Bitargeting and ambushing nanotheranostics

Abstract

The main problem in cancer chemotherapy is the cytotoxic side effects of therapeutics on healthy tissues and cells. The targeted drug delivery and nanotechnology are intensively investigated area to find new ways to solve, at least to reduce, these problems. Hereby, we have reported a new method inspired from both conventional military strategies and biorecognition in the body. In this respect, we have produced two fluorescent nano-drug systems with bitargeting and biorecognition properties, recognizing cancer cells and each other. The multiplexed nanostructures were interacted with HL-60 cells to show their efficiency for bitargeting, ambushing, timed, and double-controlled cancer cell apoptosis.

Keywords::

• bi-targeting
• cytochrome P450
• controlled cancer cell apoptosis
• fluorescent nanoparticles
• ifosfamide
• nanotheranostics
• quantum dots

Introduction

Cancer is one of the major health problems worldwide. More than 10 million people are diagnosed with cancer each year (Park et al. 2008). The incidence of the disease exponentially inclines with increase of the population age with a death account for one in four worldwide. By 2020, the authorities have estimated that there will be 16 million new cancer cases every year (Patra et al. 2010). According to huge number of the cases, the tremendous efforts have been undertaken to treat cancer, but only a small improvement has been achieved over recent years. Patients diagnosed with cancer are usually treated with a combinational therapy including surgery, chemo and radiotherapy.

Cytochrome P450 enzymes (P450) are often used in suicide cancer therapy strategies to convert an inactive prodrug such as the oxazaphosphorines cyclophosphamide and ifosfamide (IFO) into its therapeutic active metabolites (Komatsu et al. 2000, Baldwin et al. 2003, Chen et al. 2004, McFadyen et al. 2004, Kumar et al. 2005). IFO is an alkylating agent prodrug that induces DNA cross-linking in a cell cycle-independent manner, and ultimately trigger a mitochondrial pathway apoptosis after activation by P450 (Jounaidi and Waxman 2004, Schmidt et al. 2004). P450 activity is obligatorily dependent on electrons supplied by cytochrome P450 reductase (CPR) (Schwartz et al. 2003). CPR interacts with P450 as an electron donor and catalyses P450 monooxygenase reactions (Peterson et al. 1976). CPR mediates the transfer of electrons from NADPH to P450 and is required for all microsomal P450-catalyzed enzyme reactions. CPR provides an important underlying basis for P450-based cancer therapy. Drugs are activated more efficiently containing P450 and CPR as compared to P450 alone (Chen et al. 1997, Jounaidi and Waxman 2000). CPR in combination with P450 enhances the cytotoxicity of P450-activated drugs both in vitro and in vivo (Jounaidi and Waxman 2001).

The main measure for deciding the efficiency of the cancer therapeutics is the ability to reduce and eliminate the cells without damaging healthy cells or tissues (Byrne et al. 2008). The therapeutic agents that are cytotoxic drugs block cell division process and promote apoptosis of the fast growing cells (Acharya et al. 2009). In order to increase the efficiency of the treatment, these cytotoxic drugs should be specifically delivered to target cells/tissues. Also, other limitations and problems such as low water solubility and nonspecific biodistribution, short half-life in circulation, and development of drug resistance have been under investigation (Chen 2010). Hereby, specific delivery of the chemotherapeutics to the target cell is the key-step of the treatment by solving mentioned limitations.

Nanotechnology offers several possible solutions to overcome these problems by opening the doors of the new world in nanometer scale. Recently, colloidal drug-delivery systems, especially nanoparticles, have attracted great interest of the researchers. The materials in nanometer scale including dendrimers, liposomes, polymers, micelles, protein, ceramic, viral, and metallic nanoparticles and carbon nanotubes are currently used in research for drug delivery and imaging (Dobrovolskaia et al. 2008, Wu et al. 2008, Davis 2009, Gewin, 2009, Grainger et al. 2010, Liang et al. 2010, Roy et al. 2010, Liu and Gao 2011, Werlin et al. 2011). Nanoparticular carrier systems have ability to increase the stability and solubility, to reduce the toxicity, and to improve the pharmacokinetics and pharmacodynamics of these drugs (Messerschmidt et al. 2009). Formerly, drug delivery using nanoparticles have been carried out by passive targeting, but the active targeting, in which specific recognition elements have been attached on the nanoparticle's surface, has now become a vital concept in the biomedical research community. Active targeting of nanoparticles to particular sites in the body can be achieved by using specific peripheral ligands such as antibodies, growth factors, transferrin, cytokines, folates, and low-density lipoproteins (Mo and Lim 2005). In present work, we have concentrated our attention on double-controlled and actively targeted delivery of cancer nanotheranostics to interested cancer cells.

For this purpose, we have designed complementary multiplexed nano-carrier systems for ambushed, timed out, double-controlled and sustained drug delivery to cancer cell. First nanocarriers were CPR-loaded fluorescent quantum dots (CFQD) as an ambushing therapeutic agent while second nanocarriers were IFO-loaded Au/Ag nanoclusters (IAANs) as a therapeutic agent ignitor. In order to produce former complementary, a novel approach, amino acid monomer-protein cross-linking using photosensitation and conjugation approach (ANADOLUCA), was applied (Diltemiz et al. 2008, Say 2009, Say et al. 2011). This method provides a consecutively proteins conjugation strategy to prepare photosensitive, stable, and easy orientable protein cross-linking by photosensitive ruthenium-based amino acid monomers and oligomers on to micro- or nano-surfaces. The obtained products can be used targeting, imaging and theranostic purpose in a wide range of nano(bio)technology applications. As first step, CFQDs were prepared and characterized (Diltemiz et al. 2008). In order to gain smartness for active targeting and to improve biocompatibility, CFQDs were modified with amino acid-based monomers, MATrp and MACys, and transferrin, an 80 kDa glycoprotein which is a suitable ligand for tumor targeting, because its receptors are over-expressed on cancers according to increase in iron demands of the cancer cells (Elliott et al. 1993). As a second step, IAANs were synthesized and characterized (Gültekin et al. 2009), and were also modified by amino acid-based monomers for improving biocompatibility. To make a specific bridge between CFQDs and IAANs, antitransferrin antibody molecules were also immobilized on IAANs. By this way, a complementary couple-like inexplosive mine and its fire were obtained by means of biorecognition between transferrin molecules on CFQDs to target cancer cell receptors and to ignite IAANs and antitransferrin antibodies on mine layer having IAANs. To show the efficiency of the proposed complementary nanostructures, a cell line [human promyelocytic leukemia (HL-60) cells] was used as a target.

Materials and methods

Reagents

All the chemicals including L-tryptophan, L-cysteine, methacryloyl chloride, methacrylic acid, P450 reductase, transferrin, antitransferrin antibody, and other solvents were purchased from Sigma-Aldrich. All the cell culture reagents, including Roswell Park Memorial Institute-1640 medium (RPMI), fetal bovine serum (FBS), and penicillin–streptomycin, were purchased from Sigma-Aldrich unless otherwise noted. Annexin V-FITC Apoptosis Detection Kit was purchased from BD Pharmingen and Human promyelocytic leukemia cells (HL-60) were obtained from The American Type Culture Collection (ATCC).

Synthesis of modified QDs: For CdS synthesis, 0.01 M of Cd(OAc)₂.2H₂O solution (24 mL) was prepared with ethanol. Solution was stirred continuously for 30 min in a nitrogen ambient. Sodium sulfide (0.01 M, 24 mL) was slowly added, stirred under nitrogen ambient for 30 min, and, then, centrifuged to collect precipitate. It was washed with double-distilled water and dried in air. The entire synthesis was carried out at room temperature. After that, 5 mL of CdS nanocrystals were immersed in ethanol containing 0.018 M, 10 mL of MACys in order to introduce methacryloyl groups onto the surface of CdS nanocrystals (Diltemiz et al. 2008, Say 2009, Say et al. 2011). The nanocrystals were then washed with ethanol and deionized water for 10 min to remove the excess of thiol groups. A stable self-assembled monolayer of MACys was formed onto the nanocrystal surfaces after all these steps. Then, 5.08 mg of MATrp was solved in 1 mL of water. One milliliter of sample was taken from CdS-MACys solution and added to MATrp solution and mixed for 24 h.

CPR/transferrin consecutively conjugation on the QDs (CFQDs): In order to improve biocompatibility of the QDs, two different modification pathways, transferrin-decorated QD-CPR conjugate and CPR-decorated QD-transferrin conjugate, were applied to them. The aim of this approach was to determine the effect of the conjugation and cross-linking order. Firstly, 25 μL of CPR solution (100 ppm) and 50 μL of previously synthesized poly[MATrp-Ru(bipyr)₂-Cl] solution (1 mg/mL) were added onto MACys/MATrp-conjugated QDs (Diltemiz et al. 2008, Say 2009, Say et al. 2011), then, 50 μL of APS solution (100 mM) was simultaneously added. The reaction was sustained at room temperature for 24 h. Meanwhile, 500 μL of transferrin solution (5 ppm) and 50 μL of previously synthesized poly[MATrp-Ru (bipyr)₂-Cl] solution (1 mg/mL) were added onto MACys/MATrp QDs, then, 50 μL of APS solution (100 mM) was simultaneously added at the same condition mentioned previously. On the following day, 500 μL of MATrp (0.3 mg/mL) and a mixture containing 500 μL of NHS (0.1 M), 500 μL of EDC (0.4 M) were added onto first conjugated QDs suspension, respectively and then 500 μL of transferrin (5 ppm) was consecutively decorated and cross-linked on to MATrp/CPR-conjugated QDs using photosensitive polymer solution and APS solutions. A second decoration pathway, 500 μL of MATrp (0.3 mg/mL) and mixture containing 500 μL of NHS (0.1 M), 500 μL of EDC (0.4 M), were added onto transferrin-conjugated sequence QDs suspension, and then, 25 μL of CPR (100 ppm) was consecutively decorated and cross-linked MATrp/ transferrin-modified QDs using photosensitive polymer solution and APS solutions. Finally, we have obtained CPR- and transferrin-decorated multiplexed QDs as a targeting and ignition synergic system by applying two different pathways. The both QDs were used as targeting and ignition nanostructures for double-controlled active targeting of HL-60 cells.

Preparation of antibody-oriented and prodrug-having nanoclusters (IAANs)

Ag–Au nanoclusters were synthesized and characterized previously (Gültekin et al. 2009). Firstly, Ag–Au nanoclusters were modified with 1 mL of MACys solution (0.3 mg/mL). The modification reaction was carried out at room temperature for 2 h (Scheme 1). Then, 1 mL of MATrp solution (0.3 mg/mL) was added onto previous solution and mixed for 2 h. After that, 1 mL of antitransferin antibody (50 ppm, pH: 7.4, phosphate buffer), 100 μL of ruthenium-based polymer (1 mg/mL) and 100 μL of APS solutions (0.3 mg/mL) were added onto modified Ag–Au nanocluster suspension. They were mixed on magnetic mixer for 24 h. After that, ifosfamide (IFO), used like a prodrug, was dissolved (7.3 mg) in 25 μL of deionized water. Then, IFO solution, 1 mL of NHS (0.1 M), and 1 mL of EDC (0.4 M) solutions were added onto antitransferrin antibody-conjugated Ag–Au nanostructure and they were mixed on magnetic mixer for 24 h. Finally, we have obtained the second complementary of nano-mine layer system.

Cell lines and cell culture

The human promyelocytic leukemia cells (HL-60) were used in these experiments. HL-60 cells were cultured for cell suspension at 1 × 10⁶ cells/mL in RPMI medium, containing 10% (v/v) fetal calf serum and penicillin/streptomycin. The cell suspension was placed on a sterile plastic tissue culture plate and incubated at 37°C and 5% CO₂ was supplied. The use of a prodrug strategy increases the selectivity of the drug for its intended target, CFQDs were interacted with HL-60 cells in the cell culture for a different time interval range of 0.5–16 h. In order to show the effect of modification pathway, both QDs, transferrin-decorated QD-CPR conjugates and CPR-decorated QD-transferrin conjugates, were investigated. To confirm that double-controlled active targeting of HL-60 cell, IFO-decorated and antitransferrin antibody-conjugated Au–Ag multiplexed nanoclusters was added into the cell culture and they were incubated for 24 h.

Flow cytometry analysis

HL-60 cell solutions that have been incubated as explained above were used to detect apoptosis and necrosis using the Annexin V-FITC Kit (BD Company, USA) according to the manufacturer's recommendations. Cell solutions were collected and harvested by centrifugation, washed twice with cold phosphate buffer saline (PBS), and resuspended in 100 mL of binding buffer. Then, 100 mL of each cell solutions (1 × 10⁶ cells/mL) was taken and 5 μL of Annexin V- FITC and 5 μL of propidium iodide (PI) were added into the each samples and the cell solutions were incubated for staining for 15 min at room temperature (20–25°C) in the dark. Then, 400 μL of binding buffer was added into the each sample before analysing. The cells were counted with a flow cytometer (FACS Aria, BD Corporation, USA), FITC-conjugated Annexin V and PI emissions were detected using the Diva software and the percentage of cells undergoing apoptosis was determined using dual-color analysis. These stainings distinguish the cells into four subsets: viable cells (Q3) (no staining), early apoptotic cells (Q4) (annexin V positive and PI negative), late apoptotic cells (Q2) (annexin V positive and PI positive), and necrotic cells (Q1) (annexin V negative and PI positive). Experiments were repeated in triplicate.

Transmission Electron Microscopy (TEM) Analysis: Intracellular uptakes of nanoparticles were determined by Tecnai^™ G2 Spirit BioTwin Model Transmission Electron Microscope (FEI Company). Cells were deposited on formvar-coated 200–300 mesh copper grids and dried and fixed with 2% glutaraldehyde solution in 0.1 M PBS buffer. Then, they were embedded into agar and stained in 2% osmium tetroxide and dehydrated in graded ethanol. After that, cells were embedded in EPON 812 epoxy. They were sectioned thinly using a diamond knife to a maximum thickness of 100 nm. The sections were stained with lead citrate and uranyl acetate. After that cells were displayed by TEM.

Results and discussion

In present communication, we have focused our attention not only on combining nanotechnology and drug delivery via active targeting, but also double-controlled and sustained drug delivery to cancer cells, which is inspired from conventional military strategies. For these purposes, we have designed two different nano-carriers having biorecognition ability to each other for ambushed and timed cancer cell apoptosis. The methodology undertaken in an effort was to identify or design a novel CPR enzyme containing igniting nanostructures that exhibits improved catalytic activity or substrate specificity with respect to activation of the chemotherapeutic agent. CPR enzyme was conjugated onto QD and, then, transferrin was decorated onto this conjugate by consecutively decoration method, ANADOLUCA, in order to gain an ability to QDs to recognize cancer cells receptor. Meanwhile, chemotherapeutic agent was conjugated onto Au–Ag nanoclusters and, then, complementary targeting protein, antitransferrin antibody, was decorated onto this nanocluster conjugates for gaining to them to recognize the complementary QDs. In this study, we have investigated the potential utility of combining CPR conjugate delivery with IFO-based P450 therapy. P450-dependent prodrug activation and cytotoxicity can be increased by supplementation with CPR, providing a simple way to increase the efficacy of P450. Cancer cells were targeted using transferrin-decorated and CPR-conjugated fluorescent QDs. Then, IFO-decorated and antitransferrin antibody conjugated Au–Ag nano-mine layer was delivered to cancer cells. So, IFO-decorated and antitransferrin conjugated Au–Ag nano-mine layers were ignited by the targeting transferrin-decorated QD-CPR conjugates. HL-60 cells were used for apoptotic studies (Figure 1).

Scheme 1. Decoration pathways of therapeutic QDs.

The complementary nanostructure systems for ambushed, timed and double-controlled cell targeting were based on P450-based prodrug activation (enhanced by CPR) and were tested with human promyelocytic leukemia cells (HL-60) using flow cytometry measurements. As mentioned before, we have applied to two different sequences to obtain QDs: (i) transferrin-decorated QD-CPR conjugate that has transferrin on outer shell and (ii) CPR-decorated QD-transferrin conjugate that has CPR on outer shell to investigate the effect of decoration sequence. The results obtained by using former conjugates show that these conjugates are more efficient for actively targeted cell apoptosis. During production pathway, the sequence was performed as transferrin decoration after CPR conjugation. Hereby, the targeting ligand, transferrin, was oriented on the surface as more accessible manner. Therefore, they easily interact with transferrin receptors on the surface of the cells (Figure 2). In other case, the molecules remain more inside of the QD conjugates, so effective biorecognition between the cells and transferrin molecules were not achieved (the results not shown).

Figure 1. Transferrin decorated QD-CPR conjugate (leftside) and IFO decorated and antitransferrin conjugated Au-Ag nanoclusters (rightside).

As second step of the cell targeting approach, we have investigated the effect of application of second complementary nano-mine layer, antitransferrin antibody and IFO-decorated Au–Ag nanostructures. In this step, we transported the second complementary nano-mine layer to the cell samples, already interacted with CPR-decorated QD-transferrin conjugates, after a retarded time interval in a range of 0.5–16 h (Figures 1 and 2). This approach was based on the idea to reduce/overcome the limitations depending on the harmful effects of nano-destroyers carrying cancer chemo-therapeutics by double-controlled and sustained drug delivery. The flow cytometry results show the retardation effect and the performance of the complementary nanoparticulate system based on bitargeting/birecognizing nanotheranostics (Figure 3). As seen in figure, application of transferrin-decorated QD-CPR conjugates increased cell apoptosis rate. The efficiency of the method is clearly seen in the figure. After transferrin-decorated QD-CPR conjugates, the nano-ignitors, invaded into the cancer cell, they reach to their complementary nanoparticles, the nano-mine layers, and recognize each other by means of transferrin–transferrin antibody interactions. Then, the ignited nano-mine layers cause increased cell apoptosis rates. The optimum time was determined as 4 h for delaying the interaction between IFO-decorated Au–Ag nano-mine layer and transferrin-decorated QD-CPR conjugate. The necrotic cells ratio [the left-upper quadrant (Q1)], the early apoptotic cells ratio [in the right-lower (Q4)], the late apoptotic cells ratio [in the right-upper quadrant (Q2)], and the viable cells ratio [in the left-lower quadrant (Q3)] were 0.1%, 34.4%, 15.2% and 50.3%, respectively (Figure 3). The Q3 ratio (viable cells) of control experiments, in which the cells were not interacted with both nanoparticles, was measured as 93.2%.

Figure 2. Interaction of transferrin decorated QD-CPR and IFO decorated and antitransferrin antibody conjugated Au-Ag multiplexed nanostructures with a cancer cell.

The results show that the proposed complementary nanotheranostic system causes the specific cell apoptosis by active bitargeting, ambushing, double-controlled, and sustained drug delivery pathway.

The flow cytometry results demonstrated that transferrin-decorated QD-CPR conjugates target both the cells in the culture and IFO-decorated Au–Ag nanocluster antitransferrin antibody conjugates. Then, IFOs were ignited by P450 (CPR) activated through binding of antitransferrin antibody and transferrin molecules onto complementary nanostructures and were converted into their cytotoxic form. In the light of the flow cytometry analysis, TEM graph was taken from delayed interaction between complementary nanoparticles for 4 h (Figure 4). By underconsidering the Figures 3 and 4 together, it can be said that active targeting, mine layering, and delayed ignition-based, double-controlled, and sustained cancer cell apoptosis were achieved using proposed complementary nanotheranostics according to the increased death/viable ratio of the cancer cells observed from flow cytometry measurements.

Figure 3. Flow cytometry data, HL-60 cells were interacted with transferin decorated P450 conjugated QD multiplexed nanostructure for 0.5, 1, 1.5, 2, 4, 8 and 16 h. Early apoptotic cells are presented in the right-lower quadrant of the figure (Q4), late apoptotic cells in the right-upper quadrant (Q2), living cells in the left-lower quadrant (Q3), and necrotic cells in the left-upper quadrant (Q1).

Conclusion

In this study, we have demonstrated that transferrin-decorated QD-CPR conjugates has substantially increased IFO sensitivity of target/tumor cells, resulting in enhanced anticancer prodrug activation. In this strategy, we proposed two nanoparticle system having abilities to target to cancer cells and each other. First nanoparticle system, QD, was prepared by conjugation of drug-activating enzyme CPR on QDs and, then, decoration of a cancer cell targeting agent, transferrin, onto this conjugate. Second nanoparticle system, Au–Ag nanocluster, was prepared by conjugation of antitransferrin antibody on nanoclusters and, then, decoration of non-toxic prodrug, IFO, onto this conjugate. IFO molecules were ignited by P450 (enhanced by CPR) meanwhile transferrin molecules were targeted by antitransferrin antibodies. Hereby, the IFOs have been converted into a toxic drug, resulting in cytotoxic effects in cancer cells. The proposed cancer therapy is novel strategy for active bitargeting, birecognizing, ambushing, timed out, double-controlled cancer chemotherapy and classified as promising alternative for developing new complementary chemotherapeutics.

Figure 4. TEM images of (a) healthy and (b) damaged HL-60 cell interacted with complementary nanotheranostic system.

Declaration of interest

The authors report no declaration of interest. The authors alone are responsible for the content and writing of the paper.