PEG-Like Nanoprobes: Multimodal, Pharmacokinetically and Optically Tunable Nanomaterials

“PEG-like Nanoprobes” (PN’s) are pharmacokinetically and optically tunable nanomaterials whose disposition in biological systems can be determined by fluorescence or radioactivity. PN’s feature a unique design where a single PEG polymer surrounds a short fluorochrome and radiometal bearing peptide, and endows the resulting nanoprobe with pharmacokinetic control (based on molecular weight of the PEG selected) and optical tunability (based on the fluorochrome selected), while the chelate provides a radiolabeling option. PN’s were used to image brain capillary angiography (intravital 2-photon microscopy), tumor capillary permeability (intravital fluorescent microscopy), and the tumor enhanced permeability and retention (EPR) effect (111In-PN and SPECT). Clinical applications of PN’s include use as long blood half-life fluorochromes for intraoperative angiography, for measurements of capillary permeability in breast cancer lesions, and to image EPR by SPECT, for stratifying patient candidates for long-circulating nanomedicines that may utilize the EPR mechanism

Development and Screening of Contrast Agents for In Vivo Imaging of Parkinson’s Disease

Purpose: The goal was to identify molecular imaging probes that would enter the brain, selectively bind to Parkinson’s disease (PD) pathology, and be detectable with one or more imaging modalities. Procedure: A library of organic compounds was screened for the ability to bind hallmark pathology in human Parkinson’s and Alzheimer’s disease tissue, alpha-synuclein oligomers and inclusions in two cell culture models, and alpha-synuclein aggregates in cortical neurons of a transgenic mouse model. Finally, compounds were tested for blood–brain barrier permeability using intravital microscopy. Results: Several lead compounds were identified that bound the human PD pathology, and some showed selectivity over Alzheimer’s pathology. The cell culture models and transgenic mouse models that exhibit alpha-synuclein aggregation did not prove predictive for ligand binding. The compounds had favorable physicochemical properties, and several were brain permeable. Conclusions: Future experiments will focus on more extensive evaluation of the lead compounds as PET ligands for clinical imaging of PD pathology

Optogenetic Restoration of Disrupted Slow Oscillations Halts Amyloid Deposition and Restores Calcium Homeostasis in an Animal Model of Alzheimer’s Disease

Slow oscillations are important for consolidation of memory during sleep, and Alzheimer’s disease (AD) patients experience memory disturbances. Thus, we examined slow oscillation activity in an animal model of AD. APP mice exhibit aberrant slow oscillation activity. Aberrant inhibitory activity within the cortical circuit was responsible for slow oscillation dysfunction, since topical application of GABA restored slow oscillations in APP mice. In addition, light activation of channelrhodopsin-2 (ChR2) expressed in excitatory cortical neurons restored slow oscillations by synchronizing neuronal activity. Driving slow oscillation activity with ChR2 halted amyloid plaque deposition and prevented calcium overload associated with this pathology. Thus, targeting slow oscillatory activity in AD patients might prevent neurodegenerative phenotypes and slow disease progression

Multimodal imaging of EPR tumor targeting and elimination of PN(783)10.0.

<p><b>A</b>) SPECT/CT images of two mice bearing two HT-29 tumors as a function of time after injection. At 2 h post injection, agent is in the blood and interstitium. By 24 h post-injection tumors are becoming apparent as agent is being cleared. At 48 h, labeling is highly tumor selective. <b>B</b>) Surface fluorescence imaging of two additional mice bearing the same tumor. By surface fluorescence, as with SPECT, labeling is highly tumor selective at 48 h. <b>C</b>) Organ biodistribution was obtained by dissection and ¹¹¹In counting at 24 h and 48 h post-injection. Data are means and standard deviations, n = 5. <b>D</b>) A whole animal radioactivity elimination curve. Data are means and standard deviations, with extremely small standard deviations, n = 5.</p

Variable PN pharmacokinetics analyzed by the two compartment pharmacokinetic model in normal mice.

<p><b>A</b>) Two compartment pharmacokinetic model showing three microscopic rate constants. Serum fluorescence for PN(783)10.0 <b>B</b>) and PN(783)4.3 <b>C</b>) after injection are shown. Data were fit to the two compartment model with data provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095406#pone-0095406-t001" target="_blank">Table 1</a>. <b>D)</b> Time courses for blood and interstitial fluorescence of PN(783)10.0 using microkinetic constants from <b>B</b>).</p

Fluorescent imaging of three pharmacokinetic phases of PN’s with diameters of 10 nm.

<p><b>A</b>) Vascular phase, two photon microscopy of brain vasculature of normal mice. Mice underwent a craniotomy and implantation of a transparent window. Vessel intensity drops due vascular escape, but there is no interstitial fluorescence in the brain due to the blood brain barrier. Scale marker = 50 microns. <b>B</b>) Intravital confocal microscopy of the vascular and interstitial phases of an mCherry expressing HT-29 xenograft. During the vascular phase (10 min post-injection), vessels are imaged, without interstitial fluorescence. During the interstitial phase (20 h post-injection), interstitial fluorescence is prominent. Scale marker = 20 microns. <b>C</b>) Surface fluorescence/X-ray imaging of the tumor retention phase of PN(545)10.0. Shown are the HT-29/mCherry tumor with the skin removed as a white light image, mCherry tumor fluorescence (green), PN(545)10.0 fluorescence (purple) and the green/purple over lay (white). <b>D</b>) Confocal microscopy of the tumor retention phase of PN(497)10.0. Shown are a sectioned HT-29 mCherry expressing tumor with nuclei stained blue (DAPI), mCherry tumor cells (red), PN(497)10.0 (green) and a green/red overlay (yellow).</p

Reaction with PEG polymers and fluorochrome and DOTA bearing peptides yields PEG-like Nanoprobes (PN’s).

<p><b>A</b>) Syntheses of PEG-like Nanoprobe (PN’s). The peptide (DOTA)Lys-Cys(Fluor.), with an N-terminal DOTA, a variable fluorochrome (Fluor.) attached to the cysteine side chain, and a single primary amine, reacts with the NHS ester of a variable PEG. In the conventional PEGylations of proteins or nanoparticles, multiple PEG’s provide a PEG bearing surface. This approach yields materials with a PEG to protein ratio or with a PEG surface density. <b>B</b>) Synthesis of the PN denoted PN(783)4.3. A 5 kDa PEG is reacted with the (DOTA)Lys-Cys(IR-783) peptide. The diameter (by FPLC) is 4.3 nm and the absorption maximum is 783 nm, hence PN(783)4.3. <b>C</b>) A 30 kDa PEG is reacted with the (DOTA)Lys-Cys(Cy3) peptide. The diameter is 10.0 nm and an absorption maximum is 545 nm, hence, PN(545)10.0. A list of PN’s and their properties is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095406#pone-0095406-t001" target="_blank">Table 1</a>.</p

Stability of PN’s in mouse serum.

<p>The stability of PN(783)4.3 <b>A</b>) or PN(783)10.0 <b>B</b>) was examined by incubating nanoprobes for the indicated times and subjecting samples to FPLC. Arrows are the retention times of PN’s from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095406#pone-0095406-g002" target="_blank"><b>Figure 2A</b></a>, color coded as in that figure. Fluorochrome size is unchanged for both PN’s. <b>C</b>) Stability of PN fluorescence. PN(783)4.3 or PN(783)10.0 were incubated as indicated and fluorescence determined. <b>D</b>) Stability of ¹¹¹In binding to PN’s. ¹¹¹In-PN(783)4.3 or ¹¹¹In-PN(783)10.0 were incubated as indicated and radioactivity associated with the PN determined by HPLC. Exemplary chromatograms are provided; see <b>Figure S6</b> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095406#pone.0095406.s001" target="_blank">File S1</a>.</p

Summary of PEG-like Nanoprobes (PN’s).

<p>*M.W. Obs. = molecular weights were determined from mass spectrometry results.</p><p>**Volumes are expressed as diameters in nm (to enable comparison with nanomaterials) and as the equivalent volumes of proteins (to enable comparison with proteins).</p><p>***Values are means±1 S.D.</p

Role PEG in determining elimination by surface fluorescence imaging.

<p><b>A</b>) Animals were injected with unPEGylated peptide and imaged at the times indicated. Bar graph gives whole animal surface fluorescence determined as means and standard deviations, n = 6. <b>B</b>) With the 5 kDa PEG attached, PN(783)4.3 is obtained, which shows renal elimination within 20 minutes (arrow). <b>C</b>) With the 30 kDa PEG, PN(783)10.0 is obtained, which shows a long vascular phase followed by elimination. Biodistribution and elimination of ¹¹¹In-PN(783)10.0 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095406#pone-0095406-g006" target="_blank">Figure 6</a>.</p