Angiogenesis is a process wherein new vessels form in response to an ischemic or hypoxic stimuli [1, 2]. Angiogenesis is mediated through vascular endothelial growth factors, hypoxic ischemic growth factors, angiopoietic hormones, platelet derived growth factors and fibroblastic growth factors. Among all these factors VEGF plays a major role, and it exerts its effect not only by stimulation following hypoxic stimulus but also independently [3‐6]. VEGF primarily acts by phosphatidylinositol 3-kinase pathway through hypoxia inducible factor-1 transcriptional element [7]. The promoter region of VEGF is heavily influenced by hypoxic-ischemic growth factors [8]. Coronary collaterals are angiogenesis observed in response to ischemia, and it is usually a slow process [9]. In patients where coronary interventions or bypass surgery are not feasible, the growth of therapeutic collaterals would be very useful to reduce ischemic symptoms [10, 11]. Moreover, these patients are often debilitated by the ischemic symptoms. Therefore, there is a definite need for a novel therapeutic method for coronary ischemia other than angioplasty and coronary arterial bypass grafting. Hence, a method of targeted angiogenesis in the ischemic areas would be very useful as a novel and challenging therapeutic measure [11]. In the past angiogenic gene injection has shown some effects on the collateral formation with minimal benefits. Invasive angiogenic protein growth factor treatment with basic fibroblast growth factor (bFGF) or VEGF was ineffective in placebo-controlled clinical trials [12, 13]. As direct injection of proteins is ineffective, in this study, we focused on a novel therapeutic development using certain biocompatible magnetic nanoparticles as a novel carrier with vascular endothelial growth factors for growth of coronary collaterals. There is also an age-dependent impairment of angiogenesis [14]. Targeted angiogenesis is a therapeutic challenge, which is essentially useful to overcome ischemia in a focused and less invasive method. Controlled growth of collaterals in required regions or ischemic areas would be very useful in treatment strategies. The magnetic control of the particles would help to navigate or retain the particles in required ischemic regions, as isolated growth factors alone cannot be controlled.

Commercially available magnetic nanoparticles were acquired from NVIGEN Inc. USA with streptavidin on surface. Biotinylated vascular endothelial growth factor (Fluorokine) was acquired from MD systems Inc. USA. Thereafter, nanoparticles and growth factor conjugation was performed by standard techniques [15]. The size of the nanoparticles is in the range of 200 nm. To control the magnetic nanoparticles the required magnetic field gradient strength is approximately 10 T/M. Fluorescent tagging of the particles was performed using fluorescent conjugation. After completion of conjugation, the extent of release of the VEGF was studied. When the release of VEGF was confirmed the particles were taken up for tissue culture study. For setting up the experiment, standard techniques were followed [16, 17]. The experiments were setup in a vertical sandwich technique inside microfluidic chips. The tissue culture experiment was performed in a background of 5% CO₂.

HUVEC endothelial cells were modified to form clusters of HUVEC spheroids as the spheriods are better known to mimic natural cell responses and interactions [18, 19]. The extracellular matrix exerts its interaction with the cells, which again is influenced by the cellular architecture, and thereby determines the genetic and nuclear expression of the cells. This response is well observed with spheroids [20, 21]. This is especially useful for 3-D cell cultures due to its spheroidal shape. HUVECs are established to study angiogenesis or formation of capillary-like structures (CLS) in angiogenic engineering research [22, 23]. The tissue culture experiment was setup in four layers. The first layer had nutrients. The second layer was the collagen hydrogel layer; the third layer was a layer of HUVEC spheroids and the bottom layer was a layer of collagen hydrogel. The experiments were performed with nanoparticles in upper hydrogel layer, the lower hydrogel layer, and both layers and the results were compared to the control (Fig. 1).

The nanoparticles and the extent of the angiogenesis were studied using 2D-3Dimensional confocal microscopy with Z-stack fluorescent and bright field imaging. Serial pictures were acquired at regular intervals of 30 min were taken for 5 h in each experiment to study the extent of angiogenesis. Bright field imaging was used to study the extent of the spread of nanoparticles in hydrogel layer (Fig. 2). The nanoparticles in the hydrogel layer were manipulated using a bar magnet to desired locations for study. Two sets of experiment were performed in all the four cultures to study the extent and direction of angiogenesis. The concentration of the nanoparticles was the same in all the study experiments.

The nanoparticles could be used through intravascular or extravascular routes to form collaterals. Therefore, in experiment 2, which is the second phase of the study, the nanoparticles were studied for their effect on endothelial barrier crossing. The experiment was setup in a vertical sandwich structure inside a microfluidic chip with a monolayer of HUVEC cells, which was built on top of the collagen hydrogel layer. The nutrient layer with nanoparticles was placed above the HUVEC cell layer. The experiment was performed at 37 °C and 5% CO₂. The ability and the extent of the nanoparticles to cross the HUVEC layer to reach the lower hydrogel layer were studied using Z-stack confocal microscopy. The confocal pictures were taken at 10 min and at 48 h to visualize the extent of crossing by the nanoparticles. The confocal observations were also made in serial intervals of time.

The scanning electron microscopy picture of the colloidal suspension of magnetic nanoparticles is shown in Additional file 1: Figure S1. NMR images obtained are shown in Additional file 1: Figure S2.

Angiogenesis could be induced by the effect of magnetic nanoparticles conjugated with the vascular endothelial growth factor. These nanoparticles could be controlled by magnetic force, and these nanoparticles have possible potentials to cross endothelium.