Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Nanoparticles Shed Light on Cancer

By: Carol A. Rouzer, VICB Communications
Published:  April 21, 2016


Micellar nanoparticles are an efficient vehicle to deliver a targeted imaging agent for the detection of cancer and regions of inflammation.


Despite important advances in the diagnosis and treatment of cancer, many unmet clinical needs remain. Among these is the need for better methods to diagnose cancer early in the disease and to monitor the effects of therapy. To this end, an ongoing effort to discover imaging agents that are specific to targets found only in cancer cells is under way. One potential target is the enzyme cyclooxygenase-2 (COX-2), which is expressed in relatively few normal tissues but is upregulated at inflammatory sites and in many cancers. This led Vanderbilt Institute of Chemical Biology member Larry Marnett and his laboratory to launch a major effort aimed at the discovery of COX-2-targeted imaging agents. A successful outcome of this effort was fluorocoxib A (FA, Figure 1), a conjugate of the fluorescent dye 5-carboxy-X-rhodamine (5-ROX) and the nonselective COX-1 and COX-2 inhibitor indomethacin. FA is a potent and selective COX-2 inhibitor that has been validated as an optical imaging agent in numerous animal models, including carageenan-induced inflammation, spontaneous murine and canine colorectal carcinoma, human head and neck squamous cell carcinoma (HNSCC) xenografts, canine transitional cell carcinoma xenografts, non-melanoma basal cell carcinoma allografts, and murine spontaneous basal cell carcinoma. Despite these preclinical successes, however, translation of FA to the clinic has been hampered by its poor solubility in aqueous solutions suitable for drug delivery in humans. This led the Marnett lab to join with Craig Duvall of Vanderbilt’s Department of Biomedical Engineering to develop a nanoparticle-based delivery system for FA [M. J. Uddin, T. A. Werfel, et al. (2016) Biomaterials, 92, 71].


FIGURE 1. Structure of fluorocoxib A, a conjugate of indomethacin (black) and 5-carboxy-X-rhodamine (red). Figure kindly provided by L.J. Marnett and C.L. Duvall. Copyright 2016.

Polymeric nanoparticles are generating considerable interest as agents to solubilize and improve the pharmacokinetics of a wide range of drugs. Amphipathic diblock polymers can be particularly useful for this purpose. These materials, constructed of two blocks of different polymerized monomers, can include both a hydrophilic and a hydrophobic block, enabling them to form micellar nanoparticles that will entrap a hydrophobic drug in their core while remaining soluble in an aqueous environment. Additional advantages of these nanoparticles is that they can be constructed in a size range (20 to 200 nm in diameter) that both minimizes rapid renal clearance while maximizing uptake by inflamed or cancerous tissues as a result of their enhanced permeability. Construction of nanoparticles bearing inert materials, such as polyethylene glycol, on their surface reduces the likelihood that they will be opsonized and taken up by phagocytes. Finally, the chemical structure of the nanoparticle can be designed in such a way as to facilitate release of the drug cargo under the desired in vivo conditions.


The Duvall laboratory’s goal was to construct an FA-containing nanoparticle that encompassed all of these ideal characteristics. They chose a polymer of poly(propylene sulfide)106 (PPS106) to serve as the hydrophobic core of the nanoparticle. This choice introduced sulfide-containing functional groups that are susceptible to oxidation under the conditions of increased oxidative stress that are frequently found in cancer and inflammation. Sulfide oxidation leads to an increased hydrophilicity of the polymer, resulting in disruption of the micelle and release of the FA cargo. For the hydrophilic shell of the nanoparticle, the investigators selected poly[oligo(ethylene glycol)9 methyl ether acrylate]17 (POEGA17), based on prior reports that this polymer conveys excellent resistance to phagocytic uptake. The fully assembled diblock polymer (PPS106-b-POEGA17) formed micellar nanoparticles in phosphate-buffered saline that efficiently trapped either FA (FA-NPs) or the unconjugated 5-ROX fluorophore (5-ROX-NPs) (Figure 2).



FIGURE 2. Construction of the PPS106-b-POEGA17 diblock polymer and fluorophore-containing micelles. PPS106 bearing a hydroxyethyl end cap (1) was reacted with 4-cyano-4-[(ethylsulfanylthiocarbonyl) sulfanyl]pentanoic acid (2). The product (3) was then further reacted with oligo(ethylene glycol)9 methyl ether acrylate to form PPS106 -b-POEGA17 (4). Dropwise addition of a solution of PPS106 -b-POEGA17 in chloroform to phosphate-buffered saline followed by evaporation of the chloroform resulted in formation of micelles (5). Micelle formation in the presence of FA or 5-ROX led to entrapment of the fluorofore in the micelles, forming FA-NPs (6) or 5-ROX-NPs (7). FA-NPs were readily soluble in aqueous solution over a range of concentrations as indicated by the intense purple color (vials ii through vi), as compared to FA, which is insoluble at the lowest concentration (vial i). Figure kindly provided by L.J. Marnett and C.L. Duvall. Copyright 2016.



Physical analysis of the FA-NPs revealed that they contained 0.063 g of FA/g of nanoparticle. The ~82 nm diameter of the particles was within the ideal range for drug delivery, and the particles were stable in the presence of serum or whole blood. As predicted, exposure of the particles to hydrogen peroxide resulted in a concentration-dependent release of FA into the surrounding medium.


Exposure of COX-2-expressing human 1483 HNSCC cells to FA-NPs resulted in uptake of the fluorescent dye. No fluorescence was observed in the cells, however, upon exposure to 5-ROX-NPs or to nanoparticles containing no fluorophore. These results demonstrated that FA-NPs could deliver FA to COX-2 in living cells and led the investigators to further test them in vivo. They found that intraperitoneal injection of nude mice bearing xenografts of 1483 HNSCC cells with FA-NPs led to selective uptake of the fluorophore that was detected at 3 to 4 hours after nanoparticle administration. They confirmed that this uptake was COX-2-dependent by pretreating the animals with indomethacin to block the enzyme’s active site before FA-NP injection. The indomethacin pretreatment markedly reduced FA uptake by the tumors (Figure 3).


FIGURE 3. Imaging of cancer. Animals bearing xenografts of human 1483 HNSCC cells were injected with FA-NPs (left) or indomethacin followed an hour later by FA-NPs (right). Four hours following FA-NP injection, the animals were imaged. Figure kindly provided by L.J. Marnett and C.L. Duvall. Copyright 2016.



Injection of carageenan into the footpad of a rat or mouse elicits an inflammatory response that is mediated, at least in part, by COX-2. This model is particularly useful for the study of inflammation because it enables the investigator to compare the inflamed foot to the contralateral normal foot, which serves as a negative control. The researchers found that injection of the right hind paw of rats with carageenan followed two hours later by FA-NPs resulted in a selective uptake of the fluorophore in the inflamed paw as compared to the contralateral paw. Once again, pretreatment of the animal with indomethacin greatly reduced FA uptake at the site of inflammation (Figure 4).


FIGURE 4. Imaging of inflammation in the rat carageenan foot pad model. (a) The right hind foot of a rat (yellow arrow) was injected with carageenan, while the left foot was injected with vehicle. Two hours later, FA-NPs were administered. Then, imaging was performed 3 hours later. (b) The experiment was performed as in (a), except that indomethacin was administered 1 hour before FA-NPs. Figure kindly provided by L.J. Marnett and C.L. Duvall. Copyright 2016.


Although initial studies to test the feasibility of in vivo imaging with FA-NPs were done using intraperitoneal administration, the intravenous route is a much more efficient way to deliver nanoparticle-based drugs. Therefore, the researchers conducted pharmacokinetic studies following intravenous injection of mice with FA-NPs. The nanoparticles displayed a plasma half-life of 1.1 hours and were distributed primarily to the liver, kidney, and lungs at early time points. Their rate of disappearance from these organs reflected their rate of disappearance from blood plasma, and fluorescence returned to baseline by 4 hours. Intravenous injection of 1483 HNSCC tumor xenograft-bearing mice revealed that the fluorophore remained concentrated in the tumors well beyond 4 hours, indicating that the best signal-to-noise ratio for imaging occurs at the 4 hour time point or later. The investigators confirmed these findings using the mouse carageenan footpad model that demonstrated peak fluorescent signal in the footpad at 2 hours following FA-NP administration and retention of high signal-to-noise for a period of up to 8 hours. Footpad tissue from these mice also confirmed that hydrogen peroxide levels were two-fold elevated in the inflamed foot relative to the control foot, supporting the original hypothesis that oxidative stress in regions of inflammation would facilitate FA release from FA-NPs.


Although patient exposure to imaging agents is usually limited, safety remains of utmost importance during translation to the clinic. Thus, the investigators administered the minimal effective imaging dose, ten times that dose, or twenty times that dose to mice and then monitored them for signs of toxicity. They observed no toxic effects either by monitoring serum markers of liver or kidney damage or by histopathological examination of multiple organs from the mice.


These data confirm the value of FA-NPs as a delivery vehicle for the use of FA as an in vivo agent for imaging cancer and inflammation. Data support the hypothesis that the nanoparticles efficiently deliver FA to the area of interest by both taking advantage of increased vascular permeability at the site and by avoiding excessive renal clearance or phagocytic uptake. Release of FA at the target site is facilitated by elevated oxidant stress known to occur in areas where COX-2 is expressed. The successful delivery of FA in vivo in a form associated with very low toxicity is an important step forward for the future clinical translation of this optical imaging agent.


View Biomaterials article: Fluorocoxib A loaded nanoparticles enable targeted visualization of cyclooxygenase-2 in inflammation and cancer






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