Long Circulating Nanoparticles as Potential Antigen Carriers in Angiogenic Blood Vessels: Towards Tolerogenesis

Research Article

Austin J Nanomed Nanotechnol. 2014;1(1): 1005.

Long Circulating Nanoparticles as Potential Antigen Carriers in Angiogenic Blood Vessels: Towards Tolerogenesis

Amy Tekrony1, Vincent Wright2, Anne Slaney2, Usama Al-Atar2, Amy Frederick1, Teresa Rodriguez1, Jillian Buriak2, David Cramb1,4* and Lori West3

1Department of Chemistry, University of Calgary, Canada

2Department of Chemistry, University of Alberta, Canada

3Departments of Pediatrics, Surgery, and Immunology,University of Alberta, Canada

*Corresponding author: : David Cramb, Department of Chemistry, University of Calgary, 2500 University DR NW, Calgary, AB T2N 1N4, Canada

Received: November 27, 2013; Accepted: December 23, 2013; Published: December 31, 2013


Infants do not produce typical immune responses to foreign ABO-blood group antigens compared to older individuals who have a fully developed immune system. Therefore, incompatible donor organs can be transplanted into infants without rejection, which results in immunological tolerance. As a result, we hypothesized that intentional introduction of ABO-antigens would also induce tolerance and extend the period of time for safe ABO- incompatible transplantations. A proposed method to induce tolerance is by conjugating antigens to long-circulating silica nanoparticles and injecting them into blood vessels to promote maximum exposure of lymphocytes to the antigens. Here, we characterized synthesized nanoparticles for this purpose. Nanoparticle brightness and aggregation tendencies were determined for detectability and stability in a living system, respectively, using fluorescence correlation spectroscopy. Bright, non-aggregating nanoparticles were injected into the chorioallantoic membrane of chicken embryos to monitor circulation. It was determined that 100 - 200nm PEG-coated silica nanoparticles were easily detected, aggregated little, and circulated for a prolonged period of time in the blood stream.

Keywords:Silica nanoparticles; Tolerogenesis; Stealth nanoparticles; Fluorescence correlation spectroscopy; Chorioallantoic membrane of the chicken embryo.


It has been known for many years that induction of antigen tolerance is possible in animals. In 1945, Owen defined the inherent susceptibility that animals have to immune tolerance [1]. In 1949, Burnet and Fenner linked this tolerance to developmental events, using twin fetal cows [2], and in 1953, Medawar showed that this tolerance could be induced intentionally in mice [3]. Up until now, these findings were not considered to be clinically relevant to humans since human neonates have an increased maturity of their immune systems at birth in comparison to animals, and therefore were thought to be beyond susceptibility to tolerance induction. A study by West and co-workers showing successful incompatible heart transplants was the first to refute what was previously thought and provide evidence for neonatally acquired donor-specific blood type antigen immune tolerance in humans [4]. Fan et al. also provide evidence that suggests persistent exposure to donor antigens is required for tolerogenesis (induction of immune tolerance), which is dependent on the degree of antigen expression [5]. Since evidence shows that the occurrence of tolerogenesis is dependent on degree of antigen exposure, it may be possible to induce tolerance via methods other than graft exposure or transplantation, thereby possibly reducing mortality rates on transplantation wait lists. If an infant must wait for an organ past the time of susceptibility to induced tolerance, it may beneficial to induce that tolerance previously to allow for tolerance to both A and B antigens, or essentially make that infant's blood type a "universal acceptor". This would increase the likelihood of transplantation at a later point in time when a donor organ may be available, thereby decreasing the likelihood of a fatal outcome.

In order to achieve this goal, it is essential to determine novel methods of tolerance induction. Since lone antigens in the blood stream would likely be cleared quickly by the renal system, our study takes steps towards the possibility of injecting nanoparticle-antigen conjugates into the blood stream to induce tolerance. Ideal particles will be easily detected and circulate for a prolonged period of time in vasculature to ensure maximum exposure to blood stream antigens. Therefore, since polyethylene glycol (PEG) coatings have been shown to increase nanoparticle (NP) circulation time in the blood stream, the majority of the silica NPs in this study were functionalized on their surfaces with PEG [6]. Silica has demonstrated biocompatibility [7] and silica NPs are easily tuned for size [8]. In addition, most NPs synthesized contain fluorescent dyes for detection in order to monitor and assess their localization and circulation behaviour.

Previous NP circulation studies have been performed either in adult animals [9] or in tumor models [10]. Neither of these are the most relevant to model neonatal blood circulation. Neonates will have varying degrees of natural angiogenesis, which is poorly represented by tumor models and not at all by adult animals. Desirable is a simple angiogenic model into which NPs can be injected and tracked for circulation time, aggregation behavior and general stability. The chorioallantoic membrane (CAM) of the chicken embryo is an excellent model for angiogenesis. Within the CAM the blood pressure is normal and the vessels are not tortuous unlike tumor models and those of most of the blood vessels in the zebrafish embryo. The CAM serves as the respiratory system for the chicken embryo up until day 19 of the 21 day gestation period, handles any waste products from the embryo, and supplies the embryo with nutrients from the yolk [11], The CAM has been used as a model to study angiogenesis of explanted tumors and anti-angiogenic drugs [12-18], tumor vascular targeting [19], metastasis [20,21], ion transport [22], allergens [23], transplantation [24], contraceptives [25,26], effects of hyperglycemia [27], and photodynamic therapy [28]. NPs can easily be injected into blood vessels of the CAM through a window cut into the eggshell, and it has been shown that the concentration of certain particles decreases exponentially within the first few minutes of injection [29,30]. This is due to loss of the NPs from the blood stream through the angiogenic fenestrations (~500nm in size) [31]. The presence of angiogenesis in these developing vessels allows us to find ideal NPs that will circulate for a prolonged period of time, despite the "leaky" nature of the developing vessels. The NP concentration in the CAM can be monitored through the window in the shell using fluorescence correlation spectroscopy (FCS).

Two-photon excitation (TPE) -FCS is a non-invasive fluorescence technique commonly used to analyze fluorescent particles in living organisms, primarily for advantages such as: low phototoxicity, longterm analysis and ability to distinguish aggregation [29]. TPE-FCS involves analysis of fluorescent intensity fluctuations resulting from two-photon excitation of fluorescent molecules to obtain data about diffusion, concentration, and size of the fluorophores. TPE-FCS has been used to gain valuable information in many applications such as the examination of angiogenic blood vessel formation in zebrafish; the study of active transport, localization of proteins, and diffusion of receptor clusters in cells; monitoring drug delivery using photocages; and the study of DNA replication [32].

In this study, TPE-FCS is used as the primary technique to provide minimally invasive monitoring of NP behaviour in the CAM of the chicken embryo. By tracking the change in concentrations and aggregation tendencies as functions of size and surface chemistries, we may determine which NPs are most easily detected, aggregate the least, and circulate in the blood stream the longest. These data will allow us to predict the NP properties most suitable to providemaximum blood exposure to the synthesized antigens used to induce immunological tolerance in infants.


Nanoparticle design and synthesis

Various NPs were synthesized with a range of: dyes, ratios of dye to silica, surface functionalizations, and dye-incorporation methods (See Table 1 for the naming system for nanoparticles, according to their synthesis). NPs with dye incorporation were synthesized according to the Stöber process [33], which involves the hydrolysis of alkyl silicates, followed by the condensation of silica acid in alcohol using ammonia as a catalyst. This synthesis resulted in uniform particles of which sizes can be controlled from approximately50nm to 2000nm in diameter [33]. In order to synthesize the coreshell particles, a modified version of the Stöber process was used, as described by Larson et al. [34], in which the dye-rich compact core s synthesized prior to the silica shell. The NPs were functionalized by directly adding polyethylene glycol (PEG) or (3-aminopropyl) -trimethoxysilane (APTMS) to the synthesis flask, which formed covalent bonds from the polymers to the NPs as the base-catalyzed silanization occurs [35]. Once synthesized, particles were resuspended in water and stored in scintillation vials covered with aluminum foil to reduce exposure to light.