Fabrication and Characterization of Nanoparticles in Biomedical Research

Topics Covered

Introduction

The webinar sponsored by Thermo Fisher Scientific and Current Protocols, Nanoparticle Fabrication and Characterization for Biomedical Research Applications, discussed nanotechnology and its current applications in biomedicine.¹ Some of the topics discussed were

1. definition of nanotechnology and nanoparticles ( nanoparticles)
2. considerations for synthesizing and characterizing nanoparticles
3. methods used for functionalizing nanoparticles
   
4. use of nanoparticles for biomedical/clinical applications.

This webinar is suitable for bench scientists who need to know how to characterize nanoparticles, researchers who want to use nanoparticles in their studies, or anyone who wants to translate nanotechnology research into clinical applications. Key points presented at the webinar are summarized below.

What is Nanotechnology?

The National Nanotech Initiative defines Nanotechnology as “research and technology development at the atomic, molecular or macromolecular scale leading to the controlled creation and use of structures, devices and systems with a length scale of approximately 1 – 100 nanometers (nm).” Nanoparticles are used in a variety of consumer products including clothing, wound dressings, sunglasses, sunscreens, cosmetics, sporting equipment, and structural materials.

What are the main biomedical uses of nanoparticles?

Nanoparticles can be use in biomedical applications to:

• Improve solubility - nanoparticles can be used as carriers for hydrophobic drugs (e.g., Abraxane)
• Give multifunctional capability- nanoparticles with dual functionality can be used for diagnostic and therapeutic purposes (e.g., Fe₂O₃-Pt nanoparticles)
• Target tumors - nanoparticles can be used to reduce toxicity of a therapeutic drug (e.g., Aurimune)
• Perform robotic tasks-Nanorobots can be used for drug release (e.g., photo- or pH-triggered drug release), thermal ablation, and hyperthermia. For example, AuroShell is a nanoproduct that kills cells at the target tumor site by emitting heat upon absorption of NIR light.

Which nanoparticles are commonly used for biomedical/clinical applications?

• Dendrimers - Currently investigated as pharmaceutical delivery systems because of their ability to improve the solubility of drugs, facilitate drug delivery and release, and target delivery to specific sites.²
• Quantum dots (QDs) - Currently used for molecular diagnostics, cytogenetics (e.g., QD FISH), and multiplex diagnostics.³ A potential application for QDs is cancer diagnostics. For example, QDs have been covalently linked to antibodies against HER2 to visualize tumor cells by immunofluorescence.⁴
• Gold nanoparticles - Currently used for molecular diagnostic applications. For example, DNA oligonucleotides can be attached to gold nanoparticles and used to detect complementary sequences on a sensor surface.³ Other applications include radiotherapy enhancement, drug delivery, and gene delivery.
• Gold nanoshells - Optical and physicochemical properties make nanoshells ideal for cellular imaging, cancer detection, cancer treatment, and medical biosensing. In addition, nanoshells tuned to absorb nearinfrared (NIR) radiation can be used for photothermal cancer therapy.⁵
• Mesoporous silica nanoparticles (MSNP) - Currently being used for theranostic purposes. For example, MSNPs can be used as carriers for therapeutic agents. MSNPs can be functionalized with different molecular or polymer moieties, which facilitate controlled drug delivery and release.⁶

How should I characterize nanoparticles that will be used in biomedical/clinical applications?

Characterization of nanoparticles will depend on the type of NP and its intended purpose. Preclinical characterization of nanoparticles often include

• Physiochemical characterization
• In vitro characterization
• In vivo characterization for safety and efficacy

Which tools are available to characterize the physicochemical properties of nanoparticles?

Physicochemical characterization will depend on the type of NP. Some of the tools and instrumentation used to characterize nanoparticles include

• Dynamic light scattering (DLS)-Measures hydrodynamic size, size distribution, and polydispersity.
• Atomic Force Microscopy (AFM)-Allows visualization and analysis in three dimensions including determination of shape, size, and distribution. AFM also allows surface characterization.
• Zeta potential-Physical property exhibited by any particle in suspension. It is defined as the difference in potential between the bulk solution (dispersing medium) and the surface of the hydrodynamic shear (slipping plane). It can be used to optimize the NP formulations and to predict long-term stability.
• UV-Vis spectrophotometer (e.g., NanoDrop)- Allows identification, characterization, and analysis of metallic nanoparticles (e.g., silver, gold). It can be used to determine size and evaluate the dispersion and local structure of nanoparticles synthesized with metal oxides, selenides, and sulfides.⁷
• TEM-Allows analysis in three dimensions including determination of shape and size. Histograms from the TEM images can be used to count single particles.

What are the advantages of using the NanoDrop 2000/2000c for characterization of nanoparticles?

Metal NP colloids with diameters between 1 – 100 nm have a unique optical absorption, which is related to the oscillation of surface electrons. This surface Plasmon resonance (SPR) property is dependent on the size and shape of the NP and the surrounding medium.^8,9 UV-VIS instruments, such as the NanoDrop 2000|2000c, allow analysis of surface plasmon resonance (SPR) signatures of metal NP colloids.¹⁰ The SPR band depends on the NP composition and morphology, and it is often used to judge synthesis success. SPR bands are also used to monitor concentration.

Because nanoparticles (especially gold nanoparticles) have very high extinction coefficients when their diameter is > 10 nm, they have a very high absorbance. It is, therefore, difficult to measure absorbance of highly concentrated samples using a cuvette with 1 cm pathlength. In addition, metallic nanoparticles used in self- assembly studies undergo tedious functionalization and purification. As a result, very small volumes of these colloids are usually available.

Because of the high absorbance capability and low volumes measured, an instrument such as the Thermo Scientific NanoDrop 2000|2000c UV-Vis spectrophotometer is particularly useful. The instrument has a short, variable pathlength (0.05 – 1.0 mm) and a very small sample volume requirement (1 – 2 µL). For more information visit www.thermoscientific.com/nanodrop.

What type of in vitro and in vivo characterization should I do before using nanoparticles for a biomedical/clinical application?

For in vitro characterization, nanoparticles may be assayed in biological matrices such as blood, plasma, cells, or primary culture.

Some in vitro tests that may be performed include

• Sterility check (e.g., testing for the presence of bacteria, virus or mycoplasma)
• Blood contact properties (e.g., plasma protein binding, hemolysis, coagulation, complement activation, cytotoxic activity of NK cells)
• Cell uptake and distribution (e.g., cell binding, NP internalization, receptor targeting)
• Toxicity (e.g., Phase I/II enzyme induction or suppression testing, oxidative stress, apoptosis testing, necrosis testing) The amount of information obtained by in vitro testing is limited. In addition to in vitro testing, nanoparticles that will be used for biomedical/clinical applications (e.g., therapeutics, in vivo diagnostics) have to be tested in animal models. In vivo assays can provide essential information regarding what may happen when the nanoparticles are inside the body.

Some in vivo tests that may be performed include dose-response; biodistribution; acute and multidose efficacy; safety; administration route determination; and absorption, distribution, metabolism, and excretion (ADME).

The ultimate goal of in vitro and in vivo testing is to match the physicochemical parameters of the nanoparticle to its biological function.

What questions should you ask yourself before starting a nanoparticle-based project?

1. What are you trying to do?
   Start with a plan. Do you want to use nanoparticles to transfect genes, diagnose disease or kill cancer? Once you know your objective, design your NP and your assays according to your goal.
   
2. What is a reasonable size for the nanoparticle in your system?
   Know the relevant sizes of the biology you will be dealing with during your study. For example, if your goal is to transfect cells, you need to keep in mind that cells are around 10 µm in diameter; therefore, your NP should be a lot smaller (e.g., 10 – 20 nm) than your target cell type.
   
3. Does the nanoparticle need to be water-soluble?
   If you are working with a biological system, the answer is YES! Organic polymers (e.g., PEG) can be used to coat the surface of nanoparticles and enhance their solubility.
   
4. Is the nanoparticle going to be directed to its target?
   Nanoparticles can target specific (e.g., pathogenic) cells by two different approaches:
   1. Passive targeting - Commonly referred to as enhanced permeability and retention (EPR) effect. Nanocarriers can target tumors passively by taking advantage of the tumor’s leaky vasculature and its poor lymphatic drainage due to rapid angiogenesis. This enhanced permeability allows nanocarriers to extravasate the endothelial barrier and accumulate in the tumor tissue but not in the healthy tissue surrounding the tumor.
   2. Active targeting - Specific ligands (e.g., monoclonal antibodies) are attached to the surface of nanoparticles. This ligand-nanoparticle conjugate then recognizes and binds receptors found in the targeted cells but not other cells. This targeting approach requires more knowledge about the ligand-receptor interaction and expression of the particular receptor on the target cell.
   
   
5. What type of nanoparticle is appropriate for my application?
   In general, organic (e.g., polymer) or inorganic (e.g., gold, silver, silica) nanoparticles can be used for biomedical/clinical applications. For example, gold nanoparticles may be used for a variety of applications including molecular diagnostics, thermal ablation, or drug delivery.
   Nanoparticles can be synthesized in-house or can be purchased from available vendors. In-house synthesis and characterization of nanoparticles takes time and effort. Moreover, the library of nanoparticles commercially available is small and you still need to validate the physicochemical or biological data provided by the vendor.
6. How are you going to characterize your material?
   nanoparticles are messy compared to small molecules and biomolecules such as DNA and protein. Contaminants including solvents, salts, mold, and bacteria can be present after synthesis. It is essential to purify and characterize your NP extensively. It is also important to test the stability of the NP preparation at different points during the workflow.
   Your characterization will be only as good as the resolution and the detection limits of the methods and instrumentation used for characterization. It is essential to know the limits of the characterization techniques/ instrumentation available to you.
7. What will the immune system see?
   The covalent bonds used for functionalization of nanoparticles are similar to those found in nature. Depending on your goal, this could have a positive or negative effect. For example, ester bonds, which are often used to neutralize
   Nanoparticles, are readily susceptible to esterases or may interfere with the acetylcholine receptor because of their structural similarities. If you want the bond to be stable, an esterase bond may not be the best choice.
8. What is your assay readout going to be?
   What assay are you going to use to validate the biological function of the NP? Identify any solvents or reagents used during NP synthesis or functionalization that may interfere with your assay (e.g., salt can negatively affect a fluorescent-based assay). What is your benchmark? Are you going to compare your results to a standard-of-care? Are you comparing to PBS?

Conclusion

As the field of biomedical research evolves and adopts new techniques and instrumentation, the use of nanoparticles will probably increase dramatically. This companion document to the webinar Nanoparticle Fabrication and Characterization for Biomedical Research Applications¹ provides an introduction and insight into the topic of nanoparticles. Knowing the answers to the questions presented in this document will ensure

1. correct design, synthesis, and functionalization of the nanoparticle
   
2. proper analysis of the physicochemical properties of the nanoparticle
   
3. correct design and development of assays to characterize its biological function

References

1. Thermo Fisher Scientific. Nanoparticle Fabrication and Characterization for Biomedical Research Applications. Available at: www.thermoscientific.com/nd-nanoparticles. Accessed on July 6, 2012
2. Stieger N, Liebenberg W, Aucamp ME, De Villiers MM. The Use of Dendrimers to Optimize the Physicochemical and Therapeutic Properties of Drugs. In: Cheng Y, ed. Dendrimer-Based Drug Delivery Systems: From Theory to Practice. Hoboken, NJ: John Wiley & Sons Inc; 2012:93-137.
3. Jain KK. Applications of Nanobiotechnology in Clinical Diagnostics. Clin Chem. 2007;53(11):2002-2009.
4. Tada H, Higuchi H, Wanatabe TM, Ohuchi N. In vivo Real-time Tracking of Single Quantum Dots Conjugated with Monoclonal anti-HER2 antibody in Tumors of Mice. Cancer Res. 2007;67:1138-1144.
5. Hirsch LR, Stafford RJ, Bankson JA, et al. Nanoshellmediated Near-infrared Thermal Therapy of Tumors under Magnetic Resonance Guidance. Proc Natl Acad Sci USA. 2003;100(23):13549-13554.
6. Li Z, Barnes JC, Bosoy A, Stoddart JF, and Zink JI. Mesoporous Silica Nanoparticles in Biomedical Applications. Chem Soc Rev. 2012;41:2590-2605.
7. Herrera JE, Sakulchaicharoen N. Microscopic and Spectroscopic Characterization of Nanoparticles. In: Pathak Y and Thassu D, eds. Drug Delivery Nanoparticles Formulation and Characterization. New York, NY: Informa Healthcare Inc; 2009:239-251.
8. Baptista P, Pereira E, Eaton P, Doria G, et al. Gold Nanoparticles for the Development of Clinical Diagnosis Methods. Anal Bioanal Chem. 2008;391:943– 950.
9. Toderas F, Baia M, Maniu D, Astilean S. Tuning the Plasmon Resonances of Gold Nanoparticles by Controlling their Size and Shape. J Optoelectron Adv M. 2008;10(9):2282-2284.
10. Hamner K, Maye MM, Ash DL, Page AF. Quantification of Gold Nanoparticles Using the Thermo Scientific NanoDrop 2000 Spectrophotometer. Available at: www.thermoscientific.com/nanodrop. Accessed on June 20, 2012.

Contact Details

Ilsa Gomez-Curet, Ph.D., Life Science Consultant and Medical Writer,
Thermo Scientific NanoDrop Products, Wilmington, DE, USA

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