Nanotechnology and nanomaterials fuel advanced conceptual approaches in many areas of medical science and engineering. Now, biomedical nanotechnology, in which bioengineers construct nanoparticles combining polymeric and biological materials, is moving to the forefront of this rapidly advancing field of science.1 The integration of nanotechnology with biology and medicine will result in major medical advances leading to the development of multifunctional systems and smart drugs that will target only the diseased site in the body.
Our group is developing near-IR (NIR), polymeric, biodegradable/biocompatible nanoparticles that can act as both diagnostic and treatment agents for clinically superficial tumors, such as those found in skin and breast cancer (see figure 1). These nanoparticles belong to a new class of multifunctional biophotonic systems that not only serve as high-quality diagnostic fluorescence agents for noninvasive tumor detection but also as noninvasive tumor destruction systems with photodynamic anticancer properties. Such nanoparticles can also provide controlled release and targeted (tumor-specific) delivery of drugs, diagnostic agents, proteins, genes, and DNA, which highlights the multifunctional properties of the particles.
Figure 1. Nanoparticle-encapsulated ICG referentially locates in tumors to allow noninvasive imaging and destruction of superficial tumors.Diagnostics and Therapeutics
Conventional methods for cancer diagnosis use roentgenography, scintigraphy, ultrasound, and magnetic resonance imaging techniques for tissue imaging. Optical imaging provides an alternative tumor detection approach with great potential in clinical diagnostics. Among its other advantages over conventional modalities, the optical approach uses neither ionizing radiation nor radioactive materials, and can produce a wealth of information from light-tissue interaction. Fluorescence-based NIR optical imaging techniques for clinical oncology are gaining in importance, because blood and other tissues are relatively transparent across this wavelength range and the penetration depth of light in the biological tissue is highest. High-resolution NIR detectors are both available and affordable, as well.
After cancer detection, treatment usually relies on conventional approaches such as chemotherapy, radiation therapy, and hormone therapy, as well as invasive approaches such as surgery. The drawback of these approaches is mainly toxicity, serious side effects, exposure to radiation, and damage to the non-cancerous parts of the body; additionally, patients face intense physical and emotional suffering. Photodynamic therapy (PDT) offers a well-established alternative.
PDT involves the administration of a photoactive drug that preferentially locates in the tumor. Irradiation activates the drug, which then oxidizes tumor cells. Because PDT is not a systemic treatment, patients suffer minimal side effects. The use of a focused light beam for activation instigates selective killing of tumor cells. Low cost, repeatable, and less painful than conventional methods, PDT can also be effective for situations in which surgery is contra-indicated.
Indocyanine green (ICG) is an FDA-approved, NIR fluorescence contrast agent that also has the ability to kill cells by PDT.2,3 ICG provides particular benefits for in vivo imaging, since the ICG fluorescence remains relatively free of intrinsic background interference (its strongest absorption band is around 800 nm and its most intense emission is around 820 nm in vivo), which enhances signal selectivity.4
Our goal is to develop a system having both NIR fluorescence and PDT capabilities, which makes ICG an ideal starting point. The agent is not perfect, however. It shows very high protein binding in the blood and rapid elimination from the body (plasma t1/2 is 2 to 4 minutes), which leads to low tumor targeting, accumulation, and intracellular uptake of ICG. These drawbacks limit its tumor imaging and anticancer therapy applications.5 The instability of ICG in aqueous media is another major limitation, as the agent has to be administered in the form of an aqueous solution for both diagnostic and therapeutic purposes. The Nanoparticle Solution
To alleviate the above limitations of ICG, we have developed polymeric nanoparticles that encapsulate the ICG in biodegradable/biocompatible nanoshells that act as a carrier, delivery, and targeting system. The encapsulation stabilizes ICG by shielding it from the surrounding aqueous environment while enhancing the intracellular uptake of ICG by tumor cells by altering the mechanism of cell uptake. The ICG nanoparticles also enhance tumor accumulation by passive targeting (tumor entrapment as a result of the leakiness of the blood vessels associated with tumors).6 Attaching specific targeting ligands such as monoclonal antibodies, fractional antibodies, or peptides to the nanoparticles enables us to actively target delivery to various tumor sites in the body. Overall, the nanoparticles enhance the diagnostic utility and tumoritropic behavior of ICG.
Intravenous administration of the nanoparticles requires the material to have a long circulation time while remaining biocompatible and biodegradable. Poly(DL-lactide-co-glycolide) (PLGA) polymer, FDA-approved and widely used in the pharmaceutical industry, offers a good solution. We engineered PLGA nanoparticles using a modified spontaneous-emulsification solvent-diffusion method.7 First, we separately prepared PLGA solution in acetonitrile and ICG solution in methanol, then mixed the two to get a single organic phase. Adding the organic solution drop-wise into aqueous emulsifier solution, while stirring, produced the nanoparticles, which were subsequently isolated by centrifugation and washed by distilled water before freeze-drying and storage.
The resultant nanoparticles successfully entrapped ICG within polymer matrix. The use of FDA-approved ingredients and preparation processes compatible with high-volume manufacturing, combined with the biodegradability, biocompatibility, and non-toxicity of the materials, make these nanoparticles ready for widespread use in diagnostics and PDT. Testing
We characterized the nanoparticles using dynamic light scattering to determine size distribution, atomic force microscopy to determine surface and shape, and zeta potential analysis to determine the surface charge of the nanoparticles. The nanoparticles produced have about 75% ICG entrapment. They were nearly 300 nm in diameter with narrow size distribution, spherical shape, porous surfaces, and a surface charge of -16 mV.
The nanoparticle-encapsulated ICG remained stable in aqueous media. In addition, the nanoparticles provided photostability and thermal stability to ICG, with half-lives on the order of two and a half to three days as compared to half-lives of a few hours for free ICG in aqueous media.
We tested the in vitro intracellular uptake of ICG on B16-F10 and C-33A cancer cells, studying the effect of concentration and kinetics of uptake for nanoparticles and free ICG. Fluorescence microscopy confirmed the subcellular localization of nanoparticles. Delivery by nanoparticles enhanced the ICG intracellular uptake 100-fold, especially at low ICG concentrations.
Using a 70-mW diode laser operating at 786 nm, we established the photodynamic properties of the nanoparticles. The particles showed significant photodynamic activity at nanomolar ICG concentrations, even when exposed to a low light dose of 1.1 J/cm2, demonstrating enormous potential for PDT.
Finally, our group studied the biodistribution of ICG in female black mice after a tail vein injection. When given in free solution, ICG was quickly eliminated from the body. In contrast, nanoparticle encapsulation increased the in vivo retention time of ICG in different organs and plasma, increasing the concentration of the agent in various organs for up to four hourstwo to five times higher than that of ICG in solution. These tests reflected the ability of the nanoparticles to efficiently deliver, accumulate, and retain ICG in the body.
Our results demonstrate that PLGA nanoparticles provide ideal vehicles for tumor delivery of ICG. The nanoparticles stabilize ICG in aqueous media, enhance intracellular uptake of ICG by tumor cells, increase the accumulation and retention of ICG in various organs, and show promise of photodynamic anticancer activity. As engineered and tested above, the nanoparticles represent a simple, safe, and noninvasive method that can be used by doctors for early detection, monitoring, and destruction of superficial tumors. oeReferences
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3. V. Saxena, M. Sadoqi, and J. Shao, J. Pharm. Sci. 92, p. 2090 (2003).
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5. V. Saxena, M. Sadoqi, and J. Shao, J. Photochem. Photobiol. Bio. B. 74, p. 29 (2004).
6. V. Saxena, M. Sadoqi, and J. Shao, Int. J. Pharm. 278, p. 293 (2004).
7. H. Murakami et al., Int. J. Pharm. 187, 143 (1999).
Vishal Saxena, Mostafa Sadoqi, Jun Shao
Vishal Saxena is a PhD student and Jun Shao is a professor in the department of pharmacy and administrative sciences, and Mostafa Sadoqi is professor in the department of physics, at St. John's University, Jamaica, NY.
Sunil Kumar is a professor in the department of mechanical engineering at Polytechnic University, Brooklyn, NY.