Elamprakash N. Savariar

Molecular discrimination inside polymer nanotubules

Recognition of small organic molecules and large biomolecules such as proteins is of great importance in pharmaceutical as well as biological applications. Recognition inside a nanoporous membrane is particularly attractive, because of the advantages associated with ligand-receptor interactions in confined spaces. Classical nanoporous membrane-based separations simply use the difference in size of the analytes relative to pore size in the membrane. In order to bring about selectivity beyond size, it is necessary that methods for functionalizing the membrane pores are readily available. Here, we describe a simple approach to functionalize the nanopores within these membranes using self-assembling and non-self-assembling polymers. We show that these modified membranes separate small molecules based on size, charge and hydrophobicity. We also demonstrate here that proteins can be differentially transported through the nanopores based on their size and/or electrostatics.

In Vivo Targeting of Hydrogen Peroxide by Activatable Cell-Penetrating Peptides

A hydrogen peroxide (H2O2)-activated cell-penetrating peptide was developed through incorporation of a boronic acid-containing cleavable linker between polycationic cell-penetrating peptide and polyanionic fragments. Fluorescence labeling of the two ends of the molecule enabled monitoring its reaction with H2O2 through release of the highly adhesive cell-penetrating peptide and disruption of fluorescence resonance energy transfer. The H2O2 sensor selectively reacts with endogenous H2O2 in cell culture to monitor the oxidative burst of promyelocytes and in vivo to image lung inflammation. Targeting H2O2 has potential applications in imaging and therapy of diseases related to oxidative stress.

Real time in vivo molecular detection of primary tumors and metastases with ratiometric activatable cell penetrating peptides

Management of metastatic disease is integral to cancer treatment. Evaluation of metastases often requires surgical removal of all anatomically susceptible lymph nodes for ex vivo pathologic examination. We report a family of novel ratiometric activatable cell-penetrating peptides, which contain Cy5 as far red fluorescent donor and Cy7 as near-infrared fluorescent acceptor. Cy5 is quenched in favor of Cy7 re-emission until the intervening linker is cut by tumor-associated matrix metalloproteinases-2 and 9 (MMP2,9) or elastases. Such cleavage increases the Cy5:Cy7 emission ratio 40-fold and triggers tissue retention of the Cy5-containing fragment. This ratiometric increase provides an accelerated and quantifiable metric to identify primary tumors and metastases to liver and lymph nodes with increased sensitivity and specificity. This technique represents a significant advance over existing nonratiometric protease sensors and sentinel lymph node detection methods, which give no information about cancer invasion.

Fluorescence Patterns from Supramolecular Polymer Assembly and Disassembly for Sensing Metallo- and Nonmetalloproteins

Critical aggregation concentration (CAC) of surfactants is lowered when polyelectrolytes act as counterions. At a concentration in between the CACs of the surfactant and the polymer-surfactant complex, protein-induced disassemblies can be achieved. This is because, when proteins competitively bind to the polyelectrolytes, the surfactants are not capable of sustaining a micelle-type assembly at this concentration. Since these amphiphilic aggregates are capable of noncovalently sequestering hydrophobic guest molecules, the protein binding induced disassembly process also results in a guest release from these assemblies. We show here that the change in fluorescence with different proteins is dependent not only on the nature of the polymer-surfactant complex, but also on the fluorescent transducer. Two processes can be responsible for the observed fluorescence change: fluorophore guest release from the hydrophobic interior of the assembly and excited state quenching due to complementary components in the analyte. The latter mechanism is especially possible with metalloproteins. We show here that an excited state quenching is possible at nanomolar concentrations of the proteins, while the disassembly based fluorescence reduction is the dominant pathway at micromolar concentrations.

Disassembly of Noncovalent Amphiphilic Polymers with Proteins and Utility in Pattern Sensing

A simple strategy for pattern recognition of proteins through micellar disassembly is introduced. Five different noncovalently assembled receptors have been generated, and the disassembly was studied by monitoring the encapsulated dye release in response to five different proteins. The disassembly induced fluorescence change of the guest molecule produces protein-specific patterns.

Ratiometric Activatable Cell-Penetrating Peptides Provide Rapid In Vivo Readout of Thrombin Activation

In real time: thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual-emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage-dependent spatial localization. The most advanced RACPP uses norleucine (Nle)-TPRSFL as a linker that increases sensitivity to thrombin by about 90-fold.

Dual targeting of integrin αvβ3 and matrix metalloproteinase-2 for optical imaging of tumors and chemotherapeutic delivery

Activatable cell-penetrating peptides (ACPP) provide a general strategy for molecular targeting by exploiting the extracellular protease activities associated with disease. Previous work used a matrix metalloproteinase (MMP-2 and 9)-cleavable sequence in the ACPP to target contrast agents for tumor imaging and fluorescence-guided surgery. To improve specificity and sensitivity for MMP-2, an integrin αvβ3-binding domain, cyclic-RGD, was covalently linked to the ACPP. This co-targeting strategy relies on the interaction of MMP-2 with integrin αvβ3, which are known to associate via the hemopexin domain of MMP-2. In U87MG glioblastoma cells in culture, dual targeting greatly improved ACPP uptake compared with either MMP or integrin αvβ3 targeting alone. In vivo, dual-targeted ACPP treatment resulted in tumor contrast of 7.8 ± 1.6, a 10-fold higher tumor fluorescence compared with the negative control peptide, and increased probe penetration into the core of MDA-MB-231 tumors. This platform also significantly improved efficacy of the chemotherapeutic monomethylauristatin E (MMAE) in both MDA-MB-231 orthotopic human and syngeneic Py230 murine breast tumors. Treatment with cyclic-RGD-PLGC(Me)AG-MMAE-ACPP resulted in complete tumor regression in one quarter of MDA-MB-231 tumor–bearing mice, compared with no survival in the control groups. This rational mechanism for amplified delivery of imaging and potent chemotherapeutic agents avoids the use of antibodies and may be of considerable generality. Mol Cancer Ther; 13(6); 1514–25. ©2014 AACR.

Matrix-Metalloproteinases in Head and Neck Carcinoma–Cancer Genome Atlas Analysis and Fluorescence Imaging in Mice

Objective (1) Obtain matrix-metalloproteinase (MMP) expression profiles for head and neck squamous cell carcinoma (HNSCC) specimens from the Cancer Genomic Atlas (TCGA). (2) Demonstrate HNSCC imaging using MMP-cleavable, fluorescently labeled ratiometric activatable cell-penetrating peptide (RACPP). Study Design Retrospective human cohort study; prospective animal study. Setting Translational research laboratory. Subjects and Methods Patient clinical data and mRNA expression levels of MMP genes were downloaded from TCGA data portal. RACPP provides complementary ratiometric fluorescent contrast (increased Cy5 and decreased Cy7 intensities) when cleaved by MMP2/9. HNSCC–tumor bearing mice were imaged in vivo after RACPP injection. Histology was evaluated by a pathologist blinded to experimental conditions. Zymography confirmed MMP-2/9 activity in xenografts. RACPP was applied to homogenized human HNSCC specimens, and ratiometric fluorescent signal was measured on a microplate reader for ex vivo analysis. Results Expression of multiple MMPs including MMP2/9 is greater in patient HNSCC tumors than matched control tissue. In patients with human papilloma virus positive (HPV+) tumors, higher MMP2 and MMP14 expression correlates with worse 5-year survival. Orthotopic tongue HNSCC xenografts showed excellent ratiometric fluorescent labeling with MMP2/9-cleavable RACPP (sensitivity = 95.4%, specificity = 95.0%). Fluorescence ratios were greater in areas of higher tumor burden (P < .03), which is useful for intraoperative margin assessment. Ex vivo, human HNSCC specimens showed greater cleavage of RACPP when compared to control tissue (P = .009). Conclusions Human HNSCC tumors show increased mRNA expression of multiple MMPs including MMP2/9. We used RACPP, a ratiometric fluorescence assay of MMP2/9 activity, to show improved occult tumor identification and margin clearance. Ex vivo assays using RACPP in biopsy specimens may identify patients who will benefit from intraoperative RACPP use.

Detection and monitoring of localized matrix metalloproteinase upregulation in a murine model of asthma

Extracellular proteases including matrix metalloproteinases (MMPs) are speculated to play a significant role in chronic lung diseases, such as asthma. Although increased protease expression has been correlated with lung pathogenesis, the relationship between localized enzyme activity and disease progression remains poorly understood. We report the application of MMP-2/9 activatable cell-penetrating peptides (ACPPs) and their ratiometric analogs (RACPPs) for in vivo measurement of protease activity and distribution in the lungs of mice that were challenged with the allergen ovalbumin. MMP-2/9 activity was increased greater than twofold in whole, dissected lungs from acutely challenged mice compared with control mice (P=1.8×10(-4)). This upregulation of MMP-2/9 activity was localized around inflamed airways with 1.6-fold higher protease-dependent ACPP uptake surrounding diseased airways compared with adjacent, pathologically normal lung parenchyma (P=0.03). MMP-2/9 activity detected by ACPP cleavage colocalized with gelatinase activity measured with in situ dye-quenched gelatin. For comparison, neutrophil elastase activity and thrombin activity, detected with elastase- and thrombin-cleavable RACPPs, respectively, were not significantly elevated in acutely allergen-challenged mouse lungs. The results demonstrate that ACPPs, like the MMP-2/9-activated and related ACPPs, allow for real-time detection of protease activity in a murine asthma model, which should improve our understanding of protease activation in asthma disease progression and help elucidate new therapy targets or act as a mechanism for therapeutic drug delivery.

Functional Group Density and Recognition in Polymer Nanotubes

Control of the placement of functional groups within confined nanospaces is of interest because of its potential in applications such as sensing and separations. For effective recognition of analytes within nanopores, it is necessary that control over two factors—pore size and functional group presentation—be achieved. While pore-size changes in nanopores and their effects upon recognition have been reported, it is also important to investigate the relative effect of binding-site density upon molecular recognition inside these nanopores. Herein, we report the effect that the density of functional groups within polymeric nanotubes could have upon complementary analyte molecules that pass through membrane nanopores. Since dendrimers are known as a class of artificial macromolecules that can have a high density of functional groups with a significant degree of control, we hypothesized that these molecules are ideal candidates for this study. Even though dendrimer-based “testtube-like” nanostructures, where one end of the tube is closed, have been obtained using alumina membranes, the possibility of molecular recognition within such nanotubes has not been investigated. 22] Recently, we described a very simple and versatile methodology by which polymer-based functionalities can be incorporated inside nanoporous polycarbonate membranes by using polyvalent interactions. We have utilized this methodology to achieve the formation of dendrimer-functionalized nanotubes. Since polypropyleneimine (PPI) dendrimers are easily accessible, water-soluble, and charged, we used these molecules to test our hypotheses (Scheme 1). Our methodology for functionalization of the membrane nanopores uses electrostatic interactions and the PPI dendrimers are positively charged. Therefore, it is necessary that the predendrimerfunctionalized membrane nanopores be negatively charged. To achieve this, we first incorporated Sn ions into the pore walls of the membrane by using its poly(vinylpyrrolidone) functionalities. The Sn ions were used to introduce a layer of an anionic polymer, polyacrylic acid (PAA). We have shown that the vacuum-filtration-based incorporation of polymers is uniform throughout the membrane. This anionic polymerfunctionalized membrane interior was then utilized to incorporate the cationic PPI dendrimers. The schematic illustration of membrane functionalization using PPI dendrimers is shown in Figure 1. Prior to investigating how this functionalization effected recognition of the analytes that passed through the membrane, we needed to characterize the nanotubes formed in this process. We were first interested in assessing the change in pore size of the membranes upon dendrimer functionalization. While we were able to previously demonstrate drastic pore-size changes by using our self-assembling polymers, systematic control over the size of the final nanopores was not demonstrated. We envisaged that dendrimers would provide a significant amount of control over the nanopore dimensions. We tested this possibility by assessing the porediameter change induced by the dendrimer functionalization; this was carried out by using a calibration curve obtained from Scheme 1. Structures of PPI(G4), PPI(G1), PAA, rhodamine 6G, and calcein.