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PDF file - 5319K, Supplementary Materials and Methods. Supplemental Table 1: List of peptides and synthetic intermediates with corresponding molecular weights. Supplemental Figure 1: Chemical Structure of dual targeted peptides. Supplemental Figure 2: Gel images for MMP-2 cleavage assay. Supplemental Figure 3: Peptide SUVs (MDA-MB-231 tumors). Supplemental Figure 4: Injection of cyclic-RGD-PLGC(Me)AG-ACPP with excess of unlabeled cyclic-RGD. Supplemental Figure 5: Dual targeting of PLGC(Me)AG-ACPP with folate does not improve tumor uptake in SK-OV-3 tumors. Supplemental Figure 6: No primary antibody control for IHC. Supplemental Figure 7: Plasma stability and blood clearance of cyclic-RGDPLGC(Me)AG-ACPP. Supplemental Figure 8: Tumor growth curve corresponding to Figure 5B and animal body weights corresponding to Figure 5 B&C. Supplemental Figure 9: Animal body weight and tumor growth curves for therapy studies at 1.2mg/kg (~40nmol) and 0.6mg/kg (~20nmol). Supplemental Figure 10: Targeting of MMAE in Py230 syngeneic tumor model.

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.

Polycation conjugated MMAE is cytotoxic to tumor cells.

MMAE enhances γH2Ax foci formation following irradiation.

PDF file - 40K

Combination of increased delivery of ACPP-cRGD-MMAE with IR significantly improved tumor regression.

Schematic model of therapeutic strategy utilizing tumor targeted MMAE delivery with ACPP.

MMAE enhances comet tail length following irradiation in pancreatic tumor cell lines.

PDF file - 835K, Supplementary Fig 1. Structures of RACPP1,2,3 and uncleavable control. Supplementary Fig 2. Structures of synthesized RACPPs with varying acceptor fluorophores. Supplementary Fig 3. Characterization of RACPP2. Supplementary Fig 4. Ratiometric RACPP images show superior contrast compared to Cy5 fluorescence alone. Supplementary Fig 5. RACPPs show specificity for MMP2,9 cleavage in transgenic animal models. Supplementary Fig 6. RACPPs show tumor specific cleavage and uptake. Supplementary Fig 7. Non-ratiometric ACPP has poor contrast in liver metastases. Supplementary Fig 8. Ratiometric analyses using RACPPs show sensitive detection of metastatic lymph nodes. Supplementary Fig 9. LCMS characterization of RACPPs. Supplementary Fig 10. Synthetic schemes for RACPPs.

Tumor xenografts show cleavage dependent tumor contrast with radiometric ACPP.

AVI file - 1355K, Supplementary Movie - Movie of mouse bearing GFP-labeled 8119 primary auricular tumor with a cervical lymph node with partial cancer invasion 2 hours following IV injection of RACPP1. Green color is GFP labeling seen in the primary tumorand on the lateral aspect of the involved lymph node. Images with aqua pseudocolor are generated from the Cy5 emission fluorescence intensity alone superimposed on white light reflectance. Images with multihued pseudocolor varying from dark blue (low ratio) to red (high ratio) are generated from the Cy5/Cy7 ratio at every pixel. Note that region of cancer invasion (GFP positive cells seen as green) in the cervical lymph node correlate with the region of highest Cy5/Cy7 ratio (seen as red pseudocolor).

Tumour resistance to radiotherapy remains a barrier to improving cancer patient outcomes. To overcome radioresistance, certain drugs have been found to sensitize cells to ionizing radiation (IR). In theory, more potent radiosensitizing drugs should increase tumour kill and improve patient outcomes. In practice, clinical utility of potent radiosensitizing drugs is curtailed by off-target side effects. Here we report potent anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize to tumours based on surface receptor expression. While two classes of potent anti-tubulins, auristatins and maytansinoids, indiscriminately radiosensitize tumour cells, conjugating these potent anti-tubulins to anti-ErbB antibodies restrict their radiosensitizing capacity. Of translational significance, we report that a clinically used maytansinoid ADC, ado-trastuzumab emtansine (T-DM1), with IR prolongs tumour control in target expressing HER2+ tumours but not target negative tumours. In contrast to ErbB signal inhibition, our findings establish an alternative therapeutic paradigm for ErbB-based radiosensitization using antibodies to restrict radiosensitizer delivery., Drugs that sensitize tumour cells to ionizing radiation are prized because they can overcome resistance to radiotherapy. Here, the authors show that anti-tubulin drugs conjugated to cetuximab or trastuzumab can radiosensitize EGFR- or HER2-expressing tumors by increasing DNA damage and cell death due to ionizing radiation.

Author(s): Orosco, Ryan K; Savariar, Elamprakash N; Weissbrod, Philip A; Diaz-Perez, Julio A; Bouvet, Michael; Tsien, Roger Y; Nguyen, Quyen T | Abstract: Background and objectivesMolecularly targeted fluorescent molecules may help detect tumors that are unseen by traditional white-light surgical techniques. We sought to evaluate a fluorescent ratiometric activatable cell penetrating peptide (RACPP) for tumor detection in a transgenic model of PTC.MethodsThirteen BRAFV600E mice with PTC were studied-seven injected intravenously with RACPP, four controls with saline. Total thyroidectomy was performed with microscopic white-light visualization. Fluorescent imaging of post-thyroidectomy fields was performed, and tissue with increased signal was removed and evaluated for PTC. Final samples were analyzed by a pathologist blinded to conditions. Vocal cord function was evaluated postoperatively with video laryngoscopy.ResultsThe average in situ ratiometric (Cy5/Cy7) thyroid tumor-to-background contrast ratio was 2.27 +/- 0.91. Fluorescence-guided clean-up following thyroidectomy identified additional tumor in 2 of 7 RACPP animals (smallest dimension 1.2 mm), and decreased the number of animals with residual tumor from 4 to 3. All retained tumor foci on final pathology were smaller than 0.76 mm. Intact vocal abduction was present in all of the RACPP animals.ConclusionsRACPPs successfully targeted PTC in a transgenic thyroidectomy model, and allowed for residual tumor detection that reduced positive margins beyond what was possible with white-light surgery alone.

Matrix metalloproteinases-2 and -9 (MMP-2/-9) are key tissue remodeling enzymes that have multiple overlapping activities critical for wound healing and tumor progression in vivo. To overcome issues of redundancy, we created MMP-2/-9 double knockout (DKO) mice in the C57BL/6 background to examine wound healing. We then bred the DKO mice into the polyomavirus middle T (PyVmT) model of breast cancer to analyze the role of these enzymes in tumorigenesis. Breeding analyses indicated that significantly fewer DKO mice were born than predicted by Mendelian genetics and weaned DKO mice were growth compromised compared with wild type (WT) cohorts. Epithelial wound healing was dramatically delayed in adult DKO mice and when the DKO was combined with the PyVmT oncogene, we found that the biologically related process of mammary tumorigenesis was inhibited in a site-specific manner. To further examine the role of MMP-2/-9 in tumor progression, tumor cells derived from WT or DKO PyVmT transgenic tumors were grown in WT or DKO mice. Ratiometric activatable cell penetrating peptides (RACPPs) previously used to image cancer based on MMP-2/-9 activity were used to understand differences in MMP activity in WT or knockout syngeneic tumors in WT and KO animals. Analysis of an MMP-2 selective RACPP in WT or DKO mice bearing WT and DKO PyVmT tumor cells indicated that the genotype of the tumor cells was more important than the host stromal genotype in promoting MMP-2/-9 activity in the tumors in this model system. Additional complexities were revealed as the recruitment of host macrophages by the tumor cells was found to be the source of the tumor MMP-2/-9 activity and it is evident that MMP-2/-9 from both host and tumor is required for maximum signal using RACPP imaging for detection. We conclude that in the PyVmT model, the majority of MMP-2/-9 activity in mammary tumors is associated with host macrophages recruited into the tumor rather than that produced by the tumor cells themselves. Thus therapies that target tumor-associated macrophage functions have the potential to slow tumor progression.

Author(s): Hussain, Timon; Savariar, Elamprakash N; Diaz-Perez, Julio A; Messer, Karen; Pu, Minya; Tsien, Roger Y; Nguyen, Quyen T | Abstract: BackgroundWe evaluated the use of intraoperative fluorescence guidance by enzymatically cleavable ratiometric activatable cell-penetrating peptide (RACPPPLGC(Me)AG) containing Cy5 as a fluorescent donor and Cy7 as a fluorescent acceptor for salivary gland cancer surgery in a mouse model.MethodsSurgical resection of small parotid gland cancers in mice was performed with fluorescence guidance or white light (WL) imaging alone. Tumor identification accuracy, operating time, and tumor-free survival were compared.ResultsRACPP guidance aided tumor detection (positive histology in 90% [27/30] vs 48% [15/31] for WL; p l .001). An approximate 25% ratiometric signal increase as the threshold to distinguish between tumor and adjacent tissue, yielded g90% detection sensitivity and specificity. Operating time was reduced by 54% (p l .001), and tumor-free survival was increased with RACPP guidance (p = .025).ConclusionRACPP provides real-time intraoperative guidance leading to improved survival. Ratiometric signal thresholds can be set according to desired detection accuracy levels for future RACPP applications.

Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor-targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell-penetrating peptide targeting matrix metalloproteinases and RGD-binding integrins (ACPP–cRGD–MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low-passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule- and dose-dependent manner, correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double-strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with nontargeted free MMAE or tumor-targeted MMAE (ACPP–cRGD–MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor-targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell-penetrating peptides. Cancer Res; 75(7); 1376–87. ©2015 AACR.

OBJECTIVE:Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. METHODS:K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPPNleTPRSFL . Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPPNleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. RESULTS:In chronic arthritis, RACPPNleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPPNleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPPNleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. CONCLUSION:RACPPNleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPPNleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression.

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.

Amphiphilic homopolymers that self-assemble into reverse micelles in nonpolar solvents have been used by us in the context of a two-phase liquid-liquid extraction protocol to selectively extract peptides from aqueous solution for MALDI-MS detection. In this manuscript, we investigate the scope of these materials in terms of its extraction capabilities, using compounds with varying isoelectric points (pI) and pK(a) values over a range of aqueous solution pHs. We find that the aqueous solution pH and analyte pK(a) values are the major factors controlling extraction selectivity. We also find that the experimental extraction efficiencies correspond very well with the fractional compositions of species calculated using analyte pK(a) values, indicating that these extraction materials can be used to simultaneously generate titration-type curves for each individual peptide in a mixture. We predict that such titration curves, along with accurate mass measurements, could represent a new way of improving protein identification procedures.

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 non-covalently 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 not only dependent 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 or excited state quenching due to complementary components in the analyte. The latter mechanism is especially possible with metalloproteins. We show here that at nanomolar concentration of the proteins, an excited state quenching is possible while at micromolar concentrations the disassembly-based fluorescence reduction is the dominant pathway.

Biaryl dendritic micelles have been used for generating fluorescence response patterns that are used for sensing metalloproteins. By varying the generation of the dendrimer and the encapsulated dye molecules, the differential receptors for four different metalloproteins have been created. The pattern obtained from these dendritic micelles indeed confirms that the combination of dendrimer generation and electronic energy levels of the fluorescent transducers can be used for generating analyte-specific patterns.

Supramolecular complex of a cationic surfactant and oppositely charged disulfide containing polyelectrolyte was found to form micelle type aggregates at concentration much lower than the critical aggregate concentration (CAC) of the surfactant itself. We show that this difference can be utilized to generate stimulus-sensitive disassembly of these structures. This can be achieved either by converting the polyelectrolyte counterions to monovalent counterions in response to a stimulus or by simply weakening the interaction between the polymer and the surfactant in the presence of a stimulus. We have utilized three different stimuli to demonstrate these possibilities.

The influence of the microenvironment of chains on their self-organization has been studied using styrene-based linear polymers having two different pendant groups on every repeat unit. The noncovalent cross-linking of a polymer containing both carboxylic acid and alkyl (n-C10H21−) pendant groups with a multifunctional amine (PMDETA) in a nonselective solvent (DMF) resulted in thermally reversible supramolecular aggregates that were stable without interparticle cross-linking. The size of the aggregates was controlled on a sub-micrometer level by varying concentration. In the absence of the alkyl pendant group, control over the structure and properties of the aggregates was lost. These multifunctional polymers are model systems to investigate the three-dimensional organization of linear polymers by noncovalent cross-linking and the influence of chain structure on supramolecular organization.

Molecularly targeted fluorescent molecules may help detect tumors that are unseen by traditional white-light surgical techniques. We sought to evaluate a fluorescent ratiometric activatable cell penetrating peptide (RACPP) for tumor detection in a transgenic model of PTC.Thirteen BRAFV600E mice with PTC were studied-seven injected intravenously with RACPP, four controls with saline. Total thyroidectomy was performed with microscopic white-light visualization. Fluorescent imaging of post-thyroidectomy fields was performed, and tissue with increased signal was removed and evaluated for PTC. Final samples were analyzed by a pathologist blinded to conditions. Vocal cord function was evaluated postoperatively with video laryngoscopy.The average in situ ratiometric (Cy5/Cy7) thyroid tumor-to-background contrast ratio was 2.27 +/- 0.91. Fluorescence-guided clean-up following thyroidectomy identified additional tumor in 2 of 7 RACPP animals (smallest dimension 1.2 mm), and decreased the number of animals with residual tumor from 4 to 3. All retained tumor foci on final pathology were smaller than 0.76 mm. Intact vocal abduction was present in all of the RACPP animals.RACPPs successfully targeted PTC in a transgenic thyroidectomy model, and allowed for residual tumor detection that reduced positive margins beyond what was possible with white-light surgery alone.

Head and neck squamous cell carcinoma (HNSCC) is a source of significant morbidity and mortality worldwide1. Well-established risk factors for HNSCC include tobacco, alcohol, and human papilloma virus (HPV)1. Current staging and clinical management HNSCC patients are based on anatomical location and size2. If patients undergo surgical excision, management decisions are guided by presence or absence of bony invasion, lymph-node metastasis, or lymph-node extracapsular spread. Patients with locally advanced disease are treated with multimodality therapy including chemotherapy, radiotherapy and surgery. Many prevalent malignancies such as breast, colon, lung, or prostate cancer have diagnostic assays based on genomic profiling, HER-2 and estrogen receptor expression, or ALK, EGFR and KRAS mutation. HPV has recently been identified as being responsible for the increasing incidence of oropharyngeal SCC (OPSCC)3,4, and HPV positive (HPV+) tumor status is currently the strongest prognostic factor for these patients4,5. However, there are currently no widely adopted prognostic assays for HPV-negative (HPV−) HNSCC. We previously analyzed The Cancer Genomic Atlas (TCGA), which contains the largest collection of patient HNSCC tumor specimens6. In HPV− tumors, reduced survival outcomes associated with tumor protein p53 (TP53) mutation occur only in combination with loss of chromosome 3p6. In these patients, median survival decreased from >5 years for TP53 mutations to only 1.7 years for both mutational events (“double-hit”)6. These findings are independent of clinical stage and replicated in an independent HNSCC cohort6. In HPV+ tumors, we believe E6-mediated inactivation of TP53 also acts in synergy with 3p deletion to produce poor survival outcomes6. Our prior analysis of the TCGA demonstrated that of the genes on the 3p chromosome, FHIT, a known tumor suppressor gene involved in aerodigestive cancer, best correlates with 3p status6. The FHIT gene is located on a chromosomal fragile site, and loss of the 3p arm is most commonly mediated by chromosome breakage at this site7. Thus, 3p deletion is commonly associated with decreased FHIT protein expression or reduced copy number at the 3p.14.2 FHIT locus7. Given these findings, our analysis in this study uses FHIT copy number as representative of 3p status and thus a proxy for “single hit” (TP53 only) vs “double hit” (TP53 and FHIT deletion) in HNSCC cell lines. It has also been shown that FHIT and p53 status are linked in development of lung cancer, although little is known about the molecular nature of this interaction8. Here, we analyze data from the TCGA to examine whether TP53 and 3p status are linked in HNSCC patients. The etiology for worse clinical outcomes in TCGA patients with “double-hit” cancers is unclear. Potential explanations include greater resistance to radiation monotherapy or more aggressive tumor biology in cancers with the “double hit.” We do not anticipate a difference in cisplatin sensitivity given its lack of efficacy as monotherapy in clinical practice. To examine these hypotheses, we obtained four human tongue squamous cell carcinoma cell lines from American Type Culture Collection (ATCC). We utilized the Cancer Cell Line Encyclopedia (CCLE) to examine differences in their TP53 mutational and FHIT copy number status, corresponding with the prognostic markers identified in the TCGA. To compare radiosensitivity in-vitro, we utilized a comet-tail assay to measure each cell line’s ability to repair ionizing radiation-induced double-stranded deoxyribonucleotide (DNA) damage. FHIT knockout has been previously associated with increased activation of the ATR/CHK1 pathway and stronger cell-cycle checkpoint responses, resulting in greater cellular repair in response to ionizing radiation9. We would thus expect reduced radiation-induced DNA damage in “double-hit” lines with FHIT deletion compared to “single-hit” lines with intact FHIT. We also examined the aggressiveness of “single-hit” versus “double-hit” cell lines by comparing their respective matrix-metalloproteinase (MMP) activity levels. MMP expression has previously been associated with tumor aggressiveness and patient prognosis in head and neck cancer15–19. Absolute levels of MMP2/9 have been used to differentiate between benign papillomas and carcinoma of the larynx15. Increased MMP2/9 expression has been correlated with higher cancer grade16 and reduced survival in HNSCC17–18. In tongue SCC, increased MMP2/9 expression directly correlates with incidence of lymph node metastases19. Recently, our own work has demonstrated that MMP over-expression in HPV+ tumors predicts poorer survival14. Additionally, HPV− tumors, which have a more aggressive phenotype compared to their HPV+ counterparts, also express higher levels of MMP14. We compared MMP activity in our cell lines using previously described ratiometric activatable cell-penetrating peptides (RACPP)10–12. The RACPP relies on tumor-associated MMP2/9 to cleave an intervening linker, resulting in increased Cy5/Cy7 fluorescent signal ratio, and has previously been used for margin detection during surgery12–14.

It has been shown by us in a recent communication that homopolymers, in which each repeat unit contains a hydrophilic and a hydrophobic head group, are capable of forming environment-dependent micellar or inverse micellar assemblies. A systematic structure-property relationship study is carried out here to test the scope of the design. We show here that the molecular design is indeed broadly applicable and that there is a significant gain in the critical aggregation concentrations of these polymers, as compared to the small molecule counterparts. We also show that the design can be tuned to achieve vesicle-type assemblies, which further expands the repertoire of amphiphilic homopolymers in a variety of areas. Characterizations of these assemblies have been carried out using transmission electron microscopy, dynamic light scattering, static light scattering, and dye incorporation experiments.

A copolymer of N-isopropylacrylamide with the N-hydroxysuccinimide ester of methacrylic acid has found use in a variety of applications. Here we report our efforts to gain control over the molecular weight distribution of this copolymer with controlled radical polymerization methods, such as atom transfer radical polymerization, reversible addition–fragmentation transfer (RAFT), and nitroxide-mediated polymerization. We have found that RAFT is capable of affording these copolymers with a polydispersity index of 1.1–1.2. Our results for all three polymerizations are reported. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 6340–6345, 2004

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 designRetrospective human cohort study; prospective animal study.SettingTranslational research laboratory.Subjects and methodsPatient 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.ResultsExpression 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).ConclusionsHuman 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.

BackgroundThe aim of this study was to evaluate the efficacy of using matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9)-cleavable ratiometric activatable cell-penetrating peptides (RACPPs) conjugated to Cy5 and Cy7 fluorophores to accurately label pancreatic cancer for fluorescence-guided surgery (FGS) in an orthotopic mouse model.MethodsOrthotopic mouse models were established using MiaPaCa-2-GFP human pancreatic cancer cells. Two weeks after implantation, tumor-bearing mice were randomized to conventional white light reflectance (WLR) surgery or FGS. FGS was performed at far-red and infrared wavelengths with a customized fluorescence-dissecting microscope 2 h after injection of MMP-2 and MMP-9-cleavable RACPPs. Green fluorescence imaging of the GFP-labeled cancer cells was used to assess the effectiveness of surgical resection and monitor recurrence. At 8 weeks, mice were sacrificed to evaluate tumor burden and metastases.ResultsMice in the WLR group had larger primary tumors than mice in the FGS group at termination [1.72 g ± standard error (SE) 0.58 vs. 0.25 g ± SE 0.14; respectively, p = 0.026). Mean disease-free survival was significantly lengthened from 5.33 weeks in the WLR group to 7.38 weeks in the FGS group (p = 0.02). Recurrence rates were lower in the FGS group than in the WLR group (38 vs. 73 %; p = 0.049). This translated into lower local and distant recurrence rates for FGS compared to WLR (31 vs. 67 for local recurrence, respectively, and 25 vs. 60 % for distant recurrence, respectively). Metastatic tumor burden was significantly greater in the WLR group than in the FGS group (96.92 mm(2) ± SE 52.03 vs. 2.20 mm(2) ± SE 1.43; respectively, χ (2) = 5.455; p = 0.02).ConclusionsRACPPs can accurately and effectively label pancreatic cancer for effective FGS, resulting in better postresection outcomes than for WLR surgery.

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.

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 H2O2through release of the highly adhesive cell-penetrating peptide and disruption of fluorescence resonance energy transfer. The H2O2sensor selectively reacts with endogenous H2O2in cell culture to monitor the oxidative burst of promyelocytes and in vivo to image lung inflammation. Targeting H2O2has potential applications in imaging and therapy of diseases related to oxidative stress. © 2013 American Chemical Society.

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 (see figure). © 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Nonconjugated fluorogenic amphiphilic polymers containing an anthracene chromophore exhibit fluorescence quenching in the presence of metalloproteins, although the binding of the polymer to proteins is not selective. The reason for this difference is that the possible conformational changes that protein binding could bring about on a polymer do not affect the fluorescence properties of a pendent chromophore in nonconjugated polymers. This is in contrast to the nonspecific binding and response found with conjugated polymers to proteins.

Syntheses up to three generations have been achieved of biaryl-based amphiphilic dendrons with a charge-neutral pentaethylene glycol as the hydrophilic part and a decyl chain as the hydrophobic part. Studies on the temperature-dependent characteristics revealed that these dendrons exhibit a generation-dependent lower critical solution temperature (LCST). This behavior is attributed to the combination of the amphipathic nature of the hydrophilic pentaethylene glycol side chain and dendritic effect. Interestingly, this biaryl-based scaffold also maintains the ability to form a micelle-like assembly in polar solvents and an inverted micelle-like assembly in apolar solvents. Polarity of the dendritic interior was investigated using dye-based microenvironment studies. The aggregation behavior of these micelles was analyzed by fluorescence spectroscopy and dynamic light scattering. Critical micelle concentrations (CMC) of these assemblies were investigated using fluorescence excitation spectra of the sequestered guest molecule, pyrene.

This review highlights the developments in dendrimer-based micelles for drug delivery. Dendrimers, the perfectly branched monodisperse macromolecules, have certain structural advantages that make them attractive candidates as drug carriers for controlled release or targeted delivery. As polymeric micelle-based approaches precede the work in dendrimers, these are also discussed briefly. The review concludes with a perspective on possible applications of biaryl-based dendrimeric micelles that exhibit environment-dependent conformations, in drug delivery. Keywords: Micelle; drug delivery; dendrimers; controlled release; targeted delivery; polymer−drug conjugates

L14 Upregulated Matrix Metalloproteinase 2/9 Activity in Murine Asthma Detected with Activatable Cell-Penetrating Peptides Csilla N. Felsen, Elamprakash N. Savariar, Michael Whitney, Roger Y. Tsien; University of California, San Diego. RATIONALE: Asthma, an airway inflammatory disease characterized by intermittent airway obstruction, affects more than 300 million people worldwide. Matrix metalloproteinase 2 and 9 (MMP2/9) mRNA and protein levels correlate with asthma pathogenesis; however, the role of MMP2/9 in asthma is not well understood, particularly because previous studies have not evaluated protease activity. We report the application of activatable cell-penetrating peptides (ACPPs), injectable probes that highlight in vivo protease activity, to assess protease activity in murine asthma. METHODS: Asthma mice were sensitized (ovalbumin [OVA] + alum in saline [PBS]) and then challenged (OVA in PBS). Age-matched control mice were sensitized but not challenged. The day after the final OVA challenge, ACPPs (cleavable and uncleavable, n 5 3-6 mice each) were administered intravenously 6 hr before mice were sacrificed. Lungs were imaged for Cy5 fluorescence (Maestro, CRi). Lung sections (10 mm, 8 images/mouse) were imaged for Cy5 fluorescence on a confocal microscope (5Live, Zeiss) and stained with hematoxylin and eosin. RESULTS: MMP2/9 activity was > 2-fold higher in lungs from asthma mice than controls (p5 1.8310). The same pattern was observed for ratiometric ACPPs.MMP2/9 activity localized around inflamed airwayswith 1.6-fold higher ACPP uptake surrounding airways compared to the normal lung parenchyma (p 5 0.03). MMP2/9 activity detected by ACPPs co-localized with gelatinase activity measured with in situ DQ gelatin. CONCLUSIONS: MMP-activated ACPPs allow for real-time detection of protease activity in a murine asthma model, improving our understanding of protease activation in asthma progression and elucidating novel therapy targets.

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.

Obesity in postmenopausal women is a risk factor for breast cancer. Since 25-30% of women in the USA are obese, the public health implications of obesity and breast cancer are immense. Activation of inflammatory pathways is one plausible mechanism underlying the association between obesity and increased breast cancer risk. We have used a novel immunocompetent mouse model of luminal breast cancer to demonstrate that luminal tumor progression is enhanced in ovariectomized mice fed a high fat diet (HFD). The obese mammary fat pads are proinflammatory as measured by an increase in M1 polarized macrophages, increased TNF-α production and increased matrix metalloproteinase (MMP)-2 and -9 expression. To evaluate activatable cell penetrating peptide (ACPP) targeting of mammary tumors in the obese fat pad we utilized a version of the MMP cleavable ACPP that has a Cy5 fluorophore attached to the polycationic domain of the peptide and a Cy7 conjugated to the polyanionic domain. Upon cleavage of the peptide by MMPs, the fluorescence resonance energy transfer (FRET) between the two dyes is disrupted and the Cy5 emission increases while the Cy7 re-emission diminishes. This change in emission spectra is measured and the ratio of Cy5 to Cy7 fluorescence is used as a measure of peptide cleavage. The ACPP was administered at a dose of 0.4nmol/g and allowed to circulate for 2 hours prior to harvesting of the fat pads and imaging. The Cy5/Cy7 ratio was 24% higher in the obese fat pads, with a 45% increase in fluorescence when examining just the Cy5 emission. To determine if tumors could be identified in the obese fat pads with the higher background resulting from increased macrophage recruitment and MMP activity, normal chow (NC) and HFD fed C57Bl/6 mice were injected with syngeneic luminal Py230 and claudin low EMT Py8119 cell lines derived from spontaneous polyomavirus middle T (PyVmT) tumor homogenates. Mice harboring both Py230 and Py8119 tumors in their thoracic mammary fat pads were dosed with either the MMP cleavable FRET ACPP or an uncleavable control ACPP. Two hours after peptide injection, mice were sacrificed and the skin was removed to image the tumors in the context of the fat pad. In concordance with our previous results, tumor growth of the Py230 cells was significantly greater in the HFD mice. Interestingly, growth of the more aggressive Py8119 tumors was not enhanced in the HFD microenvironment. For both cell lines, the tumors were clearly delineated using the Cy5/Cy7 ratio. The Py8119 tumors had a ratio of 6.1±0.7 in NC mice and 5.4±0.3 in the HFD mice, and a similar trend was observed in the less aggressive luminal Py230 tumors, with ratios of 4.9±0.8 for NC and 3.8±0.4 for HFD fed mice. This was consistent with analysis showing increased macrophage recruitment in the Py8119 tumors compared with the Py230 tumors, but no difference in recruitment between tumors of either type in mice fed NC or HFD. Our results indicate that although inflammation associated with obesity results in higher background fluorescence in the mammary fat pad, this is not a barrier to visualizing the tumor tissue in the fat pad. Since a large amount of adipose tissue can be problematic during surgery, MMP FRET ACPPs may be useful for identifying and resecting breast cancers in obese patients leading to improved survival. Citation Format: Jessica L. Crisp, Elamprakash N. Savariar, Jonathan P. Hasselmann, Heekyung Chung, Roger Y. Tsien, Lesley G. Ellies. MMP FRET ACPP imaging of mammary tumors in a mouse model of postmenopausal obesity. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A005.

For clinical imaging, we need synthetic molecules with novel amplifying mechanisms for homing to diseased tissues. Activatable cell penetrating peptides (ACPPs) are polycationic cell penetrating peptides (CPPs) whose cellular uptake is minimized by a polyanionic inhibitory domain and then restored upon proteolysis of the peptide linker connecting the polyanionic and polycationic domains. Local activity of proteases able to cut the linker causes amplified retention in tissues and uptake into cells. ACPPs on dendrimers labeled with Cy5 and Gd-DOTA enable whole body magnetic resonance imaging (Olson et al (2010) PNAS 107: 4311–4316) followed by fluorescenceguided surgery. Such fluorescence guidance improves tumor-free survival in two animal models (Nguyen et al (2010) PNAS 107: 4317–4322). Contrast for tumor over normal tissues is amplified and accelerated when the polyanionic domain includes an acceptor of fluorescence resonance energy transfer (FRET), because loss of FRET (monitored either by multispectral emission or donor excited-state lifetime) instantly signals proteolysis without waiting for uncleaved substrate to wash out of normal tissues. Thrombin-cleavable ACPPs accumulate in atherosclerotic plaques, and their labeling intensity seems to correlate with progression towards rupture. Separately, we have developed fluorescent peptides that light up peripheral nerves to show surgeons where not to cut (Whitney et al (2011) Nature Biotech. 29: 352–356). Thus we believe that basic tumor biology and molecular design can help improve early detection and surgical resection, not just chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr IA1.