High Efficiency Diffusion Molecular Retention Tumor Targeting

<div><p>Here we introduce diffusion molecular retention (DMR) tumor targeting, a technique that employs PEG-fluorochrome shielded probes that, after a peritumoral (PT) injection, undergo slow vascular uptake and extensive interstitial diffusion, with tumor retention only through integrin molecular recognition. To demonstrate DMR, RGD (integrin binding) and RAD (control) probes were synthesized bearing DOTA (for ¹¹¹ In³⁺), a NIR fluorochrome, and 5 kDa PEG that endows probes with a protein-like volume of 25 kDa and decreases non-specific interactions. With a GFP-BT-20 breast carcinoma model, tumor targeting by the DMR or IV methods was assessed by surface fluorescence, biodistribution of [¹¹¹In] RGD and [¹¹¹In] RAD probes, and whole animal SPECT. After a PT injection, both probes rapidly diffused through the normal and tumor interstitium, with retention of the RGD probe due to integrin interactions. With PT injection and the [¹¹¹In] RGD probe, SPECT indicated a highly tumor specific uptake at 24 h post injection, with 352%ID/g tumor obtained by DMR (vs 4.14%ID/g by IV). The high efficiency molecular targeting of DMR employed low probe doses (e.g. 25 ng as RGD peptide), which minimizes toxicity risks and facilitates clinical translation. DMR applications include the delivery of fluorochromes for intraoperative tumor margin delineation, the delivery of radioisotopes (e.g. toxic, short range alpha emitters) for radiotherapy, or the delivery of photosensitizers to tumors accessible to light.</p> </div

Tumor targeting by DMR by using the GFP expressing BT-20 breast carcinoma xenograft visualized by surface fluorescence.

<p>A) Two animals bearing two tumors were PT injected with the RGD probe or RAD probe as indicated and surface fluorescence images were obtained. With the RAD injected animal, tumors were more sagittal so two views of the same animal are provided. Green = GFP. Purple = probe. White = green+purple overlay. The RGD probe diffused around the tumor and is retained while the RAD probe was eliminated. B) Quantitation of tumor surface fluorescence after injections of the RGD or RAD probes as above. Surface fluorescence was quantified through the use of standard solutions. Only the RGD probe was retained by the tumor. n = 4, values are mean ± SD.</p

Synthesis of RGD and RAD probes.

<p>The general strategy used to synthesize the RGD probe (7a) and RAD probe (7b) is shown.</p

Radiosynthesis and Evaluation of an ¹⁸F‑Labeled Positron Emission Tomography (PET) Radioligand for Metabotropic Glutamate Receptor Subtype 4 (mGlu₄)

Four 4-phthalimide derivatives of <i>N</i>-(3-chlorophenyl)-2-picolinamide were synthesized as potential ligands for the PET imaging of mGlu₄ in the brain. Of these compounds, <i>N</i>-(3-chloro-4-(4-fluoro-1,3-dioxoisoindolin-2-yl)­phenyl)-2-picolinamide (<b>3</b>, KALB001) exhibited improved binding affinity (IC₅₀ = 5.1 nM) compared with ML128 (<b>1</b>) and was subsequently labeled with ¹⁸F. When finally formulated in 0.1 M citrate buffer (pH 4) with 10% ethanol, the specific activity of [¹⁸F]<b>3</b> at the end of synthesis (EOS) was 233.5 ± 177.8 GBq/μmol (<i>n</i> = 4). The radiochemical yield of [¹⁸F]<b>3</b> was 16.4 ± 4.8% (<i>n</i> = 4), and the purity was over 98%. In vivo imaging studies in a monkey showed that the radiotracer quickly penetrated the brain with the highest accumulation in the brain areas known to express mGlu₄. Despite some unfavorable radiotracer properties like fast washout in rodent studies, [¹⁸F]<b>3</b> is the first ¹⁸F-labeled mGlu₄ radioligand, which can be further modified to improve pharmacokinetics and brain penetrability for future human studies

SPECT/CT images after PT and IV injections with the ¹¹¹In RGD probe using the BT-20 tumor model.

<p>SPECT images after injections (A–C) of the ¹¹¹In-RGD probe by the IV or PT DMR methods are shown with one or two tumors/animal (arrows). Radioactivity is shown with a green to red color scale, while CT bone density is yellow. A) Tail vein IV injection. B) Single PT injection (DMR). C) Dual PT injections (DMR). Post dissection tissue radioactivity concentrations obtained with the ¹¹¹In-RGD and ¹¹¹In-RAD probes by IV injection (D) and PT injection (E) are shown. Radioactivity was 0.3 mCi per injection for IV and PT injections in this figure.</p

Efficiency of tumor targeting by DMR or IV methods by surface fluorescence.

<p>A) Skin covering GFP-BT-20 tumors was removed. Shown are white light images, GFP fluorescence images, probe NIR fluorescence, and the overlay of GFP and probe fluorescence, plus an X-ray image. As with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058290#pone-0058290-g004" target="_blank">Figure 4</a>, green GFP plus purple NIR fluorescence yields a white overlaid image. B) By with PT DMR or IV, probe fluorescence included a stromal zone of integrin binding surrounding the tumor as was seen in (a). C) A comparison of tumor surface fluorescence intensities by PT DMR versus the IV methods is shown. Doses were 50 pmoles (per site) and 2000 pmoles (IV) for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058290#pone-0058290-g005" target="_blank">figures 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058290#pone-0058290-g006" target="_blank">6</a>.</p

IV Molecular Targeting And Diffusion Molecular Retention (DMR) Molecular Targeting.

<p>(A) IV. Retention can be due to target binding, when the probe (triangle) binds to a molecular target (black), or it can be targetless (e.g. kidney Non-specific binding). Non-tumor organs have higher probe concentrations (darker shading) than the tumor. Transport from the vascular compartment (blood) to tumor interstitium (dotted line) is slow while probe transport to normal organs (solid lines) is fast. When the probe reaches the tumor, distribution is uneven (perivascular accumulation). (B) DMR employs a peritumoral (PT) administration, followed by extensive diffusion through normal and tumor interstitium, and retention only if the probe encounters a molecular target. Because the tumor “sees” the agent first, uptake by normal organs is greatly reduced. To obtain extensive interstitial diffusion, transport from the tumor interstitium to the vascular compartment (dotted arrow) must be slow. Slow interstitial to vascular transport results from probe size and hydrophilicity.</p

Design of RGD and RAD Probes and their binding to integrins on GFP-BT-20 cells.

<p>A) Structures of the RGD and RAD probes. A 5 kDa PEG provides most of the probe volume which is a protein-like 25 kDa by size exclusion chromatography, but does not surround the RGD peptide which binds integrins, see (2B) below. B) Binding of RGD and RAD probes to GFP-BT-20 cells, and displacement by RGD and RAD peptides, by single channel FACS. Also shown is the intrinsic fluorescence of “unstained” calls. C) Displacement of RGD and RAD probes by RGD and RAD peptides. D) Dual wavelength FACS scatter plot for lentivirus transduced, GFP expressing BT-20 cells. Cells bind the RGD probe and express GFP.</p

The PEG-Fluorochrome Shielding Approach for Targeted Probe Design

We provide a new approach for fluorescent probe design termed “PEG-fluorochrome shielding”, where PEGylation enhances quantum yields while blocking troublesome interactions between fluorochromes and biomolecules. To demonstrate PEG-fluorochrome shielding, fluorochrome-bearing peptide probes were synthesized, three without PEG and three with a 5 kDa PEG functional group. <i>In vitro</i>, PEG blocked the interactions of fluorochrome-labeled peptide probes with each other (absorption spectra, self-quenching) and reduced nonspecific interactions with cells (by FACS). <i>In vivo</i>, PEG blocked interactions with biomolecules that lead to probe retention (by surface fluorescence). Integrin targeting <i>in vivo</i> was obtained as the differential uptake of an ¹¹¹In-labeled, fluorochrome-shielded, integrin-binding RGD probe and a control RAD. Using PEG to block fluorochrome-mediated interactions, rather than synthesizing <i>de novo</i> fluorochromes, can yield new approaches for the design of actively or passively targeted near-infrared fluorescent probes