Technology Behind our Nanoparticle Products

Through the years of research and development in Luna Nanotech we have developed an extensive expertise in nanoparticle synthesis, surface functionalization and characterization. In particular, we specialize in developing biocompatible nanoparticles that can be used in biological in vitro assays and administered in vivo. Please see below the description of technologies behind some of our nanoparticles with examples of their conjugation to biological ligands and suggested applications. Click one of the nanoparticle images below to go to the corresponding section.

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NanoVIVO™ Spherical Gold Nanoparticles

 
NanoVIVO™ Spherical Gold Nanoparticles

Gold nanoparticles are widely used in biomedical research due to their reproducible synthesis protocols, low batch to batch variability, high monodispersity and ease of conjugation to biological ligands. In addition, gold nanoparticles have a very strong absorbance cross section. This is the result of surface plasmon resonance (SPR) fields, which refers to collective oscillations of electrons on gold nanoparticle surface. SPR strongly couple to incident light, making nanoparticles appear extremely bright. The peak of the SPR absorbance depends on nanoparticle diameter, with larger particles preferentially absorbing at longer wavelengths.

Highly pronounced colour makes gold nanoparticles optimal contrast agents for colorimetric assays such as lateral flow pregnancy dipstick. Gold nanoparticles are also useful imaging agents for electron microscopy due their electronically dense structures. High monodispersity and precise control of nanoparticle size make gold nanoparticles useful tools to study the effects of size and surface chemistry on nanoparticle based drug delivery. One emerging application of gold nanoparticles is in formation of assembly structures that can respond to biological environment. The ability to easily functionalize gold nanoparticle surface at very high density makes them key building blocks of supramolecular architecture. 

Our gold nanoparticle line includes particles ranging in sizes from 4 nm to 200 nm diameter and a variety of surface chemistries. Figure below shows transmission electron microscopy images of the different nanoparticle sizes and their associated absorbance profiles.

Gold Nanoparticle Synthesis

We follow a well-established synthesis strategy to generate citrate coated gold nanoparticles ranging from 4 to 200 nm in diameter. 15 nm diameter nanoparticles are synthesized through Oswald ripening process using citric acid to reduce gold chloride salt. Citrate also serves as a surfactant that coats nanoparticle surface and stabilizes them in water and mild salt conditions.

15 nm gold nanoparticles are then used as seeds onto which larger nanoparticles are grown. In this process a combination of citric acid and hydroquinone are used as reducing agents for gold ions. Hydroquinone is removed from the solution by washing steps, but small amounts of this molecule might remain on the surface, which is mostly coated with citrate.

To synthesize 4 and 7 nm gold nanoparticles, a combination of citric and tannic acids are used to reduce gold ions. After synthesis the surface of these nanoparticles is further stabilized by Bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salts (BSPP). Multiple washing steps are performed to remove tannic acid, but small amounts of these molecules might remain on nanoparticle surface.

All of the particles are kept in solution containing 0.01% of Tween-20 surfactant to improve their stability and increase shelf life. Twin-20 forms a loose coat on the nanoparticle surface, which can be removed by washing steps. If your application is incompatible with the presence of Tween-20 surfactant, please contact us and we can supply gold nanoparticles without added Tween-20. However, these nanoparticles will have reduced shelf life. Table below lists the surface chemistries of the different nanoparticle sizes.

Citrate coated nanoparticles are stable in water and weak buffers at neutral or acidic pH. However, they rapidly aggregate in physiological buffers. Their stability can be significantly improved by replacing the citrate by a thiol-containing methoxy-terminated polyethylene glycol (mPEG) molecules. mPEG coated nanoparticles remain monodispersed over long periods in physiological and higher salt (up to 1 M NaCl) buffers.

Gold Nanoparticle Functionalization

The citrate surfactant can be readily displaced from the nanoparticle surface by thiol-containing molecules that form a strong thiol-gold covalent bond. This interaction has a very low footprint, which allows loading of surface ligands at very high densities that cannot be achieved with most other nanoparticle types. Functional groups, such as amines or carboxyls, can be introduced onto nanoparticle surface by using a bifunctional polyethylene glycol (PEG) linker, which includes the gold binding thiol at one end and the required functionality at the other. However, introduction of charged terminal groups tends to reduce nanoparticle stability. Therefore, functional PEG molecules are interspersed with an inert mPEG on the nanoparticles surface for stabilization. We optimized our nanoparticle design to obtain a stable configuration which maximizes the number of available functional groups. The ratio of mPEG to functional PEG is indicated in the Product Data Sheet for each design. The following functionalities are currently available.

Fluorescent Gold Nanoparticles

Luna Nanotech offers a line of fluorescently labeled gold nanoparticles. Fluorescent molecules are loaded onto nanoparticle surface using PEG spacer. To avoid the dye from being quenched by the nanoparticle, longer PEG spacer of 5 kDa or 10 kDa is used to keep the dye away from the surface. Fluorescently labeled PEG is interspersed with mPEG to ensure nanoparticle stability in physiological buffers. Currently available fluorophores include FAM, Cy3, Cy5 and Cy7 dyes. Please contact us to set up custom synthesis for any other fluorophores.

Protein Functionalized Gold Nanoparticles

Luna Nanotech products also include gold nanoparticles functionalized with protein ligands.  Available ligands include such proteins as Albumin, Streptavidin, Transferrin, Lectin, Proteins A, G, L, and a number of different antibodies. With the exception of Albumin proteins are covalently attached to the gold nanoparticle surface through long 5 kDa PEG spacers. This positions the proteins far enough from the particle surface to ensure that steric hindrance does not interfere with proteins binding to their target. In addition, nanoparticle surface is backfilled with a brush layer of shorter 2 kDa methoxy-terminated PEG. Such backfill prevents non-specific protein adsorption onto the nanoparticle, which can interfere with the targeting protein-receptor interaction and lead to reduced blood half-life in vivo. Albumin is non-covalently adsorbed onto the nanoparticle at high surface coverage density, which stabilizes the particles in physiological fluids and reduces non-specific protein adsorption.

 

Nanoparticles can also be optionally labeled with FAM, Cy3, Cy5 or Cy7 dyes, allowing them to be fluorescently tracked both in vitro and in vivo. These dyes are also covalently introduced at the terminal ends of 5 kDa PEG spacers.

 

Please contact us if you are looking for nanoparticles functionalized with a different protein currently not available in our product line. We use robust bioconjugation protocols that can be easily modified to introduce new targeting ligands.

DNA Functionalized Gold Nanoparticles

Gold nanoparticles are also available with their surface functionalized with single-stranded DNA oligonucleotides. DNA is introduced at saturating surface coverage of around 0.1 oligos per square nanometer. This is the highest surface saturation that can be achieved due to strong repulsion between negatively charged DNA backbones. To further stabilize these nanoparticles against salt-induced aggregation and to minimize non-specific protein adsorption, their surface is backfilled with PEG of 1, 2, 5, or 10 kDa. Longer PEG molecules provide higher degree of stabilization and protection, but reduce accessibility of the oligonucleotide for complement hybridization or protein binding.

NanoVIVO™ Gold Nanorods

Gold nanorods have a very high absorbance cross section and efficiently convert the energy of optical excitation into heat generation. Nanorod geometry can be varied to adjust the maximum absorbance wavelength, allowing it to extend into the near-infrared region (670 nm to 760 nm). This region is particularly useful for biological applications since it corresponds to the optical penetration window of minimal tissue absorbance. This makes gold nanorods optimal probes for photothermal therapy, dark field microscopy and plasmonic nanosensors.

The location of the peak of the gold nanorod absorption profile depends both on its dimensions and the aspect ratio (the ratio of the length and cross sectional diameter. Nanorod length can vary between 29.2 nm and 52.6 nm, and aspect ratio between 2.2 and 3.9 is possible.

 

Gold nanorods are initially synthesized using C-Tab surfactant. However, this detergent has been found to be highly toxic to living cells and tissues. In addition, C-Tab functionalized nanoparticles are not stable and have to be kept at elevated temperatures of >30ºC. Therefore, post-synthesis we replace C-Tab surfactant with polyethylene glycol (PEG) molecules of 2 kDa, 5 kDa, or 10 kDa in size. Such surface chemistry makes the nanoparticles highly resistant to charge induced aggregation, reduces non-specific protein adsorption, and maximizes biocompatibility. This surface modification also allows functional groups to be introduced at the PEG terminal ends.

 

NanoVIVO™ Magnetic CLIO Nanoparticles​

Cross-linked dextran coated iron oxide (CLIO) nanoparticles are synthesized by seed-mediated growth of iron oxide salt precursors in the presence of dextran sugar (40 kDa or 70 kDa). This process produces superparamagnetic nanoparticles with large magnetic moment. These nanoparticles have 1-3 of iron oxide magnetic cores (8-10 nm in diameter) imbedded within a polymerized dextran coat. The overall nanoparticle hydrodynamic diameter ranges between 65 nm and 100 nm, with 70 kDa dextran producing larger particles. In order to prevent dextran depolymerization leading to the nanoparticle breakdown we covalently cross-link the dextran sugar strands. After cross-linking, different surface functionalities such as Biotin, Streptavidin, or Fluorescent Dye are introduced.

 
CLIO Nanoparticle Functionalization

CLIO nanoparticles can be further functionalized with a range of different biological ligands. Molecules containing carboxylic acid (such as proteins) or its activated NHS variant can be directly conjugated to the amine group on CLIO nanoparticle surface using EDC/NHS catalyzed bioconjugation reaction.

Many commercially available antibodies have been conjugated to a Biotin molecules. Biotin has a very strong affinity for Streptavidin protein (Kd = 10-15 M). Therefore, simply mixing Biotin-conjugated antibodies with our NanoVIVO™ Magnetic CLIO nanoparticles functionalized with Streptavidin will load these antibodies onto the nanoparticle surface.

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