Small spheres, big potential

The integration of nanotechnology and biophotonics techniques has resulted in development of nanoparticle platforms that will impact biophotonics applications such as bioimaging, biosensing, genomics and proteomics, light-activated therapy, and tissue engineering.

These platforms include 20- to 40-nm-diameter silica bubbles that can have molecules attached to the outside or encapsulated inside. Silica-based nanoparticle platforms provide a number of advantages: the nanoparticles can be made under mild conditions that don't damage biological activity; the nanometer-scale size of the particles minimizes interaction with the immune system; the silica shell can be functionalized for coupling of biologics; silica nanoparticles are stable in biological environments; and silica-based nanoparticles are optically transparent.

The potential of nanoparticle technology for biophotonics can be seen in the example of a multifunctional nanoclinic, a patented concept in diagnostic and targeted therapy developed at the Institute for Lasers, Photonics, and Biophotonics at the State University of New York, Buffalo.¹ The development of a multifunctional nanoclinic illustrates the power of a multidisciplinary approach involving nanochemistry, bio-optics, and biotechnology.

Figure 1. A nano-sized silica bubble provides both an optical probe in the core and surface sites to anchor drugs or biotargeting groups.

The nanoclinic can be visualized as a mobile clinic housing various optical diagnostic tools and therapeutic agents in a silica bubble (see figure 1). Chemical modification of the nanoclinic's surface allows them to couple with appropriate carrier or biotargeting groups. The carriers have a selective affinity for the receptors present at the biological site being targeted. They direct the nanoclinic to a specific biological site, which can be a specific compartment (organelle) of a biological cell, a specific cancer cell, or a diseased tissue.

The silica shell of the nanoclinic can also be chemically modified, such as in the form of an organically-modified silica. We can make the shell hydrophobic, hydrophilic, or both so that the character of the nanoclinic is compatible with the desired biological media. Similarly, we can tailor the porosity of the surface to allow for communication between the cellular environment and the interior components of the nanoclinic. This allows for interaction with the cellular environment for biosensing.

We can also package optical probes such as fluorphores inside the nanoclinic for applications in bioimaging, biosensing, and real-time monitoring of intracellular physiological activities. Therapeutic agents can be immobilized either on the surface (as for gene delivery) or encapsulated inside the nanoclinic. Light can trigger the release of therapeutic agents, either by cleaving the bond that anchors the agent to the nanoclinic or by photoactivation as in photodynamic therapy.

A prototypic nanoclinic first synthesized at the Institute consisted of a magnetic iron oxide (Fe₂O₃) core, a two-photon optical probe, and a silica shell. We covalently coupled leutinizing hormone-releasing hormone (LH-RH), a biotargeting hormone analog, to the shell. LH-RH acts as a carrier for the receptor expressed on the surface of cells in some types of cancer, including breast, prostate, and ovarian. Multistep nanochemistry produced the initial nanoclinics. They are fabricated to encapsulate various optical, magnetic, or electrical probes and act as platforms to deliver externally activatable drugs or therapeutic agents. Prototype multi- functional nanoclinics containing the magnetic Fe₂O₃ nanoparticles also produced a new targeted therapeutic effect: the selective destruction of targeted cancer cells in a DC magnetic field (magnetocytolysis).²

Two-photon excitation is a nonlinear optical technique that allows the excitation light to pass deeper into tissue, offers the user better control of excitation location, and results in less collateral tissue damage than single-photon excitation. The two-photon probe requires two photons to be activated, rather than just one. The advantage of two-photon laser-scanning microscopy (TPLSM) is that two-photon excitation is highly localized near the focal point, allowing such systems to essentially reject out-of-focus fluorescence. The use of a near-IR or IR pulsed laser minimizes autofluorescence as well as cell damage, while simultaneously providing enhanced tissue penetration.

We performed TPLSM using a modified BioRad confocal microscope. The titanium-doped sapphire excitation source generated 90-fs pulses at 800 nm, operating at an 82-MHz repetition rate. We adjusted the average power at the sample to less than 15 mW to avoid photodamage to the cells. The system used a water-immersion objective lens for cell imaging. The incorporated two-photon probe allowed us to visualize the selective interaction of nanoclinics and cells, and the internalization of the nanoclinics into the cells in real time, using TPLSM.

Figure 2. An optical section shows an oral epithelial carcinoma cell with internalized HPPH nanoclinics. Fluorescence of nanoclinics is indicated by red pseudocolor.

Figure 3. An optical section shows a human uterine tumor generated in an athymic mouse. The mouse was injected with HPPH nanoclinics and the tumor harvested 24 hours later. Presence of nanoclinics (indicated by red pseudocolor in tissue) indicates that targeted nanoclinics are concentrated in the targeted tissue.

Images obtained from the interaction of these nanoclinics with cells confirmed the specificity of this nanoclinic for LH-RH receptor positive cell lines and tumor tissue (see figures 2 and 3). Prototypic multifunctional nanoclinics containing the magnetic Fe₂O₃ nanoparticles also produced a new targeted therapeutic effect—the selective destruction of targeted cancer cells in a DC magnetic field by magnetocytolysis.³

Photodynamic therapy (PDT) has emerged as a promising treatment of selective cancers and other diseases. A photosensitizing drug (P) accumulates in tumors, and then the tumor site is exposed to a light of specific wavelength (h*). The PDT drug absorbs this light, producing reactive oxygen species that can destroy the cells of the tumor. In most cases, the reactive oxygen species is singlet oxygen (¹O₂) produced by following set of photoprocesses:

1_P _____hυ______> 1_P* __Intersystem crossing_> 3_P*

3_P* + ³O₂ __Energy transfer__> 1_P + ¹O₂*

Significant limitations hinder the use of PDT for many types of malignancies, however. There are limits to how deeply the activating light can penetrate tissue, for example. In addition, finding biologically compatible fluids to serve as solutes for the PDT drugs is nontrivial.

Researchers are addressing the latter problem using ceramic nanoclinics to deliver water-insoluble PDT. ⁴ We have trapped water-insoluble photosensitizing anticancer drug/dye 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide (HPPH) in ultra-fine hydrated silica-based nanoparticles 30 nm in diameter. Imaging with a confocal laser-scanning microscope verified that the nanoparticles were taken into cells.

Figure 4. A localized spectrograph of HPPH nanoclinics in the cancer cell shown in figure 2 shows that fluorescence from inside the nanoclinics can provide the excitation light to activate photodynamic therapy drugs.

A comparison of fluorescence spectra of HPPH and the fluorescence spectra from cells and gathered using localized spectrofluorometry confirmed that the intracellular fluorescence originated from the nanoparticles (see figure 4). The HPPH entrapped inside nanoparticles generates singlet oxygen when irradiated with the suitable wavelength of visible light. The singlet oxygen spectra obtained from the HPPH nanoclinic and free HPPH show similar phosphoresence intensity and peak positions (1270 nm), indicating similar efficiency of singlet oxygen generation in both cases. Subsequent in-vitro studies demonstrated significant damage to nanoclinic-impregnated tumor cells upon irradiation with 650-nm light. These observations suggest the potential of using ceramic-based nanoparticles as drug carriers for PDT.

Up-conversion, in which excitation light at a longer wavelength produces emission at a shorter wavelength, may provide the key to deeper tissue penetration. The process allows lower-energy, longer-wavelength photons to provide the excitation energy through multiple photon absorption. Because two-photon up-conversion is quadratically dependent on the excitation intensity, up-converting particles can provide better spatial resolution for bioimaging applications.⁵

These up-converting nanoclinics can penetrate the cell through the process of endocytosis or by attachment to the cell surface via a silica coating functionalized with a carrier or targeting group. The up-converting nanoclinics contain rare-earth-doped yttria (Y₂O₃) nanoparticles, coated with silica to produce nanophores about 25 nm in diameter.⁶ These silica-coated nanophores can disperse in water, are extremely stable, and exhibit no photobleaching.

The Y₂O₃ nanoparticles containing Tm³⁺, Er³⁺, or Yb³⁺ ions generate up-converted emission in the blue, green, or red spectral regions. The up-conversion processes exhibit step-wise multiphoton absorption; in other words, the up-conversion produced by the rare-earth ions involves two or more sequential linear absorptions, which only require a low-intensity continuous-wave laser at 974 nm. Because the nanophores emit phosphorescence with a lifetime typically in milliseconds (which is long compared to the nanosecond lifetime of dye fluorescence), they are not suitable for applications that require short-lived emission.

The nanophores hold promise in other ways. Currently, photosensitizers in clinical applications are photoactivated by a light source in the range of 630 to 690 nm. Tissue penetration (defined by 1/e) by these wavelengths is 2 to 4 mm, but the photodynamic effect is generally seen as much as two to three times deeper than that. As a result, despite the potential for PDT-induced cellular changes to reach as deep as 15 mm, in most cases PDT depth is much less than half of that.

Near-IR excitation sources can penetrate deeper into tissue. What, then, if we harnessed the up-conversion properties of silica-encapsulated rare-earth-doped Y₂O₃ nanoparticles for PDT? We could use the IR-to-visible up-conversion in these nanophores to activate the photosensitizer drug in situ.

To demonstrate proof of this concept, our team used HPPH to test the ability of the nanophosphors to excite a PDT drug. Studies clearly demonstrated that within the experimental parameters, both the green- and red-emitting up-converting nanophosphors are capable of exciting HPPH. Coupling of the nanophosphor emission with HPPH was shown by loss of particle emission and appearance of the HPPH emission. These preliminary studies suggest the feasibility of coupling nanophosphors and PDT.

Research combining nanotechnology and biophotonics is just beginning. The fusion of nanoscience and nanotechnology with biophotonics provides a new dimension for early detection of diseases and more effective targeted therapy. oe

Acknowledgements

The authors acknowledge the valuable help of X. Wang, G. Xu., T. De, L. Krebs, J. Bhawalkar, R. Kapoor, L. Levy, L. Lin, and C. Friend. This work was partially supported by the funding through the Directorate of Chemistry and Life Sciences of the Air Force of Scientific Research (F496200110358, F496209710454, 02-S470-020-C1), and New York State Technology and Research (NYSTAR) through the Center for Advanced Technology in Ultrafast Photonics at CCNY, New York, NY.

References

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6. R. Kapoor, et al., Optics Letters 25, 338-340, 2000.