Chemotherapy of Prostate Cancer by Targeted Nanoparticles Trackable by Magnetic Resonance Imaging

Prostate cancer (CaP) is the commonest diagnosed malignancy and the second main cause of cancer mortality in males in the United States. Thus, there is an urgent need to develop novel drug delivery systems to improve the chemotherapy option for CaP patients. The goal of this paper is to describe novel moleculary guided nanoscale drug delivery system with dual functionality for treatment and MR imaging of CaP. We describe the synthesis of iron oxide nanoparticles (IONPs) which are then coated with carboxyl-ended amphiphilic polymer. We present the protocol for tethering of the CaP targeting protein, human amino terminal fragment (hATF) to the terminal carboxyls of the IONPs. We describe the drug loading and release and the methods for measuring of the internalization of the hATF-guided IONPs into CaP cells. We also describe the methods for usages of IONPs are MR imaging contrast agent and successful targeted drug carriers.


Introduction
Prostate cancer (CaP) is the most prevalent noncutaneous malignancy in the United States with an estimated 192,280 new cases and 27,360 deaths in 2008 [1].It is estimated that CaP alone accounts for approximately 29% of cancer cases in men, with one in six men expected to develop invasive CaP in his lifetime.Although the chemotherapeutic armamentarium of drugs has been a major option for treatment of advanced CaP patients [2,3], chemotherapy has several drawbacks such as the severe cytotoxic side-effect on normally proliferating tissues [4].In addition, development of multidrug resistance by cancer cells often leads to the eventual failure of treatment [5].
In the last two decades, there has been an exponential growth of interest in magnetic nanoparticles (MNPs) which offer unique physical properties and ability to function at the cellular and molecular level of biological interactions [6,7].In particular, MNPs present exciting new opportunities including dual functionality of site-specific delivery of anticancer drugs coupled with an enhanced quality of magnetic resonance imaging (MRI) [8].Thus, MNPs have the potential to revolutionize current clinical diagnostic and therapeutic strategies for CaP patients [9][10][11].
The most commonly studied MNPs are iron oxide nanoparticles (IONPs), mostly maghemite, γ-Fe 2 O 3 , or magnetite, Fe 3 O 4 [6,7,[12][13][14][15].IONPs have become popular options for potential multifunctional drug delivery vehicles for several reasons.First, the chemistry of the surface modification for these nanoparticles is well known, and thus molecules such as targeting ligands and drugs can be conveniently tethered to their surface [12,[15][16][17][18]. Secondly, they have been extensively used as MRI contrast agents due to their superparamagnetic property [6,16].Lastly and most importantly, they are known to be biocompatible and nontoxic as upon metabolism, iron ions are added to the body's iron stores and eventually incorporated by erythrocytes as hemoglobin allowing for their safe use in vivo [7,19].
To achieve optimum performance of MNPs as drug delivery vehicles, it is essential to well tailor their surface chemistry, size (magnetic core and size distribution), and magnetic properties (magnetic moment, remanence, and coercivity) [20].There are vital reasons to modify the surface chemistry for MNPs so they can serve as successful drug delivery vehicles [18,[21][22][23].First, these MNPs must be well dispersed in plasma for a reasonable period without aggregation which otherwise might cause severe embolization in vivo by blocking of capillary vessels.Second, nonspecific adsorption of plasma proteins onto MNPs surfaces should be prevented, as this would result in uptake of these nanoparticles by macrophages of the reticuloendothelial system in the liver and spleen.Finally, it is desirable that suitable functional groups are present on the surfaces of MNPs; as such groups are used to immobilize bioactive ligands for targeting or therapeutic purposes.
In order to be able to achieve dispersibility in an aqueous medium, the best surface modification for these NPs is coating them with an amphiphilic polymer system [18,24].The amphiphilic polymer will provide the hydrophobic part which can anchor to the magnetite NPs surface while the hydrophilic portion is directed towards the aqueous environment, thus providing robust water dispersibility.Moreover, to block protein adsorption to these NPs, a hydrophilic and biocompatible polymer such as poly(ethylene glycol) (PEG) is tethered to the amphiphilic polymeric coating which increases the circulatory half-life from minutes to hours or days [8,13,14].
Thus, based upon the wide variety of parameters that need to be accounted for the design, preparation, optimization, and characterization of the magnetite nanoparticles, we present here some of the techniques we have employed in our laboratory to design smart magnetic nanovehicles for targeted drug delivery.For convenience, we will refer throughout this paper to the magnetite nanoparticles as IONPs.

Synthesis and Surface Modification of Iron
Oxide Nanoparticles (IONPs) All the chemicals for the synthesis of the IONPs are obtained from Sigma-Aldrich, St. Louis, MO.

2.1.
Coprecipitation Method.This is the most common chemical method for the synthesis of IONPs [25].In this method, it is essential to control the reaction kinetics, which is strongly related with the oxidation speed of iron species.Thus, the synthesis of magnetite NPs must be done in an oxygen-free environment by passing nitrogen gas through the reaction system.Bubbling nitrogen gas through the solution not only protects critical oxidation of the magnetite but also reduces the particle size when compared with methods without removing the oxygen [12,17].The procedure involves that ferric (Fe 2+ ) and ferrous salts (Fe 3+ ) (molar ratio 2 : 1) are dissolved in deoxygenated water at a concentration of 0.1 M of iron ions.Chemical precipitation is achieved by using a 1 M deoxygenated solution of sodium hydroxide.The reaction is carried out in nitrogen atmosphere at low temperature (4-6  1) [28,31].The size and morphology of the synthesized NPs are determined from their transmission electronic microscopy micrographs.Samples for the TEM experiments are prepared by suspending a dried sample in absolute ethanol.A drop of the sample suspension is allowed to dry on a 400-mesh copper grid (Electron Microscopy Sciences, PA) coated with a carbon film.Particles are imaged using a JEOL-JEM 100 SX electron microscope (JEOL, CA) at an accelerating voltage of 200 kV.It can be observed from Figure 1(b) that the IONPs nanocrystals are relatively uniform with an average size of 10 nm [27,28,30].The magnetic properties of the synthesized IONPs under a magnetic field are evaluated by alternating gradient magnetometer (AGM) to confirm the feasibility and sensitivity as MRI nanoprobes.The field dependence of magnetization is recorded at 300 K under circulate magnetic field ranging between 2 and −2 T. The results showed that the IONPs exhibit superparamagnetism without magnetic hysteresis at room temperature (300 K) (Figure 1(c)) [27,28,30].

Surface Modification of IONPs by Amphiphilic Polymer.
All the chemicals for the surface modification of the IONPs are obtained from Sigma-Aldrich, St. Louis, MO.The amphiphilic polymer is composed of three functional parts: hydrophobic groups for anchoring onto the IONPs surface, PEG for blocking protein adsorption, and carboxylic acid to permit the introduction of bioactive molecules [31,32].First, the procedure involves the synthesis of the amphiphilic polymer as follows [18,24].Dodecyl methacrylate (4 mmol, 1.016 g), poly(ethylene glycol) methyl ether methacrylate (PEGMA, average M n = ca.475; (4 mmol, 1.9 g), and methacrylic acid (2 mmol, 0.172 g) are dissolved in 10 mL of tetrahydrofuran (anhydrous, 99.9%, inhibitor-free).This mixture is degassed for 15 min by bubbling with stream of N 2 gas.After adding 0.1 mmol of 2,2 -azobisisobutyronitrile (16.4 mg) as a radical initiator, the vial is sealed with a Teflonlined screw cap.The polymerization reaction is carried out at 7 • C for 24 h.The final product solution is cooled to room temperature and is stored at 4 • C until use.After the modification is completed, the IONPs concentration is determined by measuring absorbance at A 500 (Beckman DU 650 UV-VIS Spectrophotometer, CA) [9,33].Generally, a 10 mg/mL solution of IONPs gives an absorbance of 32.5, so generally we make sure that the absorbance values fall between 0.2 and 0.8 by using appropriate dilution, and then we use the dilution factor to estimate the final IONPs concentration based on (1): IONPs concentration mg/mL = A 500 × dilution factor 3.25 . (1)

Production of the Targeting Protein hATF
To enable the selective and specific delivery of anticancer drugs to prostate cancer cells, we have taken advantage of urokinase plasminogen activator receptor (uPAR) overexpression in prostate cancer compared to normal prostate epithelia [34,35].Specifically, we employed the human 135 amino-acid amino-terminal fragment (hATF) of urokinase plasminogen activator (uPA), a high-affinity natural ligand for uPAR.The expression of the hATF is carried out as follows.The DNA sequence encoding the first 135 amino acids (with a C-terminal His-tag) of amino terminal fragment (ATF) of the human uPA that binds to uPAR is cloned into pET-20b(+) (Novagen) between EcoRI and XhoI sites of the vector [36].These recombinant plasmids are transformed in competent TOP10F Escherichia coli cells (Invitrogen, CA) [36,37].Transformants are selected on a LB-Agar-Amp plate 50 μg/mL ampicillin (LB/amp agar) which is incubated at 37 • C overnight.A colony is selected from the plate, and it is used to inoculate 25-50 mL sterile LB-Amp medium.
The inoculate is incubated at 37 • C at 250 rpm for 12 h, and the plasmid is isolated using plasmid Midi Kit (QIAGEN, CA).To express target protein, the plasmid is transformed in expression bacteria BL21 cells (Invitrogen, CA).A small amount of BL21 transformation mixture is placed on an LB-Agar-Amp plate and is incubated at 37 • C overnight.Four colonies are selected and are inoculated in four different tubes with sterile 5 mL LB-Amp medium and then incubated overnight at 37 • C. Three hundred microliters of the overnight culture are added to 4 different fresh sterile tubes each containing 10 mL of sterile LB-Amp, and these tubes are incubated at 250 rpm at 37 • C to OD 600 of 0.6-0.8.Each culture is then split into two 5 mL cultures using fresh sterile tubes making a total of 8 tubes from 4 cultures.For induction of protein over expression, one tube is induced by adding 5 μL of 1 mM isopropyl-thio-β-D-galactopyranoside (IPTG), and the second tube is kept as a control (un-induced).Both the induced and uninduced tubes are incubated at 250 rpm at 37 • C for 4 h.At each hour during this incubation period, 500 μL aliquot from each culture (0 h samples) is removed into a microcentrifuge tube and is pelleted at 13000 rpm for 30 sec.The supernatant is removed by aspiration, and the crude extract and purified proteins from the cultures are analyzed by SDS-PAGE followed by Coomassie brilliant blue staining.After confirming the successful expression of the hATF protein, the process is scaled up as follows.A single colony of the transformant is grown in 2.5 liter of LB medium containing 100 mg/mL of ampicillin and is incubated at 37 • C at 250 rpm until a measured OD 600 of about 0.6-0.8 is achieved.To induce the production of recombinant protein, IPTG is added to a final concentration of 1 mM.The cells are incubated for an additional 3 h at 37 To determine the concentration of the protein in each sample, the Bradford assay (Bio-Rad, CA) is used.

Conjugation of Infrared Dye to Targeting Moiety.
In order to increase the imaging capability of our targeted IONPs, the fluorescent Cy5.5 dye is conjugated to the targeting moiety, hATF, and it is recommended to wash the protein before proceeding with any conjugation.Three hundred microliters of the stock protein solution are placed into a Nanosep 3 K centrifugal column (Pall, Ann Arbor, MI), and 300 μL of sterile water is added to it, and the mixture is centrifuged at 5000 rpm for 20 min.This washing step is repeated one or two more times, and generally about 300 μL of the protein solution is collected into a fresh microcentrifuge tube.The conjugation of the dye to the protein is carried out based on protocol from the dye's manufacturer (GE Healthcare, NJ).In an eppendorf tube, 200 μL of sterile water and 200 μL of 10x PBS buffer (pH 7.8) are added to 100 μL of protein solution (concentration 1 μg/μL) and mixed for 5 min.To this solution, 1.2 μL of TCEP (0.05 M) (Sigma Aldrich, MO) is added and mixing is continued for an additional 5 min.The dye 2.4 μL of Cy5.5 monomaleimide stock solution 1 mg of dye dissolved in 1 mL dimethylsulfoxide (Sigma Aldrich, MO), is added to this mixture and is gently rotated at RT for 1 h in dark environment.Free dye molecules are separated using Nanosep 3 K centrifugal column, and the hATF-Cy5.5conjugate is washed with sterile 1x PBS and is immediately stored at 4 • C.

Conjugation of Targeting Protein to Amphiphilic Polymer-Coated IONPs.
The hATF-Cy5.5 complex is conjugated to the surface of IONPs via cross-linking of carboxyl groups to amino side groups of the protein [9].The concentration of IONPs needed for each reaction is calculated based on the size of the reaction, and a ratio of 1 : 20 of IONPs to protein-dye complex is used.Two hundred microliters of the activation buffer 20 mM borate buffer, pH 5.0 (Ocean Nanotech, AR) are added to 100 μL of the amphiphilic polymer-coated IONPs (concentration 5 mg/mL).Fifty microliters of freshly prepared 1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide (EDAC) and Nhydroxysulfosuccinimide (Sulfo-NHS) (Sigma Aldrich, MO) solutions are added to activate the IONPs, and the mixture is rotated gently for 20 min.Four hundred microliters of the reaction buffer (10 mM borate buffer (pH 8.5), Ocean Nanotech, AR) are added to the above mixture, and the hATF-Cy5.5complex is immediately added, and the mixture is reacted by gentle mixing in a rotary mixer at RT for 2 h and overnight at 4

Loading and Release of Drug from IONPs.
We have chosen noscapine (Nos) as our chemotherapeutic drug but the following procedure applies to any water-soluble drug.
An aqueous solution of noscapine (1 mg/mL) is added to NT-IO or hATF-Cy5.5-IONPs at a ratio of 1 mg drug to 3 mg of iron (Fe), and the mixture is rotated at RT for 4 h and at 4 • C overnight.After filtration of NPs through the Nanosep 100 K centrifugal column, the free drug in the flow-through is analyzed by high-performance liquid chromatography (HP Agilent 1100 HPLC).A standard curve for the drug is generated using concentration range of 5−25 μg/μL.A reverse phase C 18 symmetry column (Symmetry C 18 , 5 μm, Waters) is used as the stationary phase, and the mobile phase is a mixture of 20 μM ammonium acetate solution and acetonitrile (65 : 35 v/v).The injection volume is 10 μL, and the flow rate of the mobile phase is 1 mL/min.The column effluent is monitored at 232 nm with a UV detector.The encapsulation efficiency of noscapine is calculated as the mass ratio of the amount of drug entrapped in NPs to the initial amount which is used in drug loading (Table 1).
Next, the release of the adsorbed drug from the surface of the amphiphilic polymer coated IO NPs is determined as follows.To simulate physiological conditions, we measure the amount of Nos that can be released from the NPs at different pH.Slightly acidic pH conditions simulate the environment of the tumor interstitium while more acidic pH is more representative of the pH in intracellular vesicles such as endosomes and lysosomes.So, Nos-containing NPs are incubated in solutions of different pH (4, 5, 6, or 7) for 2 h.The free drug molecules in the buffer are then separated from the NT-IO and hATF-IO-Cy5.5NPs using Nanosep 100 K centrifugal column.The amount of free Nos is calculated from a standard curve of drug concentration and HPLC signal intensity.740 ± 98 558 ± 74 * Total number of IONPs in each sample was derived from measuring absorption of OD 500 of Fe concentration and then calculating the numbers of IONPs using (1).For example, 1 mg of Fe equals to 0.906 nmol of 10 nm IONPs.The numbers of Nos molecules in each IO nanoparticle were calculated by dividing total number of Nos with the total number of IONPs in each sample.Data from Abdalla et al. [30].

Internalization of IONPs by CaP Cells
4.1.Prussian Blue Staining.Prussian blue staining is used to confirm presence of iron in cells treated with targeted or nontargeted IONPs.In 12-well culture plates, PC-3, uPAR siRNA transfected PC-3 cells and LNCaP cells are plated and are grown in RPMI-1640 medium (supplemented with 10% fetal bovine serum (FBS)) at 37 • C with 5% CO 2 for 24 h.When the cells are about 80% confluent, the medium is removed and replaced with FBS-free RPMI medium which contains 30 pmole/mL of NT-IO or hATF-Cy5.5-IONPs and incubated for an extra 4 h.The medium is removed, and the cells are washed twice with 1x PBS and fixed using 4% formaldehyde solution.The fixed cells are stained using Prussian blue solution prepared by mixing equal amounts of 5% potassium ferrocyanide and 5% HCl for 4 h at RT. Bright field images are obtained, and blue-stained cells were quantified and represented as percentage of positive cells compared to total number of cells (Figure 2(a)).

In Vitro MRI Scan.
In 100 mm cell-culture plates (Corning, Corning, NY), PC-3 and LNCaP are incubated in RPMI-1640 medium (supplemented with 4 mM L-glutamine and 10% FBS) at 37 • C with 5% CO 2 .HEK 293 cells are used as an additional control and are incubated with Dulbecco's modified Eagle medium (DMEM) (supplemented with 4 mM Lglutamine and 10% FBS) in the same conditions.When the cells are at 80% confluent, the medium for the three cell types is removed and is replaced with medium treated with 40 picomole/mL of hATF-Cy5.5-IO.The NT-IO NPs are used as control.The cells are incubated at 37 • C for an additional 4 h.As the incubation is complete, the medium is removed, and the cells are then washed twice with 1x PBS.Then, cells are trypsinized and are collected with minimum amount of liquid.The cells are then embedded evenly in a freshly prepared 1% agarose in 1.5 mL eppendorf tubes.The samples are scanned using a 3-T MRI scanner (Siemens Healthcare, PA) using T 1 -weighted gradient echo and multiecho T 2weighted fast-spin echo imaging sequences.T 2 values of each sample are calculated from obtained multiecho (TE i , i = 20, range from 10 to 200 ms, interval = 10 ms).Transverse relaxation times, T 2 , of each sample are calculated by fitting decay curve on a pixel-by-pixel basis using the nonlinear monoexponential algorithm of M i = M 0 * exp (−TE i /T 2 ), in which M 0 is the MRI signal intensity at TE of 0, and M i is the MRI signal intensity at a selected TE (Figure 2  for one hour at RT.The membrane is washed three times for 5 min each with 15 mL of TBS/T.The immunoreactive proteins are detected by chemiluminescence (Cell Signaling Technology, Inc., Danvers, MA) as follows.The membrane is incubated with 10 mL LumiGLO (0.5 mL 20x LumiGLO, 0.5 mL 20x Peroxide and 9.0 mL deionized water) with gentle agitation for one minute at RT.The membrane is drained of excess developing solution, and it is wrapped in plastic wrap and is exposed to X-ray film.

In Vitro Cytotoxicity Assay
In a 96-well culture plate (Corning, Corning, NY), PC-3 and uPAR siRNA transfected cells are seeded at a density of 1 × 10 4 cells per well and are incubated at 37 • C and 5% CO 2 for 24 h.Normal prostate epithelia, RC-77N/E, are used as an additional control, and they are seeded at the same density and are incubated at the same conditions.RC-77N/E cells are maintained in Gibco Keratinocyte-SFM medium supplemented with EGF (human recombinant) and bovine pituitary extract (Invitrogen, Carlsbad, CA).Cells are treated with serum-free medium containing NT-IO-Nos or hATF-Cy5.5-IO-NosNPs, or free Nos (10 μM).
Additional control groups are treated with NT-IO or hATF-IO-NPs without Nos, but these control groups have equal amounts of IO as test samples.Cells are incubated with these treatments for 4 h then the serum is added to the medium, and incubation is continued for an additional 48 h.Percentage of tumor cell death is determined by crystal violet assay as follows.The medium is carefully removed from all the wells, and they are washed with 200 μL 1x PBS which is warmed to room temperature.Then, carefully the PBS is removed, and 50 μL of crystal violet solution (0.2 g crystal violet, 2 mL ethanol, 98 mL dH 2 O) is added to each well, and the cells are incubated for 10 min at RT.The plates are gently washed twice in tap water by immersion in a large beaker.The excess water is drained by placing the plates on papers and left to air dry. 100 μL of 1% SDS is added to each well to solubilize the stain, and the plates are agitated on orbital shaker until color is uniform with no areas of dense coloration in bottom of wells.The optical density is at 590 nm using a microplate reader (SpectroMax, Molecular Devices).Absorbance values were normalized to control values to obtain the percentage of viable cells.The uPAR-targeted NPs have 6-fold enhancement in cell death compared to the free drug (Figure 3).

Figure 3 5.
Figure2 separated by centrifugation for 30-45 min at 15000 rpm.The supernatant is filtered using syringe and needle through 0.2 or 0.45 μm filters into a fresh 50 mL tube.Then, 1.8 mL of QIAGEN Ni-Nitrilotriacetic acid (Ni-NTA) agarose beads is added to the lysate and is left to mix in a rotary mixer for 1 h at 4 • C. The supernatant is applied to a QIAGEN His-tag affinity column which is equilibrated with Ni-NTA buffer (50 mM Na 3 PO 4 , 1 M NaCl, pH 7.9).Prior to elution, the column is first washed with 35 mL of 35 mM imidazole-containing Ni-NTA buffer, and the protein is eluted twice with 1.2 mL of elution buffer (Ni-NTA buffer contains 250 mM imidazole).The elutions are collected in a fresh microcentrifuge tube each time.After confirming by SDS-Page, the elutions containing the desired protein are combined, and the excess imidazole and other salts are removed from the elutions by passing through Nanosep 3 K centrifugal column twice at 6000 rpm for 20 min.The solution in the top of the column is collected in a fresh microcentrifuge tube.Sufficient amount of 10x PBS buffer (pH 7.4) (if the sample is 1.2 mL, then 120 μL is added) and protease inhibition cocktail (1-2 μL/mL) is added to the concentrated elution sample.The purity of the protein is determined by analyzing the protein solution by SDS-PAGE.
• C and finally are harvested by centrifugation at 5000 rpm for 15 min, and the pellet is stored at −80 • C. To start the purification of the protein, the cell pellet is suspended in a lysis buffer (50 mM Na 3 PO 4 , 1 M NaCl, 8 mM Imidazole, 500 μL Sigma's PIC (protease inhibitor cocktail)) and is cooled for 10 min before it is subjected to sonication for 15 min (10 sec on, 20 sec off, 40% amplification).The soluble and insoluble fractions are • C. The resulting hATF-Cy5.5-IONPs are separated from the reaction buffer using Nanosep 100 K centrifugal column.The NPs are purified by washing with sterile 1x PBS buffer followed by resuspension in sterile distilled water and stored at 4 • C. The mean hydrodynamic particle size and zeta potential of the various NPs are determined using dynamic laser scattering (Malvern ZetaSizer Nano ZS, Malvern Instruments, UK).Samples are prepared in a 10 mM NaCl to a final concentration of 2 mg/mL and pH 7.4.The sample is transferred to a zeta cell (Malvern Instruments, UK), and all measurements are made at a scattering angle of 90 • at 25 • C.