Targeting Negative Surface Charges of Cancer Cells by Multifunctional Nanoprobes
2016. 2016

Bingdi Chen
The Institute for Translational Nanomedicine
Shanghai East Hospital
The Institute for Biomedical Engineering & Nano Science
Tongji University School of Medicine
200120ShanghaiChina

Wenjun Le
The Institute for Translational Nanomedicine
Shanghai East Hospital
The Institute for Biomedical Engineering & Nano Science
Tongji University School of Medicine
200120ShanghaiChina

Yilong Wang
The Institute for Translational Nanomedicine
Shanghai East Hospital
The Institute for Biomedical Engineering & Nano Science
Tongji University School of Medicine
200120ShanghaiChina

Zhuoquan Li
The Institute for Translational Nanomedicine
Shanghai East Hospital
The Institute for Biomedical Engineering & Nano Science
Tongji University School of Medicine
200120ShanghaiChina

Dong Wang
Department of Biomaterials
College of Materials
Xiamen University
361005XiamenChina

Lei Ren
Department of Biomaterials
College of Materials
Xiamen University
361005XiamenChina

Ling Lin
Institutes of Biomedical Sciences
Fudan University
200032ShanghaiChina

Shaobin Cui
The Institute for Translational Nanomedicine
Shanghai East Hospital
The Institute for Biomedical Engineering & Nano Science
Tongji University School of Medicine
200120ShanghaiChina

Jennifer J Hu
Public Health Sciences
University of Miami School of Medicine
33136MiamiFLUSA

Yihui Hu
The Institute for Translational Nanomedicine
Shanghai East Hospital
The Institute for Biomedical Engineering & Nano Science
Tongji University School of Medicine
200120ShanghaiChina

Pengyuan Yang
Institutes of Biomedical Sciences
Fudan University
200032ShanghaiChina

Rodney C Ewing
Department of Geological Sciences
School of Earth, Energy & Environmental Sciences
Stanford University
94305-2115StanfordCAUSA

Donglu Shi firstname.lastname@example.com
The Institute for Translational Nanomedicine
Shanghai East Hospital
The Institute for Biomedical Engineering & Nano Science
Tongji University School of Medicine
200120ShanghaiChina

Zheng 6 email@example.com
Materials Science and Engineering Program
Department of Mechanical and Materials Engineering
College of Engineering and Applied Science
University of Cincinnati
CincinnatiOhioUSA

Cui
The Institute for Translational Nanomedicine
Shanghai East Hospital
The Institute for Biomedical Engineering & Nano Science
Tongji University School of Medicine
200120ShanghaiChina

Department of Pathology
Wake Forest University School of Medicine
Winston-SalemNorth CarolinaUSA

Targeting Negative Surface Charges of Cancer Cells by Multifunctional Nanoprobes

Theranostics
6112016. 201610.7150/thno.16358Received: 2016.06.02; Accepted: 2016.07.11;1887 T Th he er ra an no os st ti ic cs s Research Paper * These authors contributed equally.  Corresponding authors: http://ivyspring.com/terms for terms and conditions.TargetingBiomarkerNanoprobeSurface ChargeCell MetabolismLactate SecretionGlycolysis
A set of electrostatically charged, fluorescent, and superparamagnetic nanoprobes was developed for targeting cancer cells without using any molecular biomarkers. The surface electrostatic properties of the established cancer cell lines and primary normal cells were characterized by using these nanoprobes with various electrostatic signs and amplitudes. All twenty two randomly selected cancer cell lines of different organs, but not normal control cells, bound specifically to the positively charged nanoprobes. The relative surface charges of cancer cells could be quantified by the percentage of cells captured magnetically. The activities of glucose metabolism had a profound impact on the surface charge level of cancer cells. The data indicate that an elevated glycolysis in the cancer cells led to a higher level secretion of lactate. The secreted lactate anions are known to remove the positive ions, leaving behind the negative changes on the cell surfaces. This unique metabolic behavior is responsible for generating negative cancer surface charges in a perpetuating fashion. The metabolically active cancer cells are shown to a unique surface electrostatic pattern that can be used for recovering cancer cells from the circulating blood and other solutions.

Introduction

A great challenge in cancer diagnosis is to identify molecular biomarkers specific and universal to all cancer cells [1,2]. A key application includes detection of the circulating tumor cells (CTC) from whole blood. The current CTC detection based on molecular biomarkers has been hampered by limitations in specificity and sensitivity of the methods. Therefore, new targeting strategies need to explore other bio-physical properties of cancer cells. Furthermore, the underlying mechanism responsible for the common bio-physical behavior of cancer cells, distinctively different from the normal cells, will not only provide the fundamental understanding of disease processes of cancer but also for clinical diagnosis and treatment.

The cell surface charges are the net electricity, which is different from the trans-membrane potentials [3]. In the last several decades, the studies on the electrostatic properties of somatic cell surfaces have been inconclusive and even contradicting. The contributing factors affecting the cell surface net charges include the levels of the charged components Ivyspring International Publisher on the plasma membranes, the activities of the ion channels on the plasma membranes, and the neutralizing systems in the body fluids. The plasma membrane contains the charged molecules, such as glycolipids and glycoproteins. Therefore, the cell surface net charge is a dynamical process and complicated by the competing processes between the immobile components and the mobile ions with opposite charges. For instance, the serum proteins and the buffer systems are capable of neutralizing surface charges of the solid components in the body fluids. The ion channels and pumps present on the plasma membranes all play major roles in cell surface charge behaviors by transporting a significantly larger amount of ions across the plasma membranes.

Traditionally, the surface charges and trans-membrane potentials were extensively studied by using single-cell electrodes in the excitable neurons and muscle cells [4,5]. In these cells, the changes of cell surface charges were driven primarily by the transient ion flows across the plasma membranes via ion pumps and ion channels without changing the immobile molecular composition. However, all these old methods, including single-cell electrodes and cell electrophoresis, were unable to evaluate the entire cell populations [6][7][8][9][10][11].

Nanoprobes (NPs) have emerged in recent years to study the cellular surface properties. One apparent advantage of using NPs for the assays is the improved sensitivity. However, cells in general can take up NPs nonspecifically via the general biochemical reactions with the receptors or via endocytosis of foreign objects [12]. The interactions between cells and small particles are therefore not limited only to the electrical forces. This may be highly significant when the NPs are conjugated with the functional groups, such as amino acids or proteins. Much of the inconsistent results could also be attributed to lacking of the proper means for minimizing and controlling the nonelectrical reactions. As a result, the cancer cell surface charges have never been well understood and utilized for targeting purposes.

The goals of this study are: 1) to develop a set of electrically charged, fluorescent, and superparamagnetic NPs for the assessment of cell surface charges without using any molecular biomarker; 2) to electrostatically target and magnetically capture cancer cells in solution as a quantitative assay for the overall charge status of the cell population; 3) to investigate the electrical charge behaviors of both cancer cells and normal cells, and 4) to identify the factors that can influence the cellular surface charges.

Materials and Methods

Nanomaterials

All initial reagents were obtained commercially. Iron (III) chloride hydrate (FeCl3•6H2O), ethylene glycol, sodium acetate, ammonium hydroxide (NH4OH, 28 wt%), and hydrochloric acid (37 wt% aqueous solution) were purchased from Shanghai (China) Reagent Company. Tetraethyl orthosilicate (TEOS), (3-Aminopropyl)triethoxysilane (APTES) and fluorescein isothiocyanate (FITC) were obtained from Sigma-Aldrich (USA). Branched poly(ethylene imine) (PEI, 99%, MW=10,000) were acquired from Alfa Aesar. Deionized water (DIW, 18.2 MΩ•cm resistivity at 25°C) was produced by a Thermo Easypure II UF System throughout the entire experiment.

Cell culture materials

RPMI-1640 medium, the heat-inactivated fetal bovine serum, penicillin-streptomycin and 0.25% trypsin-EDTA were purchased from Gibco Corp. Dulbecco's Modified Eagle's medium (DMEM) and phosphate-buffered saline (PBS) were purchased from Hyclone Corp. All rest of media for cell culture were acquired from Corning Corp.

Construction of electrically charged NPs

The magnetic microspheres were prepared by a solvothermal reaction [13]. Briefly, 2.56 g of sodium acetate and 0.81 g of FeCl3•6H2O were dissolved in 30 mL of ethylene glycol under magnetic stirring. The obtained homogeneous yellow solution was transferred to the Teflon-lined stainless-steel autoclave and heated at 200°C for 10 h. The autoclave was then cooled to the room temperature. The products were washed 3 times with ethanol and 3 times with DIW.

For the construction of the magnetic nanocore (Fe3O4@SiO2), 1.5 mL of Fe3O4 aqueous dispersion (100 mg/mL) was treated in 0.15 M HCl aqueous solution under sonication for 15 min. After being treated by acid, the treated Fe3O4 microspheres were dispersed in the mixture of ethanol and water (v/v = 70/30) for 10 min. Then ammonium hydroxide was added to the dispersion to adjust to pH 9.5. A certain amount of TEOS was added into the reactor with vigorous stirring and the reaction lasted for about 24 h at the room temperature. The extra reactants were removed from the Fe3O4@SiO2 after being washed 2 times with ethanol and 2 times with DIW.

For the preparation of the negatively charged NPs, 10 µL of APTES and 1mg of FITC were reacted under a dark condition overnight in 2 mL ethanol. Then, 100 mg of Fe3O4@ silica composite microspheres were dispersed in 45 mL 70% ethanol for 15 min and 1.3 mL of ammonium hydroxide and 30 µL of TEOS were added into the second reaction system. After incubation for 3 h, APTES-FITC compound was added and reacted for another 20 h in dark. The reaction mixture was washed with 3 times with ethanol and 3 times with DIW.

For the preparation of the positively charged NPs, the negatively charged NPs were used as the seeds and modified by PEI under gentle stirring and ultrasonication for 2 h. Final products were washed 3 times with DIW.

Materials characterization

Transmission Electron Microscopy images were obtained by a Philips Tecnai 20 microscope. Scanning Electron Microscopy images were taken using a JEOL SM4800 microscope. The surface zeta potentials of the magnetic NPs were obtained on a Dynamic light scattering particle size analyzer (Zetasizer Nano-ZS90, Malvern, UK). Fluorescence images were recorded with a CCD camera (Nikon DS-Ri1) mounted on an inverted fluorescence microscope (Ti-U, Nikon, Japan). A spinning-disk confocal microscope (Andor Revolution XD) was used for the confocal images.

Cell culture

K562 Cells, PC-3 cells, LNCaP cells, and BGC-823 cells were grown in RPMI 1640 medium supplemented with the 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (PS) at 37°C in a 5% CO2 humidified atmosphere. Other cells were cultured at 37 °C in DMEM supplemented with 10% FBS and 1% (v/v) PS in a humidified atmosphere in the presence of 5% CO2. Typically, cells were passaged by trypsinization and maintained in medium accordingly.

Preparation of primary cells

The research was approved and performed under the ethical and legal standards of Ethics Committee of Shanghai East Hospital. Primary hepatocytes were isolated from rats as previously reported [14,15]. Cell viability was > 90% as determined by the trypan blue exclusion test. For primary culture in 24-well plates, 1×10e5 Cells per well were plated. Two hours after seeding, cells were incubated in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin.

Mouse primary renal cells were isolated and cultured as previously described with minor modification [16]. Upon isolation, the renal cells were resuspended in DMEM containing 1% antibiotic/antimycotic solution (Sigma-Aldrich, 10,000 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 µg/mL amphotericin B), with 10% horse serum (Invitrogen Corp.). Cultures were incubated at 37°C with 5% CO2. PMN and MNC cells were isolated by density gradient centrifugation from human whole blood according to a previously published method [17] with some modifications. Briefly, peripheral blood was collected in a vial containing EDTA as an anticoagulant. Ten mL of fresh blood was collected and red blood cells were sedimented. The buffy coat was collected and layered on top of a discontinuous percoll gradient. The buffy coat was separated into the residual red blood cells, granulocytes, mononuclear cells, platelets and plasma. Viability of collected cells was analyzed by trypan blue dye exclusion test.

Cells fluorescence microscopy

Hela cells were grown at 37°C in DMEM medium under normal cell culture conditions to approximately 70% confluency. The culture wells were washed thoroughly with PBS and incubated with PBS containing 100 µg/mL NPs on ice with gentle shaking for 60 min. The wells were then washed thoroughly with PBS to remove unattached NPs and examined by fluorescence microscope.

Cells seeded in 96-well plates were incubated with the negatively or positively charged NPs at 4°C for 15 min. After incubation, the wells were rinsed gently with PBS to remove the non-interacting NPs. The cell samples were then imaged using fluorescence microscopy (Ti-U, Nikon, Japan) equipped with an electron multiplying charge-couple device (EMCCD) camera (Nikon DS-Ri1).

Electrical charge evaluation of NPs

NPs were suspended in water at neutral pH 7 and zeta potential was measured by Zetasizer Nano-ZS90. Data were plotted using the percentages of cells in y axis against the average potential in mV in x axis.

The surface zeta potential distributions of the magnetic NPs in aqueous solution were measured using dynamic light scattering.

Cell capturing by magnetic field

Hela cells were cultured at 37°C in DMEM medium under normal cell culture conditions to approximately 70% confluency. The culture wells were washed thoroughly with PBS, trypsinized, washed again with PBS, and resuspended in PBS. NPs were added at the indicated concentrations to the cell suspensions and incubated on ice for 60 min with gentle agitation. After incubation, the NP-bound cells were captured by a permanent magnet placed against the side wall of the tube and free cells were removed by washing with PBS for 3 times. The captured cells were released by removing the magnet and resuspended in PBS. Both captured and free cells were counted as 100%. The data were presented as the percentages of magnetically captured cells and free cells.

Charged NPs of different concentrations were added to cell suspension (1×10e6) cells per mL and incubated with gentle shaking on ice for 2 min. They were then isolated and washed with a magnet. The captured and uncaptured cells were counted by hemocytometer.

Effects of glucose on cancer cells

To study the effects of glucose in culture medium on the surface charges of cancer cells, K562 cells were first grown under the normal grown condition at 10 mM glucose till 30% plate confluency. The culture media were then replaced with the DMEM media of different glucose concentrations as indicated in Figure 6A and incubated with cells at 37°C for 48 h. The magnetic capture efficiency of cells by the positive NPs was subsequently determined. The concentration of lactic acid secreted by K562 cells was measured by a lactic acid detection kit.

Effects of indirect glycolysis inhibitor DCA on cancer cells

Dichloroacetate acid (DCA) is an indirect inhibitor of glycolysis. DCA does not directly inhibit any enzyme of glycolysis pathway, but rather, promote oxidation of pyruvate in mitochondria, and in turn, shunt the pyruvate away from becoming lactate, thus inhibiting the conversion of pyruvate to lactate. K562 cells were grown under normal culture conditions to 30% confluency. Various concentrations of DCA indicated in Figure 7B were added to cell culture media and incubated at 37°C for 48 h. The magnetic capture efficiency of cell by the positive NPs was subsequently determined. The concentration of lactic acid secreted by K562 cells was measured by a lactic acid detection kit.

Effects of direct glycolysis inhibitor 3BP on cancer cells

3-bromopyruvate (3BP) is a direct inhibitor of glycolysis. 3BP blocks the activities of the first enzyme, hexokinase, and the glycolysis pathway. K562 cells were grown under normal culture conditions to 30% confluency. Various concentrations of 3BP as indicated in Figure 7D were added to cell culture media and incubated at 37°C for 24 h. The magnetic capture efficiency of cell by the positive NPs was subsequently determined. The concentration of lactic acid secreted by K562 cells was measured by a lactic acid detection kit.

Effects of extra lactate in culture medium on cancer cells

K562 cells were grown under normal culture conditions to 30% confluency. Various concentrations of extra lactate as indicated in Figure 8A were added to cell culture media and incubated at 37°C for 24 h. The pH value of the culture media was maintained at 7.4 by adding lactic acid and ammonium lactate with the same molar ratio. The magnetic capture efficiency of cell by the positive NPs was subsequently determined. The concentration of lactic acid secreted by K562 cells was measured by a lactic acid detection kit.

Treatment of cell surface with sialidase

Hela cells were grown under normal conditions to 50% confluency and trypsinized into suspension solution serum-free DMEM. S180 cells were grown in suspension directly, washed and resuspended in serum-free DMEM. Neuraminidase (sialidase, 1.28 mg/mL) was added and co-incubated at 37°C for 30 min. The magnetic capture efficiency of cells by the positive NPs was subsequently determined S180 cells were first grown in DMEM under normal culture conditions. The cells were washed and resuspended in serum-free DMEM. Neuraminidase (sialidase) was used to remove sialic acid residues from the surfaces of cancer cells. Briefly, neuraminidase (1.28 mg/mL) was co-incubated with S180 cells at 37°C in serum-free DMEM and 5% CO2 for 30 min. The cancer cells were then magnetically captured by the NPs and counted.

Results

Design, Synthesis, and characterization of the NPs

Our first experimental consideration was to enable more efficient interactions between NPs and target cells. The second consideration was to minimize nonspecific interactions between NPs and unwanted cells. With these considerations in the NP design [18][19][20], superparamagnetic Fe3O4 nanocores were utilized for effective magnetic capture and separation of the attached cells. FITC fluorescence dyes were used for direct imaging of the NPs binding on the cells. The electrically charged surface functional groups on the NPs provided electrostatic force driven capability for cancer cell binding and consequent capture without using any biomarker. With this design, only the positively charged NPs were attached to cancer cells, but not those negatively charged ones.

The schematic diagram of the NP synthesis is shown in Figure 1. As shown in Figure 1A, the superparamagnetic Fe3O4 NPs are conjugated with APTES to form a thin layer of SiO2 shell on the NPs' surface upon reaction with TEOS and NH4OH. FITCs are embedded in the SiO2 shell, thus exposing the Si-linked hydroxyl groups and creating the negative surface charge. For the positively-charged NPs, PEI molecules are used not only to cover the SiO2-OH groups in a non-covalently manner but also to expose the additional amine groups that carry the positive charges.

The zeta potential of the NPs was used as a measure of surface charges. Figure 1B and 1C show the zeta potential distributions of the negative and positive NPs under the same experimental condition.

Most of the negative NPs exhibited a peak zeta potential of -20 mV, while that of the positive ones was +35 mV. Both negatively and positively charged NPs were evenly dispersed in water or buffer, such as PBS without clumping, and found stable for at least 12 months. Consistent with the nano-structure depicted in Figure 1A, the transmission electron microscopy showed a uniform SiO2 coating (~ 60 nm) on the NPs' surface with an average particle diameter of 320 nm ( Figure 1D). Upon surface functionalization, the hydrodynamic diameter was in the range of 400 nm, as shown in Figure 1E. The NPs were conjugated with FITC, exhibiting an emission at 525 nm ( Figure 1F). The pH dependences of zeta potentials for the negative and positive NPs are depicted in the Figure  S1.

Interaction of the NPs with cancer cells

To compare the levels of cell surface charges, we developed a unique method for characterizing the surface charges of cells in suspension. In order to eliminate endocytic events, all cell-NP incubations were performed on ice. At this low temperature, the cells were incubated with NPs of different electrical charges ( Figure 2). Adherent cells were mobilized by trypsin and incubated separately with the positive and negative NPs. Upon electrostatic interaction with a sufficient number of NPs, the captured cells are "pulled" to the side of the tube by a magnet. Figure 3 shows the phase contrast and florescent images of cell binding with NPs of different changes. Figure 3A shows the phase-contrast image of a Hela cell. Figure 3B is the fluorescence image of the positively-charged NPs attached on the very cell surface shown in Figure 3A, while Figure 3C is the superimposed image of Figure 3A and 3B. As shown in these images, based on the NPs' fluorescence intensity, a large number of the positive NPs were electrostatically bound to the surface of the Hela cell, due to opposite charges.

The interaction of NPs with multiple Hela cells is shown in Figure 3D. The left and right panels are the phase contrast and fluorescent images, respectively. The images in the top row are the control Hela cells without NPs. The images in the middle and bottom rows are Hela cells reacted respectively with positive and negative NPs. The strong fluorescence in the middle row indicates a high level binding of the positive NPs onto the cell surfaces.

To further characterize the dynamics of the cell/NPs interactions, we incubated Hela cells at a constant number with various concentrations of NPs ranging from 5 to 150 µg/mL. The NP-bound cells were then magnetically captured and separated. The magnetic capture efficiencies of Hela cells by NPs of different charges are plotted at various NP concentrations ( Figure 4A) and incubation times ( Figure 4B). Positive NPs and negative NPs showed a completely different pattern of interaction with cancer cells. The number of cells captured by the negative NPs is not significant under both conditions, especially at low NP concentrations ( Figure 4A).

In order to determine the interaction between Hela cells and the NPs over time, we incubated the cells with the positive NPs for periods ranging from 0.1 to 60 min on ice. As shown in Figure 4B, the NP-cell interactions are complete within 0.1 min, the shortest time within which we could start and stop the reactions. The extremely rapid reaction between the positive NPs and Hela cells is consistent with a charge-based mechanism, since cellular endocytic and biochemical mechanisms would have required a much longer interaction time, minutes to hours at low temperatures.

NPs of both signs were incubated with 22 different randomly-selected cancer cell lines and 4 types of primary normal cells freshly purified from healthy subjects of human, mouse, and rat. The experimental results showed a preferential binding of the positive NPs to all the 22 tested cancer cell lines but not the normal cells ( Figure 5A). Initially, only a low level binding of negative NPs to the cancer cells was observed. However, upon additional washes, the cells binding to the negative NPs diminished to an insignificant level whereas the cells bound to the positive NPs remained largely unchanged ( Figure S2).   To further confirm that the surface charges of cancer cells were indeed different from the primary normal cells in blood, we mixed 2.7×10e5 human breast cancer cells (MDA-MB-231) with 500 µL whole blood (containing at least 3×10e9 blood cells). These cells were captured by the positive NPs, washed and counted. An aliquot of cells was spread onto slides and stained with HEMA 3 ® . Figure 5B and 5C respectively show the typical field of normal blood smear and the normal blood spiked with MDA-MB-231 cells. The image of MDA-MB-231 cells is shown in Figure 5D. Figure 5E shows an image of MDA-MB-231 cells recovered by the positive NPs as the small dots that surround the cells. The efficiency of cancer cell recovery reached over 90% (2.5×10e5 of 2.7×10e5). The purity of recovery was found to be over 80% with a small number of granulocytes that were apparently interacting with the cancer cells. The magnetic capture resulted in at least 10,000 folds of cancer cell enrichment from the blood. These experimental results showed that the negatively charged surfaces of cancer cells tested in this study were indeed unique in comparison to primary normal cells.

Mechanism of surface charge generation via lactate secretion

All known bioelectric phenomena in mammalian cells are generated by the cross-membrane movement of mobile ions [21]. Most human cells are actively maintained as charge-neutral or slightly positive at the surfaces via the ion pumps and channels on the plasma membrane. If cells possess a significant amount of surface charge, it must be generated by an abnormal movement of mobile ions across the plasma membrane. If the surface charge is negative, this can only be generated by either an inward movement of cations or an outward movement of anions across the plasma membrane. By examining all known properties of cancer cells, one metabolic pattern is consistent with this hypothesis. Nearly all metabolically active cancer cells, whether in vivo or in vitro, are known to secrete a large amount of lactate as mobile anions [22]. This is a result of increased glycolysis in which the levels of glucose uptake and lactate secretion can be up to 30 times greater than that of normal cells. Therefore, we propose that the negative charges on the surface of cancer cells are largely generated by the increased glycolysis and the associated lactate secretion across the plasma membrane.

To test this hypothesis, we measured the levels of lactate secretion from cancer cells and the related changes in the negative surface charge when the glycolysis pathway or lactate secretion alone was altered by several different methods. First, we reduced the supply of glucose in the culture media of the cancer cells for 48 h. The lactate secreted from the cells into the culture media was measured directly. The negative surface charge was correlated to the percentages of the K562 cells captured magnetically. We found a 17% drop in the cancer cell capture efficiency ( Figure 6A) when decreasing the glucose concentration from 10 to 0 mM within 48 h. As expected, secretion of lactate from the cancer cells was also considerably reduced ( Figure 6B). Furthermore, we used either an indirect inhibitor DCA of glycolysis or a direct inhibitor 3BP for the same purpose of varying the glucose levels of culture media. Both DCA and 3BP are effective glycolysis inhibitors as described in Figure 7A. DCA has been found to inhibit aerobic glycolysis and promotes pyruvate oxidation [23]. 3BP are synthetic brominated derivatives of pyruvic acid that have been reported as a highly reactive alkylating agent and a direct glycolysis inhibitor [24]. Figure 7B shows a 35% decrease in the captured cells when 120 mM of DCA is added for 48 h. The corresponding lactate secretion variation can be seen in Figure 7C. Consistently, as shown in Figure 7D, a much greater reduction of 75% is found in the captured cells when 100 µM 3BP is present for 24 h. The effect of 3BP on secretion of lactate is also pronounced as shown in Figure 7E.

Lactate secretion occurs from a cancer cell only when extracellular lactate is dissipated away into the interstitial space. If there is a lactate buildup outside of the cell, the secretion of lactate would be specifically disrupted. If the surface negative charges are generated solely by lactate secretion, an elevation of extracellular lactate alone without blocking the glycolysis pathway would result in a decrease of lactate secretion and the surface charge of cancer cells. Based on this rationale, we designed a set of experiments to measure the surface charge and lactate secretion of cancer cells with increased extracellular lactate concentrations. When extracellular lactate concentration increased from 0 to 100 mM, the charge-based capture of cancer cells was reduced from 92% to 55% ( Figure 8A), and the lactate secretion decreased from 15 mM to 2 mM ( Figure 8B). These results are consistent with the generation of cell surface charge via anionic lactate movement across the membrane.

The electrical charge has been associated with the charged molecules on the cell surfaces. For example, sialic acid moieties of glycolipids and glycoproteins are believed to contribute to the electric properties of cancer cell surfaces [25,26]. However, a previous study indicated that the surface sialic acid moieties were mostly removed from the cell surfaces by sialidase [27]. By using the same treatment, as shown in Figure S3, there is no significant change in cell capture efficiency, suggesting that immobile sialic acids at the surface do not contribute significantly to the negative surface charges of the cancer cells.

Discussion

We have shown the negative surface charges as a unique pattern of cancer cells. In contrast, the normal cells, including all blood cells, do not possess this property. Using the uniquely designed NPs, we have recovered cancer cells from human blood with high efficiency and purity. The negative charges were found to be generated from the large quantity of lactate secretion, a known property of all metabolically active cancer cells. These findings are summarized in Figure 9.

Despite of the decades-long efforts, there has not been any theoretical consensus or clinical application based on the electrical properties of cancer cells. This study has, for the first time, provided unequivocal evidence for a unique electrical pattern of cancer cell surfaces from as many as 22 different types of cancer cells in comparison to primary normal cells. The experimental results of this study significantly differ from the previous reports on how to measure the cell surface charges. For example, the human granulocytes were reportedly to have the negatively charged surfaces whereas the data of this study clearly indicate their positive charged surfaces [28]. We attribute this contrast to the different methodologies employed by these studies. The multifunctional NPs developed in this study enabled direct assessment of the cell surface charge via electrostatic targeting, binding, and magnetic capturing of cancer cells from various solutions including blood. The findings of this study reveal a fundamental property of cancer cells and have profound potentials in clinical applications.

The phenomenon of the elevated glycolysis is unique for cancer cells and has been used widely for cancer diagnosis for decades. The level of glucose uptake and lactate secretion in cancer cells can be up to 30 folds higher than the normal cells, giving a basis for the cancer detection technologies, such as PET (Positron Emission Tomography) scan [29]. Radioactively labeled glucose can be preferentially taken up by cancer cells for imaging. Thus, the elevated glucose metabolism is a significant hallmark of cancer cells. In comparison to the glucose uptake, the secretion of lactate is also an inevitable outcome of glycolysis [30]. Our results show that lactate secretion is a direct cause for the negative charges on the cancer cell surfaces. It is not unreasonable to assume that the surface charges are consequentially linked to another well-established hallmark of cancer cells. Due to instability of genomes, the protein profiles can be vastly different even in the adjacent cancer cells of the same lesion. The search for consistent biomarker molecules on the surfaces of cancer cells has proven to be very difficult. In comparison to any molecular ligand, the biophysical property of cancer cells associated with the unique metabolism in cancer cells may prove to be a much more reliable property for detection.

Conclusions

All movements of ions across plasma membranes cause significant changes of surface charge. Cancer cells are well-known to secrete a large amount of lactate anions across plasma membranes as a hallmark. Our studies have now revealed that this hallmark may have led to a generation of unique negative charges on the surfaces of cancer cells. Without an elevated glycolysis, the surfaces of normal cells remain charge-neutral or slightly positive. This unique biophysical property can allow cancer cells to be identified and captured using a nanotechnology-based method without having to use any specific molecular marker. This technology could also be used to study the electrical property of all cells, such as neurons and other excitable cells. In clinical settings, our findings and technologies may have significant implications in developing novel diagnostic assay and probably therapies for cancer.

Supplementary Material

Figure 1 .
1Design and characterization of the nanoprobes. (A) Schematic diagram showing the design of the Fe3O4 multi-functional nanoprobe which is rendered fluorescent with FITC. (B) Zeta potential distributions of negative NPs. (C) Zeta potential distributions of positive NPs. (D) Transmission electron microscopy showing the image of NPs with a thin layer of silica coating. (E) Light scattering histogram showing the hydrodynamic diameter distributions of NPs. (F) Fluorescence spectroscopy showing the emission of NPs having a peak around 525 nm.

Figure 2 .
2Schematic diagram showing the procedure of magnet capturing of the cells. The cells in suspension are mixed with NPs of different electrical charges. Cell/NP bindings take place due to the opposite charges. The captured cells are magnetically attracted to the side of the tube by a permanent magnet.

Figure 3 .
3Optical and fluorescent images of Hela cells with binding of NPs on the surfaces. (A) the phase-contrast image of a Hela cell; (B) the fluorescence image of the positively-charged NPs attached on the very cell surface shown in (A), and (C) the superimposed image of (A) and (B). (D) Optical and fluorescent images of NPs with multiple Hela cells. The left and right panel images are respectively the phase-contrast and fluorescent fields for Hela cells upon reacting with the NPs. The images in the top row are Hela cells without the NPs controls. The images in the middle row are Hela cells upon reacting with the positive NPs. The images in the bottom row are Hela cells upon reacting with the negative NPs.

Figure 4 .
4(A) Magnetic capture efficiencies of Hela cell by both positive and negative NPs at various concentrations indicated. (B) The percentages of cells captured by the positive and negative NPs at the times indicated.

Figure 5 .
5(A) Net surface charges of cells. The calculated net surface charges expressed in this figure is the net captured percentage of cells by one type charge sign NPs subtracted by the cells captured by the NPs with opposite charge sign as controls. Microscope photographs of blood smear (Green arrow to MNC cell & orange arrow to PMN cell) (B), blood smear with breast cancer MDA-MB-231 cells (Red arrow to MDA-MB-231 cell) (C), MDA-MB-231 cells smear (D) and positive NPs captured MDA-MB-231 cells from blood (E).

Figure 6 .
6(A) K562 cancer cell capture efficiency vs. glucose concentration when reacting with the positive NPs. (B) Lactic acid secreted by K562 cancer cells vs. glucose concentration.

Figure 7 .
7(A) Mechanisms of glycolysis inhibition via dichloroacetate (DCA) and bromopyruvate (3BP) for varying the glucose levels of culture media. (B) K562 cell capture efficiency vs. the concentration of glycolysis inhibitor DCA when reacting with the positive NPs. (C) Lactic acid secreted by K562 cancer cells vs. the glycolysis inhibitor DCA. (D) K562 cancer cell capture capacity vs. the glycolysis inhibitor 3BP when reacted with the positive NPs. (E) Lactic acid secreted by K562 cancer cells vs. direct glycolysis inhibitor 3BP.

Figure 8 .
8(A) K562 cell capture capacity vs. extra lactic concentration when reacted with the positive NPs. (B) Lactic acid secreted by the K562 cells vs. extra lacate concentration.

Figure 9 .
9Schematic diagram showing secretion of lactate anions leading to a loss of cations from cancer cell surface and leaving behind the negative charges. (A) Glucose metabolism via glycolysis and oxidative phosphorylation in normal cells. (B) Glucose metabolism via highly elevated glycolysis and suppressed oxidative phosphorylation in cancer cells.

table: List of cell lines used in the experiments. Supplementary figure S1-S5. http://www.thno.org/v06p1887s1.pdf Abbreviations NP: nanoprobes; CTC: circulating tumor cells; phosphate-buffered saline; DCA: Dichloroacetate acid; 3BP: 3-bromopyruvate.TEOS:
Tetraethyl
orthosilicate;
APTES:
(3-Aminopropyl)triethoxysilane; FITC: fluorescein
isothiocyanate;
PEI:
polyethyleneimine;
DIW:
deionized water; EDTA: ethylenediaminetetraacetic
acid; FBS: fetal bovine serum; PMN: polymorphic
nuclear cells; MNC: mononuclear cells; DMEM:
Dulbecco's
modified
Eagle's
medium;
PBS:

http://www.thno.org
AcknowledgementThis study was supported by Natural Science Foundation of China (No. 51302190), an internal funding of Tongji University, and a charitable donation from Mr. Jin Nie to ZC. The author DS from the University of Cincinnati is partially supported by a National Science Foundation grant EEC-1343568.Competing InterestsThe authors have declared that no competing interest exists.
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Response to issues on GM agriculture in Africa: Are transgenic crops safe?

Ademola A Adenle
United Nations University Institute of Advanced Studies (UNU-IAS)
6F International Organisations Centre
Pacifico Yokohama, 1-1-1 Minato Mirai Nishi-ku220-8502YokohamaJapan

Response to issues on GM agriculture in Africa: Are transgenic crops safe?
C O R R E S P O N D E N C E Open Access
The controversies surrounding transgenic crops, often called Genetically Modified Organisms (GMOs), call for a need to raise the level of public awareness of Genetic Modification (GM) technology in Africa. This should be accomplished by educating the public about the potential benefits and risks that may be associated with this new technology. In the last 15 years, GM crop producing countries have benefited from adoption of this new technology in the form of improved crop productivity, food security, and quality of life. The increased income to resource-poor farmers is a key benefit at the individual level especially as most countries using this technology are in the developing world, including three African countries (South Africa, Burkina Faso and Egypt). Despite clear benefits to countries and farmers who grow GMOs, many people are concerned about suspected potential risks associated with GMOs. This sparks debate as to whether GM technology should be adopted or not. Given the concerns regarding the safety of GMO products, thorough scientific investigation of safe application of GMOs is required. The objective of this paper is to respond to the issues of GM agriculture in Africa and some of the issues surrounding the adoption of GM crops between developed and developing countries. In this article, I analyse relevant papers relating to the adoption of GM technology particularly in developing countries including the few African countries that have adopted GM crops. The issues discussed span a wide range including: safety; potential benefits and risks; disputes between the United States of America (USA) and the European Union (EU) over adoption of GM crops with a focus on Africa continent. This article is concluded by summarising the issues raised and how GM technology can be adopted for agricultural development in Africa.

Discussion

Why must GMOs be given a chance in Africa?

A growing body of evidence-based reports continue to document increased crop yields, increased farm income, and health and environment benefits associated with GM crops [1][2][3][4][5][6][7][8][9][10]. In 1996, when GM crops were first officially commercialised, six countries planted a total of 1.7 million hectares of these crops [9]. By 2010 this had grown to 148 million hectares in 29 countries, of which 19 are from the developing world. This 87-fold growth makes GM the fastest crop technology to be adopted in the history of modern agriculture. Of 15.4 million farmers that planted GM crops in 2010, over 90% (14.4 million) are resource-poor farmers in developing countries, including three African countries (Burkina Faso, South Africa and Egypt), that benefited from the adoption of GM crops [9]. For example, almost 100,000 farmers in Burkina Faso cultivated GM cotton on 260,000 hectares in 2010 with 126% increase from 115,000 hectares planted in 2009. In terms of contribution to the economy of Burkina Faso, it was estimated that a benefit of over US$100 million per year could be generated based on almost 30% increase in yield [11]. Additionally, close to a 50% reduction in insecticides sprays may be realised, thereby saving fossil fuels and lowering greenhouse gases emission, hence fighting against climate change. South Africa, the first and biggest producer of GM crops in Africa, has benefitted from growing GM crops over the past decade. They are the only African country among the five principal countries (also India, Argentina, Brazil and China) producing GM crops, with 63 million hectares planted in 2010 alone. The economy of South Africa has benefited a great deal from the adoption of GM technology and it has been reported to have enhanced farm income from GM maize, GM soybean and GM cotton by US$156 million in the period 1998 to 2006, with 2006 alone estimated to be US$67 million [12]. Egypt was the first North African country to grow GM crops commercially in 2008 with 700 hectares of GM maize planted and the Egyptian government planning to increase the hectarage of GM crops in future [13].

In contrast to the documented benefits of GM crops, there are few documented cases of potential health effects [14] or economic drawbacks [15,16] to GM crops. However, the issues of potential risks of GMOs continue to be raised, particularly in Europe, and the media is being used to attract attention to the question of whether GMO products are safe. For example, the "Flavr Savr" tomato is a genetically modified tomato that has altered DNA to delay ripening, thereby prolonging shelf-life [17]. It was the first GM food to be authorised for human consumption in the United States and received broad coverage by the media even though it is not a poison or a food known to cause illness. A GM soybean that contained a Brazil nut allergen [18] is arguably the only incident that got more attention from the media when GM crops were first commercialized. The intense media coverage resulted in cessation of further development of this particular variety due to potential health risks. In contrast, another incident that attracted media attention was that of a scientist that claimed a genetically engineered potato caused a depressed immune response in rats [19], but subsequent investigation showed that the experiment was scientifically flawed.

It should be noted that eating conventional foods is not risk free as they are known to contain allergies. For example, there was no known allergy when kiwifruit were introduced into the European and United States markets in the 1960s, but the fruits are now known to cause allergic reactions [20]. GM technology is like any other new technology and has its merits and drawbacks. Over the period of time that commercially available GM foods have been produced, no studies have indicated that GM foods are less safe than traditional counterparts. Although merit may be given to concerns of unintended gene flow from genetically engineered agricultural products, further studies are required to establish the reality and/or scope of this and other potential environmental risks due to GMOs. Unintended adverse effects of GMOs on non-target species (e.g. butterflies) have been reported to be similar to what currently exists in traditional agricultural products [21]. While there are other individual claims that GMOs could pose health risks to human beings, most of these findings are not peer-reviewed in international scientific journals or by any officially recognised standard. Rather, these are individual works that are being promoted on websites or by non-governmental organisations (NGOs). Unfortunately, when issues like these are raised and debated, many people condemn GMOs outright based upon unverified sources and also fail to see if there are advantages associated with the application of GMOs.

Several international regulatory bodies including the World Health Organisation (WHO), the Food and Agriculture Organisation of United Nations (FAO) have concluded that there is no scientific evidence that the application of GM technology has resulted in substantial human health effects or environmental problems [22,23]. For example, a British government report released by a panel of experts found out that no verifiable ill effects have been reported from consumption of GM food products and that the risks to human health are very low for current GM crops on the markets [23]. In spite of the lack of evidence for potential adverse effects of GMO production, most European countries do not grow GM crops as precautionary principle to ban GM crops has been adopted in European legislation. This precautionary principle can be invoked when: "potentially dangerous effects deriving from a phenomenon, product or process have been identified, and ...scientific evaluation does not allow the risk to be determined with sufficient certainty" because of the insufficiency of the data or their inconclusive or imprecise nature" [24]. In other words, this precautionary principle can be stated as: action can be taken against potential hazards if the scientific information does not prove lack of risk. While the use of this precautionary principle may be based upon a lack of scientific assessments of the risks of adopting GM crops, it has been criticised due to lack of assessment of economic, social and ethics aspects of GM adoption [25]. Additionally, this precautionary principle has been interpreted differently in international treaties, court decisions, academic literature and government policy reports [26]. This has effectively made it difficult to refer to the precautionary principle as a single or collective term.

While the precautionary principle has been presented in several versions, there is no clear existing scientific consensus within the scientific community with regards to any of the risks that may have been found or associated with GMO products. But the question remains: Are there books or peer-review journals that have recorded the number of illnesses or deaths as a result of consumption of GMO products in any part of the world? It is unfortunate that many people and organisations including government officials in Africa see GM technology as a poison that must be avoided and not to be introduced into their decaying traditional farming systems. Some of these problems can be attributed to lack of awareness and education on the application of modern biotechnology among Africans. For example, it was recently reported that scientists have not effectively communicated the benefits and risks of modern biotechnology to the Kenyans [27], hence leading to a high level of public concern and protests against GM crops. The failure of the scientists to play a role in public education might be attributed to negligence on the part of Kenyan government or may reflect a lack of communication and/or confidence between government and scientists over GMOs issues. These issues should be addressed and the public must be educated on both the potential economic benefits of GM crops and also the general lack of scientific evidence for health related issues when GM crops are consumed.

Africa must be saved from hunger

Given the high level of poverty, malnutrition, hunger, food security problems, and low agricultural productivity in Africa, advanced technology like GM technology has the potential to offer solutions to some of these problems. However, the controversy over the use of GM technology remains one of the biggest threats in adopting this new technology. The current regulatory approaches of the United States of America (USA) and the European Union (EU) can play an important role over adoption of GM technology in developing countries, particularly in Africa [22,28,29]. For example, the USA can allow commercial release of GMO products based upon standard tests such as allegenicity, digestivity and toxicity without any regulatory barrier, whereas in the EU, GMO products can be stopped based upon scientific uncertainty alone without any associated evidence of risks and sufficient testing according to the so called ''precautionary principle''.

The precautionary principle has become a political tussle, without clearly defined concepts, among giant nations [22]. This tussle has a serious impact on the African continent as to whether to adopt GM technology or not. Reports have shown that developing countries, including Africa, consider their trade relationships with the EU before adopting GM crops due to the fear of losing this market [30,31]. As a result, African governments tend to exhibit a ''go slow approach'' toward the adoption of GM technology. This is often reflected in delayed approval of biosafety laws required to grow GM crops, with the majority of African countries lacking a functional biosafety system [32]. An attempt to ascertain what causes delay in biosafety law approval has attributed the delay to political lobbying, change of administration, and the issue not being a high priority for the policymakers [33].

Why should we fold our arms when many people are starving, and children are dying as a result of malnutrition, in Africa and there is an innovative technology to solve part of the problem? For example, when about 14 million people in Southern African countries were faced with a food crisis due to drought, the food aid provided by the USA was rejected by Zimbabwe and Zambia simply because the maize was GM. People in these nations were left to suffer from starvation as a result. Although Zimbabwe reversed its decision after diplomatic intervention from pro-GM countries, Zambia placed an outright ban on GM maize from USA, stating potential health risks associated with the food. Severe drought in East Africa, Kenya, has resulted in recent import of GM maize due to the shortage of maize. However, many people, led by anti-GM activists, protested against the importation of GM maize as they feared it would contaminate the soil and could be harmful to the health of the citizens. Fortunately, the importation of GM maize was cleared by the Kenyan Cabinet after a proper safety assessment and clearance from the National Biosafety Authority.

Apart from constraints such as inadequate infrastructure and facilities, most channels of influence that are associated with adoption of GM technology in Africa come mainly from EU, and their tough regulatory precautionary principle can be a major threat to commercial export sales when African countries start producing GM crops [22,28,31]. This is not only restricted to African countries but other developing countries are also suffering a similar fate. Recently, the Director of the Beijing Genomics Institute strongly advised that EU should change its current GMO policies that hinder China and other developing countries from enjoying full benefits of these new agricultural technologies [34]. European Greenpeace is arguably the most organised group in the world that is campaigning against GMOs; they played a key role in influencing Zambian government officials to reject any GMO products coming into the country [22]. Any attempt to allow GMO products could mean that organic agricultural product export sales to the EU may be lost. While the USA considers GMO products safe enough for human consumption, after going through compulsory testing with their conventional counterparts, the EU regulatory approach is probably not in best interest of GMO products [29]. While no clear evidence has been established against GM crops, the EU remains sceptical about the use of GM crops despite spending more than $425 million on studying the safety of GM crops over the past 25 years [35].

Moreover, it is unclear what roles international organisations such as the World Trade Organisation (WTO) and the Convention on Biological Diversity (CBD) are playing in mediating the disputes between the USA and the EU as there does not seem to be cooperation on contending issues. Rather, it appears that multiple factors (political, economic, institutional, legal, historical and cultural) have combined together in a manner that undermines potential cooperation and mutual benefit between the two parties [36]. This lack of cooperation affects many poor countries in decision making on these new agricultural technologies. Why is there such a divide or lack of common interest to promote and encourage the fastest technology in the history of modern agriculture to fight poverty, malnutrition, hunger and food insecurity in Africa? Answering this question is beyond the scope of this article. Perhaps, this question can be answered as more knowledge about GM technology becomes available and when assessing the global benefit of this new technology becomes a reality. Current evidence shows that GM technology has a great potential to improve agricultural productivity and farmers' livelihood in developing countries. Therefore, GM technology must be allowed to play a part in alleviating hunger and poverty in Africa.

Safety is paramount: America and others leading the way

It is important to ensure the safety of all citizens that consume GM foods either in Africa or any part of the world. Not many countries are using GMOs in agricultural production around the world due to health and environmental concerns associated with the application of GMOs. The USA is one of the few developed countries growing GM crops on a commercial basis. More than 70 percent of some foods in the USA contain GMOs [37], yet no American citizen or others that have consumed their foods have been confirmed seriously ill or dead as a result of GMO consumption.

In view of the fact that no global consensus has been reached over completely safe application of GMOs and as the need to regulate or control GMO products become more important, the USA can be a good example for Africa and the rest of the world. The USA appears to be a lead country in safe application of GMO products with the United States Department of Agriculture (USDA) reporting that 93 percent of all soybeans, 78 percent of all cotton, and 70 percent of all corn grown in the USA in 2010 were genetically modified to be herbicide tolerant [38]. In fact, USA regulatory bodies have determined that GMO foods are "substantially equivalent" to conventional foods. Before any GMO products are released for human consumption, they are compared with traditional counterparts in a variety of chemical, genetic, biochemical, compositional, nutritional and environmental tests as well as comparison with known allergens [39]. The USA Food and Drug Administration (FDA) monitors every product made up of GMOs for comprehensive safety and environmental testing. Other GM producing countries, including Canada, Australia, China, and Argentina are carrying out similar tests to ensure that GMO products are safe for human consumption.

Although, the USA's FDA cannot be compared with majority of food & drug regulation departments in African countries which are without sophisticated equipment to do thorough testing and analysis, some of these countries have effective regulatory system with excellent track records in food safety. With support from developed countries, it should be possible to increase local capacity for testing in Africa and this should ensure safe delivery of GMO products while taking necessary precautions. The USA and other countries are good examples to follow when it comes to adoption of GM technology. After all, it is how this technology is used that determines whether it is risky, good, or beneficial. Therefore, as with any technology, continued evaluation of GM technology in terms of safety and benefits will be important as more knowledge becomes available. African governments can learn to follow the same procedures that other countries are already employing to ensure the safety of their citizens when GMO products become available in Africa.

Working towards common interest: embracing the Gene Revolution in Africa

Over four decades the Green Revolution was implemented and African countries were bypassed without any documented benefits. In contrast, Asian countries have benefited in terms of improved seeds, chemicals, pesticides and inorganic fertilizers. This has brought about phenomenal successes in boosting crop production in corn, wheat and rice [40]. The emergence of the Gene Revolution is assumed to play a significant role whereas the Green Revolution was not able to deliver in Africa. The slow embracement of the Gene Revolution in Africa, particularly GM technology, is the lack of access for beneficiaries such as farmers and scientists to GM crops. This lack of access is, at least in part, due to international influence against GMOs and this international influence remains fundamental to the successful adoption of GM technology in Africa.

There are a lot of issues affecting the adoption of GM technology. These require a concerted effort from African governments and developed countries to ensure successful transfer of the technology to the beneficiaries. In this regard, one of the major challenges which can delay access to GM crops is dealing with intellectual property right (IPR) issues. GM technology is currently being applied in production of the main commercial crops such as cotton, corn and soybean. Clearly this creates the largest profit for the biotech companies (e.g. Monsanto). However, it is difficult to access the technology in developing countries as biotech companies are often reluctant to invest in the development of this new technology in Africa. Most of the developing countries (especially Africa) do not have strong and effective laws to protect IPR and the legal systems can be very poor, this may partially explain why biotech companies are not attracted to African countries. Given the problem of the IPR logjam, one can imagine a lengthy period before innovative technologies that have potential benefits for orphan crops (e.g. millet, cassava, potato, sweet potato, yam, banana, cowpea and sorghum) can be realized as GM crops in African countries. Some hope may be on the horizon as the African Agricultural Technology Foundation (AATF) is working towards facilitating and promoting partnerships between the public and private sector to ensure that resource-poor farmers have free access to agricultural technologies including GM technology. Efforts are being made by AATF in partnership with other institutions towards developing GM crops that can benefit African farmers. For example, African scientists in collaboration with other scientists in developed countries are carrying out research on orphan crops such as GM banana, GM cassava and GM cowpea with field trials being carried out in Uganda and Nigeria respectively.

Other than IPR issues, the adoption of GMOs is strongly influenced by developed countries particularly the USA and EU. The USA and EU clearly demonstrate unequal levels of trust of the safe application of GMOs. This can be an obstacle for adopting this new technology in Africa. While the European influence in Africa seems stronger than the American influence, particularly in trade partnership, economic and political power should not stand in the way of new technology. Apart from foreign aid that comes from the EU, the majority of African countries have closer cultural ties with Europeans (stemming from the colonial era) than Americans. Thus, the majority of African countries are more likely to stick to the views and practices of European systems [22]. However, such practices or systems must not dominate agricultural policies in Africa. Because agricultural practices are a matter of life and death in Africa, they must be sustained and improved to feed the poor continent. The developed countries like the USA, the EU and others should work in common interest of delivering GM technology that has potentials to improve agricultural productivity, health and food security in Africa.

Africa must take action

Agriculture plays a significant role in the economy of African countries in terms of gross domestic product (GDP), promoting international trade, industrial development and creation of job opportunities. Therefore, there is an urgent need to improve agricultural production. GM technology can be a part of the solutions in cases where traditional methods of farming have been less efficient. The potential benefits of GM crops toward alleviating poverty and hunger, improving agricultural productivity, health, food security and creating a friendly environment cannot be overemphasised. The adoption of GM technology is at its initial stages in Africa and is currently faced with several constraints such as lack of infrastructures, inadequate human resource capacity, poor education, biosafety regulation, intellectual property rights and many others. A concerted effort from developed countries including international organisations must be put in place to ensure that Africa benefits from this new technology. African governments must also be involved in solving these problems themselves and they should come up with coherent strategy to adopt modern biotechnology including educating the public, farmers and government institutions, the media and private companies, and to increase understanding of GM technology. Part of the strategy must include adoption of common policies and a regional platform through which African governments can engage in dialogue and develop a common biotechnology regulatory approach. Africa might pay a huge price in many years to come if the continent continues to depend on outsiders before making decisions that determine their future. Europeans are well fed and may not necessarily require GM technology to boost their crop productions, but African farmers need fast technology that can solve part of their agricultural problems.
 Adenle BMC Research Notes 2011, 4:388 http://www.biomedcentral.com/1756-0500/4/388
Acknowledgements and FundingAdemola A. Adenle would like to acknowledge the support of the Japan Society for the Promotion of Science. The author gratefully acknowledges anonymous reviewers for their comments and suggestions. Special thanks to Dr. Nathaniel Szewczyk for editing the manuscript. The views expressed are those of author and do not necessarily reflect the official views of UNU-IAS and GRIPS.Author detailsAuthors' contributions AAA solely designed and reviewed all the papers used for developing the manuscript.Competing interestsThe author declares that they have no competing interests.
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Cite this article as: Adenle: Response to issues on GM agriculture in Africa: Are transgenic crops safe? BMC Research Notes. 388Cite this article as: Adenle: Response to issues on GM agriculture in Africa: Are transgenic crops safe? BMC Research Notes 2011 4:388.