Antibodies (Abs) are used in a variety of applications among which immunoblotting, immunoprecipitation, affinity purification until microscopy applications, immunohistochemistry and flow cytometry.
An especially important role is covered by fluorescent antibodies. The excellent signal to noise ratio makes fluorescence the perfect tool for the study of the structure and the dynamic of the matter and living systems on molecular or nanometric scale (H. Kim 2013). However, the most used fluorophores, such as organic molecules and fluorescent proteins, are not photostable and the emitted light is affected by the action of pH and external interfering agents (L. Song 1995).
A strategy has been drawn up to overcome this issue: the inclusion of fluorescent molecules in silica nanoparticles (NPs). The silica matrix acts as a shield protecting the sensitive molecules against photobleaching (photochemical degradation of the fluorophore). This feature, added to the possibility to include more fluorescent molecules in a single nanoparticle, results in an increased fluorescent signal (C. Pellegrino 2018).
In addition, in the field of fluorescence, silica has proven to be an excellent platform, thanks to the fact that it is photophysically inert, it is transparent to ultraviolet and visible radiation, and it is not involved in energy or electron transfer processes. Consequently, all the photochemical properties of silica nanoparticles are conferred by the molecules doped inside the system or grafted on its surface. Another advantage of silica is that it is intrinsically non-toxic, in particular if compared to other nanomaterials such as quantum dots (A. M. Smith 2010), which are still widely employed in bioanalytical applications, despite the well know toxicity of its constituent materials and their blinking behaviour (M.Bottrill 2011). Preliminary experiments seem to support the harmless nature of silica nanoparticles.
Lastly, from a synthetic point of view, besides the fact that it is rather inexpensive, silica is a very appealing material: the preparation of nanoparticles usually requires mild conditions and does not involve complicated purification procedures, while size is tunable with few adjustments in the reaction settings. Moreover, the design of these systems can be based on a modular approach, making them adaptable for diverse applications, thanks to their extreme flexibility and versatility.
Thanks to the nanoparticles surface coating with polyethylene glycol (PEG), they are water soluble and highly biocompatible because of the ability to avoid the opsonization and with consequent increase of their plasma half-life. This feature put forward silica nanoparticles for in vivo applications such as in vivo diagnosis (e.g. PET, MRI) and drug delivery.
On this basis we compared the results obtained in flow cytometry using three different types of anti CD4 conjugates: with fluorescein (FITC), with AlexaFluor®488 and with the nanoparticle-based dye NTB520 all three with the same clone, EDU-2.
MATERIALS AND METHODS
Anti-human CD4 (EDU-2) production
The monoclonal antibody against human CD4 is obtained by affinity purification on protein G (Sepharose 4 Fast Flow, GE Healthcare) of cellular supernatant produced by highly selected hybridoma. The hybridoma EDU-2 is cultured in cell culture flasks with RPMI-1640 medium with L-Glutamine (Sigma-Aldrich), 5% foetal bovine serum (ThermoFisher) and 1% penicillin-streptomycin (Sigma-Aldrich). It is cultured in standard conditions at 37°C and 5% CO2 to allow the secretion of full Igs in the supernatant. The supernatant is harvested once a week and purified by affinity chromatography (figure 1).
Figure 1: Standard purified monoclonal antibodies production process
It is flushed across the Protein G column thanks to a Fast Protein Liquid Chromatography (FPLC) system (Model EP1, Bio-Rad) at 2mL/min in order to maximize the antibody binding to the resin. The adsorbed antibody is then detached using a pH gradient provided by an acid glycine solution (pH 2.4). The collected antibodies are buffered with a basic (pH 9.0) solution of Tris-HCl. The antibody concentration is estimated by means of the absorbance at 280 nm read on a spectrophotometer (Cary 60, Agilent Technologies). The storage buffer is exchanged by dialysis against phosphate buffer for, at least, 48 hours.
NTB520 are core-shell dye doped silica nanoparticles synthesized through a one-pot, two-steps reaction, deriving from Stöber synthesis, known as micelle-assisted method (figure 2). All the reagents are mixed together in a solution of water and n-butanol to allow the creation of micelles from the surfactant. All the hydrophobic reagents used in the reaction, spontaneously arrange inside the micelles. Thanks to the addition of ammonia and silane precursor, the base-catalysed hydrolysis of all the trialkoxysilanes takes place.
The variation of surfactant characteristics affects the nanoparticles size. The initial mixture contains 2 different hydrophobic fluorophores arranging inside the micelles and becoming part of the nanoparticle, thanks to the covalent modification with a trialkoxysilane group: as part of the structure, dyes are not released over time. Even without a covalent binding among the 2 fluorophores, they can generate an efficient fluorescence resonance energy transfer (FRET).
The number of fluorophores is modulated to obtain the maximum efficiency of the cascade transfer without inducing the self-quenching phenomenon, thanks to these devices NTB520 reach over 90% of FRET efficiency. NTB520 has the same excitation and emission wavelengths of the commercial dyes FITC and AlexaFluor®488 (C. Pellegrino 2018).
Figure 2: Schematic representation of NTB520 synthesis
The conjugations with fluorescein and with AlexaFluor®488 involve a preliminary step performed in a basic solution to expose the e amino groups on which the fluorescent molecules will be linked. The molar ratio fluorophore/antibody used is the standard one (25). The incubation happens for 60 minutes at room temperature (RT: 18-25°C) and the obtained mixture is purified by means of gel filtration (Sephadex G25, cut-off 1000-5000 Da, GE Healthcare).
Figure 3: Schematic representation of the anti CD4 conjugation to NTB520
A different conjugation approach is used for NTB520 as this kind of conjugation, is comparable to the one with big fluorescent proteins. The preliminary steps to be followed are two: the first one involves the activation of the nanoparticles by means of their conjugation with a crosslinker molecule; the second one concerns the exposition of the sulphidrilic groups in the hinge regions of the antibodies obtainable by a treatment with a reducing agent (figure 3).
The incubation conditions are the same used for the traditional fluorophores’ conjugations: 60 minutes at RT. The silica matrix allows a harder two-steps purification procedure after whom the purity degree of the conjugates is higher than 96%. The first purification stage is a size exclusion chromatography to eliminate the free antibodies from the mixture of the other specie (antibody-nanoparticles and free nanoparticles) the second purification is an affinity chromatography on protein G: in this case the unreacted nanoparticles will be flushed away and the conjugate construct will be retained by the resin. This second purification step is feasible thanks to the silica shell which protects the fluorescent molecules from the effects of the low pH used to detach the conjugate from the resin (figure 4).
Figure 4: Elution profiles after each one of the purification steps: the blue line is the absorption at 280nm, the red one the adsorption at 498 nm (nanoparticles maximum absorption). The green line represents the glycine buffer gradient used for the elution.
Flow Cytometry Analysis
The analysis in flow cytometry has been performed on whole blood from healthy donor pre-treated with EDTA as anti-coagulant. The conjugated antibodies have been incubated, at the same concentrations, for at least 15 minutes at RT in the dark. 2 mL of lysing solution (Biolisante, AcZon) have been added to the mixture and the tube has been vortexed at low speed. The following step is a 10 minutes incubation at room temperature in the dark.
The tubes are then centrifuged 5 minutes at 300 x g at 2-8°C. The supernatant is discarded, and the pellet is resuspended in 2mL of phosphate buffer. The samples have been read on the flow cytometer (FACSCanto™ II, BD Biosciences) within 1 hour, for each tube at least 10000 events have been examined. The data have been analysed on Kaluza Analysis software (Beckman Coulter).
RESULTS AND DISCUSSION
The analyses on peripheral whole blood with anti CD4 conjugates with 3 different typologies of fluorophores, highlighted a good discrimination among negative and positive populations in each of the three cases. The test performed comparing the 3 conjugates at the same concentration, shows differences in the fluorescence intensity (figure 5). The higher fluorescence intensity of the positive population, due to the use of NTB520 instead of traditional fluorophores, is related to a lower fluorescence level of the negative population, increasing once more the signal-to-noise ratio.
Figure 5: Comparison among the three different conjugates at the same concentration
The conjugation of fluorescent silica nanoparticles (like NTB520) to antibodies, combines the properties of nanoparticles to the inherent properties of antibodies such as the ability to specifically recognize antigens. Thanks to the isolation of the sensitive fluorescent molecules from the external, interfering, environment, the signal provided by this new class of fluorophores is more stable over time. This isolation solves another issue due to the non-specific binding of fluorophores to specific cellular populations such as monocytes, resulting in a reduced background.
The concentration capability allows the inclusion of more fluorophores in each nanoparticle, permitting the binding of more fluorescent molecules for single signalling unit (e.g. antibody) giving an enhanced fluorescence intensity (a decade respect of FITC and half a decade respect of AlexaFluor®488).
To confirm the superiority of nanoparticles-based reagents respect of those using conventional fluorophores, the stain index has been calculated for all the conjugates (data not shown). The stain index is the difference between the mean fluorescence of the positive population minus the central tendency of the negative population. This is divided by twice the standard deviation. Thanks to this measure, the fluorescence intensity of a fluorophore is normalized against all possible factors affecting it and can be compared among different fluorophores.
The stain index of NTB520 resulted higher than those of other fluorophores. These results confirm fluorescent silica nanoparticles as a promising tool for biomedical applications thanks to their high sensitivity, enhanced stability and reduced background noise.
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