman No. 1 filter paper (ITLC) and each of the stripes was placed in the solvent (1 mM NaCl) from the drop point region was counted by means of a γ-counter [32, 33]. 2.6. Cell culture
4
Human breast cancer MCF-7, mouse breast cancer and myoblast normal C2C12 cell lines were purchased from the Pasture Institute of Iran and maintained in RPMI and DMEM (Gibco, UK) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 μg/ml streptomycin and 100 IU/ml penicillin. 2.7. Cell internalization Internalization studies of P-P-FA-99mTc were conducted using PBS 1X and 0.1 M NaOH. C2C12, 4T1 and MCF-7 cells were harvested at 5 × 104cells/300 μl in medium and cultured in 24 well plates overnight. Then, 500 nM (175 μCi), 250 nM (90 μCi) and 125 nM (50 μCi) of PP-FA-99mTc were added to each well and incubated. At various time intervals (1, 3 and 24 h), RPMI and DMEM medium of wells were removed and medium activity, as a non-uptake drug conjugates, was read by γ-counter. In addition, the cultured cells in any of the wells were washed with filtered PBS 1X, incubated for 15 min and 400 μl of 0.1 M NaOH to separate cells and cell activity was counted using γ-counter as uptake and receptor conjugated carriers. Differences between the medium and cell activities were considered as an internalized P-P-FA-Suc-99mTc [32]. 2.8. MTT assay The growth inhibitory effects of PAMAM G4, P-P, P-P-FA, P-P-FA-5FU, P-P-FA-Suc-99mTc and P-P-FA-5FU-suc-99mTc with 1000, 500, 250, 125 and 62.5 nM concentrations on MCF-7 and C2C12 cells were determined using the MTT assay with 9 repeat (at 24, 48 and 72 hour after treatment). Briefly, the cells were cultured on 96-well microtiter plates and were allowed to attach overnight. Subsequently, cells were treated with different type of drugs. After the posttreatment times, MTT solution (5 mg/ml in PBS) was added to each well and incubated for another 4 h. Consequently, the liquid suspension was removed and cells were resuspended in DMSO. The optical density (OD) of these DMSO solutions was read at 570 nm, and OD differences of treated and non-treated cells revealed the cell viability. 2.9. Mouse model with breast tumor Five- to-six week-old female BALB/c mice (avg. body weight 16.0 ±1.5 g) were used for the in vivo studies. The tumor was established by subcutaneous injection of 4T1 tumor cell line in the right side of the post neck region. The mouse breast carcinoma 4T1 cell line was grown continuously as a monolayer at 37 °C and 5% CO2. The 4T1 cells were prepared for the injection in the female BALB/c mice. A 2 ml of 0.25% trypsin/1 mM EDTA solution was added to the plate and swirled to cover the plate and incubated at room temperature for 2 min, followed by addition of 5-6 ml serum-free media to harvest the trypsinized cells from the plate and transfer to a conical tube. These cells were centrifuged, the supernatant were discarded and the pellet was resuspended in serum-free medium. The cells were diluted with a serum-free medium to the desired concentration (1 × 106 cells/0.2 ml). These 4T1 tumor cells in serum-free medium suspension were inoculated subcutaneously. Then, the mice were monitored daily for tumor onset by palpating the injection area with the index finger and thumb for the presence of tumor. After 12 to 15 days of inoculation, the mice injected with 4T1 cells developed palpable solid tumors (619.2 ± 98.5 mm3 in volume). 5
2.10. In vivo imaging Imaging was performed on female, 7–8 weeks old tumor bearing BALB/c mice (n=3). The planar γ-camera imaging was performed by means of an in vivo single head γ-camera. The mice were treated with 5.4 MBq (200μCi) of 99mTc labeled dendrimer (P-P-FA-Suc-99mTc) through the tail intravenous (IV) injection. The mice were studied for imaging evaluation of radiolabeled smart nano-complex at 1, 3 and 24 h of post-injection time. 2.11. Biodistribution study Normal and tumor bearing BALB/c mice were divided into two groups (n=12). Practically, 200 μCi of the radiolabeled dendrimer (P-P-FA-Suc-99mTc) in PBS was injected, intravenously. After 1, 3, and 24 h post-injection interval times, the mice were euthanized and the main organs including the heart, lung, adrenal, stomach, intestine thyroid, liver, spleen, kidney, muscle, bone, bladder, pancreas, blood, skin, tail, whole body and tumor were removed and weighted (stomach and intestine were washed with PBS, firstly). The radioactivity of P-P-FA-Suc-99mTc accumulated organs were detected by a γ-counter and the results were expressed as percentages of the injected dose per gram (% ID/g) organs. 2.12. Statistical analysis Comparisons between the groups were expressed as mean ± standard error of the mean. Statistical differences were evaluated using one way ANOVA and Tukey’s HSD with the level of statistical significance at p < 0.05. 3. Results and Discussion 3.1. Characterization of synthesized nano-complexes FT-IR and 1H NMR spectroscopies were applied to confirm the synthetic nano-complexes. In FT-IR analysis (Fig. 1), the pure PAMAM G4 characteristic absorption peaks were obtained at 3306 cm-1 (N–H stretch of amines); 3180 cm-1 (antisymmetric N–H stretch of substituted primary amine) and 1252 cm-1 reflecting the C–O stretching vibration; 1112 cm-1 (C–C bending) [34, 35]. Absorption peaks at 1643 cm-1 (–C=O stretch); 2887 cm-1 (C–H stretch); 3408 cm-1 (N– H stretch of amide) and 1734 cm-1 (C=O stretch of carbonyl group) were revealed for P-P nanocomplex [36, 37]. There was an upshift of C=O stretch of acid group of carboxymethyl mPEG of amide linkages. For P-P-FA nano-complexes, important peaks of folic acid were at 3545, 3415 cm-1 (N–H stretch of primary amine and amide); 3328 cm-1 (alkyl C–H and C=C stretch); 1695 cm-1 (aromatic C=C bending and stretching); 1492 cm-1 (CH–NH–C=O amides bending); 837 cm-1 (aromatic C–H bending and benzene 1,4-disubstitution); and for the NHS conjugated folic acid, 3690 cm-1 (amide N–H and C=O stretching); 2993 cm-1 (carboxylic acid C=O and O–H stretching unconjugated); 1712 cm-1 (ketones C=O unconjugated stretch), which confirmed the synthesis of NHS ester of folic acid [35, 38]. The 5-FU loaded P-P-FA nano-complex FT-IR analysis revealed absorption peaks at 3238 cm-1 (N–H stretching vibration); 3068 cm-1 (C–H stretching vibration); 2929 and 2829 cm-1 (–CH2– symmetric and asymmetric stretching vibration). Also, other peaks such as 1725, 1648 and 1249 cm-1 that are related to the carbonyl 6
stretching vibrations were determined; 1232 cm-1 (C–F stretching vibration); 991 cm-1 (N–H and C–H wagging vibration); 811 cm-1 (C–F stretching vibration) [39]. The peaks of conjugated linker were achieved at 3038 cm-1 (CH2 stretching vibrations); 1701 cm-1 (COO– stretching vibration); 3693 cm-1 (amide N–H stretching); 835 cm-1 (aromatic C–H bending), which indicated the HYNIC conjugation to our nano-carrier. Absorption peak at 935, 920, 892 and 898 cm-1 were related to TcO4– in P-P-FA-99mTc and P-P-FA-5FU-Suc-99mTc [40]. 1 H NMR spectra of the synthetic nano-complexes were also implemented (Fig. 2). The P-P-FA nano-complex aromatic folate proton peaks were obtained at δ 6.71, δ 7.60, and δ 8.69 ppm, and PEG proton peak was revealed at δ 3.70 ppm [34, 35]. The multiple proton peaks of PAMAM were found at δ 1.29, δ 1.34 ppm and and δ 2.45 to δ 2.68 ppm [35, 38]. In addition, 5FU proton peak was ascertained at δ 7.58 ppm. Proton peaks in the range of δ 0.83 to δ 2.06 ppm and δ 3.29 ppm were assigned to aliphatic protons of CH2NHC=O and amide bond formation. Size diameter determination of synthetic nano-complexes were ascertained by means of TEM (Fig. 3), and final nano-complexes size were determined between 30 to 80 nm in diameter. The surface charges of the samples were also ascertained and -2.10 ± 1.11 and -1.69 ± 0.51 mV were obtained for P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc nano-complexes, respectively (Table 1). The values of 99mTc were also measured by the Energy Dispersive X-ray Spectroscopy (EDS or EDX). The EDS is a chemical microanalysis technique used in conjunction with scanning electron microscopy (SEM) to provide elemental identification and quantitative compositional information. The weight and atomic percentage of 99mTc, F and other elements were determined in P-P-FA-5FU-99mTc and P-P-FA-99mTc samples via EDS. The weight percentage of carbon and 99mTc were ascertained as 93.66 and 6.34% for P-P-FA-Suc-99mTc, respectively. Also, the weight percentage for carbon, F and 99mTc elements were determined as 64.76, 25.37 and 9.87% in P-P-FA-5FU-Suc-99mTc, respectively (Fig. 3). 3.2. PBS and serum stability Radiochemical stability of P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc was evaluated by using the ITLC at different time intervals (1, 2, 4 and 24 h) (Fig. 4 A and B). After incubation of 200 μCi of P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc (radiolabeling yield >95%) in PBS and serum at 37 °C for 24 h, more than 76 % conservation of total radioactivity was revealed. The P-P-FA-Suc-99mTc stabilities were determined as 85.28±1.99 % and 86.67±2.11 % in PBS and serum after 1 h, respectively. It was the same for P-P-FA-5FU-Suc-99mTc nano-complex in the same time (85.32±1.15 % in PBS and 83.75±0.88 % in serum). Also, it was 77.2±0.76% and 77.98±0.74 % in the PBS and 76.27±1.02 % and 78.35±1.47 % in the serum samples after 24 h for P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc, respectively. In comparison with antibody labeling efficiency, PAMAM G4 nanoparticle could be considered as good candidate for radiolabeling and imaging [41]. In general, stability decreases after 24 h which could be attributed to protein interactions such as albumin in the serum [42].
3.3. Cell internalization Cellular uptake of FA targeted nano-carrier was evaluated on normal and cancer cell lines to investigate FR-mediated endocytosis (Fig. 4 C and D). Higher internalization of P-P-FA-Suc7
99m
Tc was observed in MCF-7 cell line with respect to the normal cells. The results indicated that 64.11±0.88 % of nano-complex (500 nM) was internalized by MCF-7 cancer cell line after 24 h, while in the same condition, 45.74±0.48 % for C2Cl2 as a control cell line was determined. A comparison of the first and last uptake values demonstrated a considerable uptake difference between the normal and cancer cell. For example, MCF-7 cells internalized 41.11% of the smart nano-complex after 24 h at 500 nM; however, it was 12.52 % for normal cell lines in the same condition which crucially revealed the importance of FA targeting [11, 21]. Generally, the cellular uptake of P-P-FA-Suc-99mTc was depicted as time and concentration dependent behavior in a direct relation. 3.4. Cell viability assay Cell viability was implemented by MTT assay on human breast cancer (MCF-7) and normal (C2Cl2) cell lines for all synthetic nano-complexes (Fig. 5). Synthesis of the novel nanocomplexes triggered our interest to evaluate the cytotoxic effect of surface modified and engineered PAMAM G4 dendrimer as a carrier system for the delivery of chemotherapeutic radiolabeled agent on cell lines. All compounds included pure PAMAM G4, P-P, P-P-FA, P-PFA-5FU, P-P-FA-Suc-99mTc and P-P-FA-5FU-Suc-99mTc in different concentrations (62.5, 125, 250, 500 and 1000 nM). It is worth mentioning that PEGylation of PAMAM G4 demdrimer can considerably decrease the cytotoxic effects of positive charge PAMAM G4 and effectively increase the biocompatibility and stability of polymer. For instance, in concentration of 1000 nM the PEG reduced the cytotoxicity of PAMAM G4 from 43.75±0.55 % to 79.86±1.56 % of cell viability in MCF-7 cell line (p<0.01) [13, 15]. Also, at up to 500 nM concentration, the P-P-FA strengthens the percentage of the cell viability which may be due to augmentation of PEGylated PAMAM G4 and also usage of cell from FA as a nutritional compound (p<0.05). The cell viability was enhanced from 53.95±0.61% to 66.68±0.73% after PEGylation at 250 nM (after 24 h in MCF-7) which increased to 84.07±0.54 % after FA addition to P-P [9, 10]. A comparison of the C2Cl2 normal cell line with MCF-7 regarding the effects of various types of nano-complex types showed that pure PAMAM G4 had cytotoxic effects on normal cell in comparison with cancer cell lines and PEGylation mainly reduced the side effects of PAMAM G4 on the normal cell lines. The P-P-FA nano-complex in both cell lines demonstrated nearly the same effects, but mainly different from those of concentration. Drug loaded nano-complex (P-P-FA-5FU) has been extremely cytotoxic in comparison with others. It showed concentration dependent cell viability assay, as 23.82±0.56% and 14.69±0.84% cell viability were obtained at 62.5 and 1000 nM 1000 nM concentration for MCF-7, and 27.51±0.65% and 23.42±0.46% of cell viability were obtained for C2Cl2 at the same concentration after 24 h. These revealed that drug loaded nano-complex has more effective cytotoxicity on cancer cell in contrast with normal cell lines. Radiolabeled samples were tested on cell viability through both P-P-FA-Suc-99mTc and P-PFA-5FU-Suc-99mTc nano-complexes. Due to the low half-life of 99mTc, radiolabeled complexes were also assessed at 3 and 24 h post treatment. Synergistic effects of drug and 99mTc were observed; however, major cell inhibition effect was related to 5FU. Apparently, high cell inhibition effect of radioactive agent was obtained at high dose of 99mTc. Values of 69.80±1.98 % and 86.19±0.51 % of cell viability were obtained at 1000 nM of P-P-FA-Suc-99mTc after 3 and 24 h of treatment, while 13.10±1.4 % and 87.34±2.35 % of the cell viability were observed for P8
P-FA-5FU-Suc-99mTc at 3 and 24 h for 1000 nM, respectively (Fig. 5). Normal cell as a control was also the same for 99mTc radiolabeled nano-complexes. 3.5. In vivo imaging Fig. 6 A to C shows the γ-camera taken images of female BALB/c mice bearing breast carcinoma tumor at 1, 3 and 24 h interval times as a molecular imaging technique in clinical and basic research fields. P-P-FA-Suc-99mTc nano-complex as novel radiotracer was completely localized at site specific area of tumor after IV injection as indicated in the images. 3.6. Biodistribution study For biodistribution study, we attempted to label functionalized PAMAM G4, targeting highly metastatic cells with 99mTc, for potential imaging of tumors and metastases. Fig. 6 D and E demonstrates the biodistribution profile of P-P-FA-Suc-99mTc radiotracer in the mice with breast tumor at 1, 3 and 24 h after intravenous administration and was expressed as percentage of injected dose per gram (%ID/g) of organs. Biodistribution studies at 1, 3 and 24 h of postinjection revealed the specific localization of nano-complex at the tumor site. The liver, spleen and tumor were indicated as a substantial accumulation of radioactive nano-complex, respectively. Tumor uptake of P-P-FA-Suc-99mTc radiotracer showed time-dependent manner against the liver and spleen uptake especially at the last time. The tumor uptake (%ID/g) was 4.66±0.64 % at 1 h post-injection which increased to 13.76 ± 1.39 % after 24 h revealing excellent tumor targeting. As the tumor uptake of nano-complex was interestingly higher than that of other organs except the liver and spleen, we could conclude that these organs did not actively uptake P-P-FA-Suc-99mTc given that they did not over-express folate receptor that leads to a high retention of radioactivity in the tumor. Also, high plasma concentration of the nanocomplex indicated good circulation of radiolabeled dendrimer as the site specific targeting delivery systems, and with respect to peptide and antibody based radiotracers, late clearance from plasma were shown [9, 11, 43]. The in vivo biodistribution profiles demonstrate that the radioactivity in the kidney was beyond the normal levels 1 h after nano-complex IV administration, illustrating that P-P-FA-Suc-99mTc was mainly excreted through the kidney. These data were in line with the results of Chen et al [41, 44, 45]. In this study, we evaluated smart PAMAM G4 labeled with a radionuclide as a tumor-targeting imaging agent for the first time. P-P-FA-Suc-99mTc biodistribution and γ-camera imaging demonstrated its ability to target highly metastatic tumors. The uptake values of the other organs of the normal and tumor bearing BALB/c mice are presented in Table 2. 4. Conclusion Herein, we report PEG modified and FA functionalized PAMAM G4 as a smart low toxic nano-carrier asset to be used in therapeutic, imaging and biodistribution domains. Our results demonstrate that a novel nano-complex has excellent potentials for the delivery of 5-FU chemotherapeutic agents for breast cancer cell lines and tumor accumulation with best internalization to cancer cells. This nano-complex was investigated for quantitative in vivo biodistribution according to the accumulation into the tumor, liver and spleen following intravenous administration in the breast tumor bearing mice. The tumor accumulation indicated 9
the targeting capacity of the smart nano-complex. Moreover, imaging study of the site specific targeting radiotracer was ascertained and the results confirm the excellent power of nanocomplex in the tumor bearing mice model. In conclusion, the novel smart synthetic nanocomplex is applicable for the treatment, tracing and imaging of tumors at clinical levels. Acknowledgments This paper has been extracted from an MSc thesis. The authors would like to acknowledge all the supports of University of Tehran. Tehran, Iran (Grant no. 28701/06/09).
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Fig 1. FT-IR spectra of PAMAM G4 nano-complexes. The main locations of any compounds and linkage formation in conjugates were illustrate in text via their specific absorption peaks.
Fig 2. 1H NMR spectra of P-P-FA-Suc-99mTc (up) and P-P-FA-5FU-Suc-99mTc (down) nanocomplexes. The folate proton peaks were obtained at δ 6.71, δ 7.60, and δ 8.69 ppm, and PEG proton peak was revealed at δ 3.70 ppm. The 5FU proton peak was ascertained at δ 7.58 ppm.
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Fig 3. TEM image (up) and EDS (down) graph of P-P-FA-Suc-99mTc (left) and P-P-FA-5FUSuc-99mTc (right) nano-complexes. The size of both nano-complex was determined between 30 to 80 nm in diameter.
95
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Fig 4. The PBS and serum stability and cell uptakes. PBS and serum stability of 99mTc radiolabeled nano-complexes at various time intervals (A and B). Cellular uptake of C2Cl2 and MCF-7 cell in 125 nM, 250 nM and 500 nM concentrations after 1, 3 and 24 h post treatment time intervals (C and D).
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P-P-FA-5FU P-P-FA-Tc
60
P-P-FA-5FU-Tc 40 20 0 0
62.5
125
250
500
1000
Concentration (nM) 120
P
C2Cl2 (24 h)
Cell viability (%)
100
P-P P-P-FA
80
P-P-FA-5FU 60
P-P-FA-Tc P-P-FA-5FU-Tc
40 20 0 0
62.5
125
250
Concentration (nM)
18
500
1000
120
P
MCF-7 (48 h)
Cell viability (%)
100
P-P P-P-FA
80
P-P-FA-5FU 60 40 20 0 0
62.5
125
250
500
1000
Concentration (nM) 120
P
C2Cl2 (48 h)
P-P
Cell viability (%)
100
P-P-FA 80
P-P-FA-5FU
60 40 20 0 0
62.5
125
250
Concentration (nM)
19
500
1000
120
MCF-7 (72 h)
P
Cell viability (%)
100
P-P P-P-FA
80
P-P-FA-5FU 60 40 20 0 0
62.5
125
250
500
1000
Concentration (nM)
120
C2Cl2 (72 h)
P
Cell viability (%)
100
P-P P-P-FA
80
P-P-FA-5FU 60 40 20 0 0
62.5
125
250
Concentration (nM)
20
500
1000
120
MCF-7 (3 h)
P-P-FA-Tc
Cell viability (%)
100
P-P-FA-5FU-Tc
80 60 40 20 0 0
62.5
125
250
500
1000
Concentration (nM) 120
C2Cl2 (3 h)
P-P-FA-Tc
Cell viability (%)
100
P-P-FA-5FU-Tc
80 60 40 20 0 0
62.5
125
250
500
1000
Concentration (nM)
Fig 5. Cytotoxicity assessments by MTT assay in MCF-7 and C2Cl2 cell lines after 3, 24, 48 and 72 h of post-treatment with P, P-P, P-P-FA, P-P-FA-5FU, P-P-FA-Suc-99mTc and P-P-FA-5FUSuc-99mTc nano-complexes.
21
90
D 80 70
ID/g (%)
60
1h
50
3h
40
24h
30 20 10 0
22
90
E 80 70
1h
ID/g (%)
60
3h
50
24h 40 30 20 10 0
Fig 6. The imaging study and biodistribution of synthetic nano-complex in mice body. γ-Camera image of tumor bearing BALB/c mice after 1 h (A), 3 h (B) and 24 h (C) of P-P-FA-Suc-99mTc tail IV injection. Evaluation of P-P-FA-Suc-99mTc biodistribution in normal (A) and tumor bearing (B) BALB/c mice (%ID/g) (D and E).
23
Table 1. Particle diameter and their surface charges determined via DLS for dendrimer-modified nanoparticles PAMAM modification
Dispersed in
Diameter/nm
Surface charge (mV)
PAMAM PAMAM-PEG PAMAM-PEG-FA PAMAM-PEG-FA-5FU PEG-PAMAM G4-FA-5FU PEG-PAMAM G4-FA-5FU-Suc PEG-PAMAM G4-FA-Suc PEG-PAMAM G4-FA-Suc-Tc PEG-PAMAM G4-FA-5FU-Suc-Tc
H2O H2O H2O H2O H2O H2O H2O H2O H2O
4.49 17.28 58.27 58.45 58.45 52.09 59.33 69.88 76.58
25.38 ± 2.8 -24.38 ± 5.16 -25.63 ± 1.62 -21.71 ± 0.71 -21.71 ± 0.71 -2.58 ± 0.60 -7.89 ± 2.96 -2.10 ± 1.11 -1.69 ± 0.51
Table 2. In vivo biodistribution values of P-P-FA-Suc-99mTc nano-complex in various organs (%ID/g). Organs
Blood Heart lung Adrenal Stomach intestine Thyroid Liver Spleen Kidney Muscle Bone Bladder Tail Pancreas Skin Whole body
Normal mice
Tumorous mice
1h
3h
24 h
1h
8/21±1.68 1/86±0.17 7/46±1.01 2/65±0.43 15/31±4.14 1/66±0.27 4/45±0.98 82/08±5.63 26/42±1.56 6/48±0.8 0/79±0.26 0/94±0.38 0/75±0.34 2/37±0.09 3/27±0.78 1/67±0.02 0/98±0.25
7/06±1.59 1/62±0.03 5/7±0.2 0/64±0.05 6/71±0.34 1/69±0.06 3/51±0.84 72/21±1.98 25±0.98 5/24±0.73 0/43±0.31 0/76±0.23 0/82±0.28 3/03±0.31 1/26±0.06 1/12±0.1 1/25±0.41
4/21±0.49 0/77±0.06 3/6±0.22 7/21±0.37 0/83±0.06 0/69±0.01 0/37±0.16 46/74±2.07 16/73±0.45 4/38±0.71 0/26±0.19 0/46±0.19 0/44±0.22 2/36±0.9 0/79±0.12 0/61±0.07 3/67±0.69
9/89±1.21 2/49±0.15 6/51±0.53 2/69±0.05 7/18±0.85 2/01±0.36 2/44±0.65 83/21±3.72 13/31±1.1 6/16±0.25 1±0.04 0/79±0.07 1/41±0.03 4/39±0.25 2/07±0.21 2/75±0.27 1/57±0.29
7/68±0.89 1/72±0.02 3/68±0.5 0/87±0.27 6/32±0.59 1/99±0.25 1/19±0.14 69/34±1.36 14/43±0.79 4/09±0.2 1/51±0.03 0/5±0.03 0/71±0.02 2/55±0.16 0/72±0.07 1/24±0.03 1/57±0.35
5/46±0.94 4/21±0.8 1/54±0.07 1/35±0.65 2/97±0.67 1/96±0.29 3/59±0.46 13/62±1.29 3/28±0.28 4/45±0.43 2/13±0.91 1/12±0.13 0/84±0.17 1/79±0.61 2/25±0.1 3/01±0.23
4/66±0.64
10/61±0.48
13/76±1.39
Tumor
24
3h
24 h
6/02±0.78