Surface functionalization of Feraheme (FH, 1) with azide or alkyne groups and radiolabeling functionalized NPs by HIR.

<p>a) Using FH (<b>1</b>)<b>,</b> the Azide-FH (<b>4</b>) and Alkyne-FH (<b>5</b>) were synthesized. Portions of Azide-FH <b>(4)</b> and Alkyne-FH (<b>5</b>) were then radiolabeled by HIR, yielding ⁸⁹Zr-Azide-FH (^<b>89</b><b>Zr-4</b>) and ⁸⁹Zr-Alkyne-FH (^<b>89</b><b>Zr-5</b>). To determine reactive azide or reactive alkynes, NPs were reacted with the appropriate click reactive Cy5.5 fluorochromes, with Cy5.5s shown as the yellow stars of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172722#pone.0172722.g001" target="_blank">Fig 1</a>. After removal of the unreacted Cy5.5s (DBCO-Cy5.5, <b>6</b> or Azide-Cy5.5, <b>7</b>), the number of Cy5.5’s per NP was determined from absorption spectra examples of which are shown in Fig 2b–2e. Controls for covalent binding were a reaction of FH (<b>1</b>) and DBCO-Cy5.5 (<b>6</b>) and a reaction of Azide-FH <b>(4)</b> and DBCO-Cy5.5 (<b>6</b>) preoccupied with DBCO-NH₂. Values in parentheses are the numbers of reactive groups per NP with values summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172722#pone.0172722.t001" target="_blank">Table 1</a>.</p

Outline of heat induced radiolabeling (HIR) and click chemistry surface functionalization used to obtain multimodal, targeted NPs.

<p>Our previous HIR of FH NPs (top) yielded non-surface functionalized, radioactive NPs (red core). The alkyne and azide functionalized FH intermediates were synthesized (Azide-FH, <b>4</b> and Alkyne-FH, <b>5</b>) and labeled by HIR reaction, to yield ⁸⁹Zr-Azide-FH (^<b>89</b><b>Zr-4</b>) and ⁸⁹Zr-Alkyne-FH (^<b>89</b><b>Zr-5</b>). Imaging detection modalities for the NPs are in bold. NPs targeted to folate receptors (⁸⁹Zr-Folate-FH, ^<b>89</b><b>Zr-11</b>), integrins (RGD-FH, <b>14</b>) or NPs with protamines (⁸⁹Zr-Cy5.5-Protamine-FH, ^<b>89</b><b>Zr-16</b>) were then synthesized. Detailed synthetic schemes are given in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172722#pone.0172722.g002" target="_blank">2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172722#pone.0172722.g004" target="_blank">4</a>.</p

Summary of HIR NP’s and Surface Functionalization Reactions.

<p>Summary of HIR NP’s and Surface Functionalization Reactions.</p

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

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

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

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

Synthesis of RGD and RAD probes.

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