Activatable Cell-penetrating Peptides Research Articles

Enzyme‐Responsive Cell‐Penetrating Peptide Conjugated Mesoporous Silica Quantum Dot Nanocarriers for Controlled Release of Nucleus‐Targeted Drug Molecules and Real‐Time Intracellular Fluorescence Imaging of Tumor Cells

Here, a set of novel and personalized nanocarriers are presented for controlled nucleus-targeted antitumor drug delivery and real-time imaging of intracellular drug molecule trafficking by integrating an enzyme activatable cell penetrating peptide (CPP) with mesoporous silica coated quantum dots nanoparticles. Upon loading of antitumor drug, doxorubicin (DOX) and further exposure to proteases in tumor cell environment, the enzymatic cleavage of peptide sequence activates oligocationic TAT residues on the QDs@mSiO2 surface and direct the DOX delivery into cellular nucleus. The systematic cell imaging and cytotoxicity studies confirm that the enzyme responsive DOX-loaded CPP-QDs@mSiO2 nanoparticles can selectively release DOX in the tumor cells with high cathepsin B enzyme expression and greatly facilitate DOX accumulation in targeted nucleus, thus exhibiting enhanced antitumor activity in these cells. As contrast, there is limited nuclear-targeted drug accumulation and lower tumor cytotoxicity observed in the cells without enzyme expression. More importantly, significant antitumor DOX accumulation and higher tumor inactivation is also found in the drug resistant tumor cells with targeted enzyme expression. Such simple and specific enzyme responsive mesoporous silica-QDs nanoconjugates provide great promise for rational design of targeted drug delivery into biological system, and may thus greatly facilitate the medical theranostics in the near future.

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.

Upregulated Matrix Metalloproteinase 2/9 Activity in Murine Asthma Detected with Activatable Cell-Penetrating Peptides

RationaleAsthma, 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.MethodsAsthma 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 = 3-6 mice each) were administered intravenously 6 hr before mice were sacrificed. Lungs were imaged for Cy5 fluorescence (Maestro, CRi). Lung sections (10 μm, 8 images/mouse) were imaged for Cy5 fluorescence on a confocal microscope (5Live, Zeiss) and stained with hematoxylin and eosin.ResultsMMP2/9 activity was > 2-fold higher in lungs from asthma mice than controls (p = 1.8×10−4). The same pattern was observed for ratiometric ACPPs. MMP2/9 activity localized around inflamed airways with 1.6-fold higher ACPP uptake surrounding airways compared to the normal lung parenchyma (p = 0.03). MMP2/9 activity detected by ACPPs co-localized with gelatinase activity measured with in situ DQ gelatin.ConclusionsMMP-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. RationaleAsthma, 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. 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. MethodsAsthma 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 = 3-6 mice each) were administered intravenously 6 hr before mice were sacrificed. Lungs were imaged for Cy5 fluorescence (Maestro, CRi). Lung sections (10 μm, 8 images/mouse) were imaged for Cy5 fluorescence on a confocal microscope (5Live, Zeiss) and stained with hematoxylin and eosin. 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 = 3-6 mice each) were administered intravenously 6 hr before mice were sacrificed. Lungs were imaged for Cy5 fluorescence (Maestro, CRi). Lung sections (10 μm, 8 images/mouse) were imaged for Cy5 fluorescence on a confocal microscope (5Live, Zeiss) and stained with hematoxylin and eosin. ResultsMMP2/9 activity was > 2-fold higher in lungs from asthma mice than controls (p = 1.8×10−4). The same pattern was observed for ratiometric ACPPs. MMP2/9 activity localized around inflamed airways with 1.6-fold higher ACPP uptake surrounding airways compared to the normal lung parenchyma (p = 0.03). MMP2/9 activity detected by ACPPs co-localized with gelatinase activity measured with in situ DQ gelatin. MMP2/9 activity was > 2-fold higher in lungs from asthma mice than controls (p = 1.8×10−4). The same pattern was observed for ratiometric ACPPs. MMP2/9 activity localized around inflamed airways with 1.6-fold higher ACPP uptake surrounding airways compared to the normal lung parenchyma (p = 0.03). MMP2/9 activity detected by ACPPs co-localized with gelatinase activity measured with in situ DQ gelatin. ConclusionsMMP-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. 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.

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.

Abstract A005: MMP FRET ACPP imaging of mammary tumors in a mouse model of postmenopausal obesity

Abstract 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.

Fluorescence‐Guided Thyroidectomy in a Transgenic Murine Model of Papillary Thyroid Carcinoma

Objectives: A transgenic murine model for thyroid cancer was used to evaluate the effectiveness of molecularly targeted ratiometric activatable cell-penetrating peptides (RACPPs) for surgical navigation. We will analyze the implementation of fluorescence guided surgery in this model and discuss future applications. Methods: Thirteen transgenic BRAFV600E mice were studied prospectively. Seven were injected with RACPP, and 4 were injected with saline (controls). Pre- and postoperative laryngoscopy was used to assess vocal cord function. Total thyroidectomy was performed with microscopic white-light visualization. Fluorescence imaging of the post-thyroidectomy field was performed, and fluorescent tissue was removed for pathology. One surgeon carried out all procedures. Data underwent blinded review by a board certified pathologist and a laryngologist. Results: There were 7 successful fluorescence guided surgical procedures and four successful controls. Average operative times were 15.8 (RACPP) and 14 minutes (controls). RACPPs improved thyroid tumor visualization with an average intensity ratio of 9.1, versus 1.5 with white-light alone. Intact function was present in 12 of 12 vocal cords in the study group and 1 of 2 vocal cords of the controls. Postoperatively, 5 RACPP animals showed fluorescent signal, but on histology there was no tumor. Thyroid tumor was present in the final surgical beds of 3 animals, 2 RACPP animals, and 1 control. Conclusions: In this transgenic murine model of papillary thyroid cancer, RACPPs improved surgical visualization without compromising post-resection vocal cord function. There was no proven benefit for completeness of resection. Surgical molecular guidance with RACPPs warrants further study.

Tumor targeting and microenvironment-responsive nanoparticles for gene delivery

A tumor targeting nanoparticle system has been successfully developed to response to the lowered tumor extracellular pH (pHe) and upregulated matrix metalloproteinase 2 (MMP2) in the tumor microenvironment. The nanoparticles are modified with activatable cell-penetrating peptide (designated as dtACPP) that's dual-triggered by the lowered pHe and MMP2. In dtACPP, the internalization function of cell-penetrating peptide (CPP) is quenched by a pH-sensitive masking peptide, linking by a MMP2 substrate. The masking peptide is negatively charged to quench the cationic CPP well after systemic administration. Hence, dtACPP-modified nanoparticles possesses passive tumor targetability via the enhanced permeability and retention (EPR) effect. Once reaching the tumor microenvironment, the pre-existing attraction would be eliminated due to the lowered pHe, accompanying the linker cleaved by MMP2, dtACPP would be activated to expose CPP to drive the nanoparticles' internalization into the intratumoral cells. The studies of plasmid DNA loading, toxicity assessment, cellular uptake, tumor targeting delivery, and gene transfection demonstrate that dtACPP-modified nanoparticle system is a potential candidate for tumor targeting gene delivery.

Tumor-Targeting and Microenvironment-Responsive Smart Nanoparticles for Combination Therapy of Antiangiogenesis and Apoptosis

Tumor microenvironment, such as the lowered tumor extracellular pH (pHe) and matrix metalloproteinase 2 (MMP2), has been extensively explored, which promotes the development of the microenvironment-responsive drug delivery system. Utilizing these unique features, an activatable cell-penetrating peptide (designated as dtACPP) that is dual-triggered by the lowered pHe and MMP2 has been constructed, and a smart nanoparticle system decorating with dtACPP has been successfully developed, which could dual-load gene drug and chemotherapeutics simultaneously. After systemic administration, dtACPP-modified nanoparticles possess passive tumor targetability via the enhanced permeability and retention effect. Then dtACPP would be activated to expose cell-penetrating peptide to drive the nanoparticles' internalization into the intratumoral cells. As angiogenesis and tumor cells might be mutually improved in tumor growth, so combining antiangiogenesis and apoptosis is meaningful for oncotherapy. Vascular endothelial growth factor (VEGF) is significant in angiogenesis, and anti-VEGF therapy could decrease blood vessel density and delay tumor growth obviously. Chemotherapy using doxorubicin (DOX) could kill off tumor cells efficiently. Here, utilizing dtACPP-modified nanoparticles to co-deliver plasmid expressing interfering RNA targeting VEGF (shVEGF) and DOX (designated as dtACPPD/shVEGF-DOX) results in effective shutdown of blood vessels and cell apoptosis within the tumor. On the premise of effective drug delivery, dtACPPD/shVEGF-DOX has demonstrated good tumor targetability, little side effects after systemic administration, and ideal antitumor efficacy.

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.

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.

Applications of Cell-Penetrating Peptides for Tumor Targeting and Future Cancer Therapies

Cell-penetrating peptides provide a highly promising strategy for intracellular drug delivery. One relevant clinical application of cell-penetrating peptides is cancer therapeutics. Peptide based delivery could increase the uptake of drugs in tumor cells and thereby increase the efficacy of the treatment, either of conventional small molecular drugs or oligonucleotide based therapeutics. This review is focused on the cancer applications of cell penetrating peptides as delivery systems; different aspects of drug loading, cargoes and delivery are discussed together with methods for targeted delivery, activatable cell-penetrating peptides and transducible agents coupled to cell-penetrating peptides.

Thrombin Activity Associated with Neuronal Damage during Acute Focal Ischemia

Mechanisms of ischemic neuronal and vascular injury remain obscure. Here we test the hypothesis that thrombin, a blood-borne coagulation factor, contributes to neurovascular injury during acute focal ischemia. Stroke was induced in adult Sprague Dawley rats by occluding the middle cerebral artery. Intra-arterial thrombin infusion during ischemia significantly increased vascular disruption and cellular injury. Intravenous infusion of argatroban, a direct thrombin inhibitor, alleviated neurovascular injury. Immunostaining showed thrombin on neurons in the ischemic core. Using an activatable cell-penetrating peptide engineered to detect thrombin activity, we discovered that thrombin proteolytic activity was specifically associated with neuronal damage during ischemia. Protease activated receptor-1, the presumptive thrombin receptor, appeared to mediate ischemic neurovascular injury. Furthermore, rats receiving thrombin during ischemia showed cognitive deficit, whereas rats receiving argatroban retained intact learning and memory. These results suggest a potential role for thrombin contributing to neurovascular injury and several potential avenues for neuroprotection.

In vivofluorescence imaging of atherosclerotic plaques with activatable cell-penetrating peptides targeting thrombin activity

Thrombin and other coagulation enzymes have been shown to be important during atherosclerotic disease development. Study of these proteases is currently limited because of lack of robust molecular imaging agents for imaging protease activity in vivo. Activatable cell penetrating peptides (ACPPs) have been used to monitor MMP activity in tumors and, in principle, can be modified to detect other proteases. We have developed a probe that incorporates the peptide sequence DPRSFL from the proteinase activated receptor 1 (PAR-1) into an ACPP and shown that it is preferentially cleaved by purified thrombin. Active thrombin in serum cleaves DPRSFL-ACPP with >90% inhibition by lepirudin or argatroban. The DPRSFL-ACPP cleavage product accumulated in advanced atherosclerotic lesions in living mice, with 85% reduction in retention upon pre-injection of mice with hirudin. Uptake of the ACPP cleavage product was highest in plaques with histological features associated with more severe disease. Freshly resected human atheromas bathed in DPRSFL-ACPP retained 63% greater cleavage product compared to control ACPP. In conclusion, DPRSFL-ACPP can be used to study thrombin activity in coagulation and atherosclerosis with good spatial and temporal resolution. Thrombin-sensitive ACPPs may be developed into probes for early detection and intraoperative imaging of high risk atherosclerotic plaques.

Abstract IA1: Building molecules to guide anticancer therapy

Abstract 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.

Abstract 5279: Imaging of pancreatic cancer with activated cell penetrating peptide dendrimers

Abstract We have recently described novel activatable cell penetrating peptides attached to dendrimers (ACPPDs) which can selectively label tumors for real-time imaging during surgery. The aim of this study was to evaluate the ability of Cy5-labeled ACPPDs to visualize primary tumors and their margins in an orthotopic mouse model of pancreatic cancer. BxPC-3 or BxPC-3-RFP-expressing human pancreatic cancer cells were injected into the pancreas of 6-week old athymic mice to establish the orthotopic mouse models. ACPPs were synthesized on an automated peptide synthesizer by using standard protocols for fluorenylmethoxycarbonyl solid-phase synthesis. Peptides were cleaved off the resin, purified by HPLC, and conjugated to a generation 5 PAMAM dendrimer. Cy5 labeling was performed directly on the dendrimer. Dendrimers were then capped completely with Peg4 NHS ester. Tumor-bearing mice were injected with 20 nmoles of ACPPDs via tail vein 48 hours prior to imaging. Mice were first imaged non-invasively using OV-100 (Olympus) or Maestro (CRI) small animal imaging systems. Mice then underwent laparotomy. Resection of the primary pancreatic tumor using fluorescence guidance was performed using a MVX-10 fluorescence-dissecting microscope (Olympus) customized to allow the surgeon to see white-light reflectance, fluorescence, or the two superimposed in real time. At the conclusion of the experiments, H&E staining was used to confirm the pathological diagnosis of cancer in the resected specimens. We found that in the orthotopic BxPC3 mouse model of pancreatic cancer, intravenous injection of ACPPD showed bright fluorescence labeling of tumors compared to surrounding normal tissue. Co-localization studies showed a good correlation between ACPPD uptake and RFP tumor expression. Tumor margins were more clearly delineated with fluorescence imaging using ACPPDs compared to with white light reflectance alone. Post resection, the tumor bed was examined and imaged for residual tumor. With fluorescence-guided resection, the tumor bed was free of residual tumor, which was confirmed histologically. In this study, we have shown that ACPPDs can accurately and effectively label primary pancreatic tumors in an orthotopic mouse model of pancreatic cancer. This technology has the potential to improve surgical outcomes in patients with pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5279. doi:10.1158/1538-7445.AM2011-5279

Abstract 3627: Targeted delivery of gelonin to claudin-expressing cancer cells and use of activatable cell penetrating peptides to enhance potency

Abstract Treatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosome/lysosome compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. Although their functional role in cancer progression remains unclear, the differential expression of these proteins between tumor and normal cells, in addition to their membrane localization, makes them prime candidates for targeted cancer therapy. This study utilized the C-terminal 30 amino acid fragment of C. perfringens enterotoxin (CPE), that binds to claudins 3 and 4, to deliver the toxin gelonin (rGel) and examined a new strategy for enhancing delivery to the cytoplasm. CPE was fused to rGel at its N-terminal end via a flexible G4S linker. This molecule was internalized into vesicles from which location it produced little cytotoxicity. Arginine-rich peptides have been extensively used to enhance cellular uptake of various types of cargo, and there is a reasonable presumption that they can deliver cargo out of subcellular compartments as well. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R9) was introduced between the CPE and the rGel. CPE-R9-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. Given that the R9 moiety was so effective at enhancing uptake, we reasoned that it would likely also be good at releasing rGel from the compartment into which CPE delivered it. However, to make use of the R9 sequence in this manner it is necessary to mask the R9 until after CPE-mediated and claudin-dependent endocytotic uptake. To this end an E9-G4S-R9-rGel fusion protein was produced and tested for cytotoxicity. The addition of the E9 through a G4S linker to R9-rGel increased the IC50 by 5.3-fold from 38 to 202 nM indicating that the inhibitory E9 domain can largely neutralize the R9. Addition of CPE to the E9-G4S-R9-rGel fusion protein further reduced the IC50 to 482 nM consistent with the concept that the CPE directs the CPE-E9-G4S-R9-rGel to intracellular vesicles from which it has difficulty in fully escaping. However, addition of endosomolytic reagent chloroquine at a non-toxic concentration increased the inhibition of growth produced by 31.25 nM CPE-E9-G4S-R9-rGel from 10% to 76%. Thus, while CPE provides targeting to claudin-expressing tumor cells, R9 plus chloroquine provide a mechanism for getting out of intracellular compartment, and E9 offers an approach to masking the non-specific toxicity of R9-rGel. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3627. doi:10.1158/1538-7445.AM2011-3627

Challenges associated with the targeted delivery of gelonin to claudin-expressing cancer cells with the use of activatable cell penetrating peptides to enhance potency

BackgroundTreatment of tumors with macromolecular toxins directed to cytoplasmic targets requires selective endocytosis followed by release of intact toxin from the endosomal/lysosomal compartment. The latter step remains a particular challenge. Claudins 3 and 4 are tight junction proteins that are over-expressed in many types of tumors. This study utilized the C-terminal 30 amino acid fragment of C. perfringens enterotoxin (CPE), which binds to claudins 3 and 4, to deliver a toxin in the form of recombinant gelonin (rGel) to the cytoplasm of the human ovarian carcinoma cell line 2008.ResultsCPE was fused to rGel at its N-terminal end via a flexible G4S linker. This CPE-G4S-rGel molecule was internalized into vesicles from which location it produced little cytotoxicity. To enhance release from the endosomal/lysosomal compartment a poly-arginine sequence (R9) was introduced between the CPE and the rGel. CPE-R9-rGel was 10-fold more cytotoxic but selectivity for claudin-expressing cells was lost. The addition of a poly-glutamic acid sequence (E9) through a G4S linker to R9-rGel (E9-G4S-R9-rGel) largely neutralized the non-selective cell membrane penetrating activity of the R9 motif. However, introduction of CPE to the E9-G4S-R9-rGel fusion protein (CPE-E9-G4S-R9-rGel) further reduced its cytotoxic effect. Treatment with the endosomolytic reagent chloroquine increased the cytotoxicity of CPE-E9-G4S-R9-rGel. Several types of linkers susceptible to cleavage by furin and endosomal cathepsin B were tested for their ability to enhance R9-rGel release but none of these modifications further enhanced the cytotoxicity of CPE-E9-G4S-R9-rGel.ConclusionWe conclude that while a claudin-3 and -4 ligand serves to deliver rGel into 2008 cells the delivered molecules were entrapped in intracellular vesicles. Incorporation of R9 non-specifically increased rGel cytotoxicity and this effect could be masked by inclusion of an E9 sequence. However, the putative protease cleavable sequences tested were inadequate for release of R9-rGel from CPE-E9-G4S-R9-rGel.

Tumor Targeting of MMP-2/9 Activatable Cell-Penetrating Imaging Probes Is Caused by Tumor-Independent Activation

Activatable cell-penetrating peptides (ACPPs) are a new class of promising molecular imaging probes for the visualization of enzymes in vivo. The cell-penetrating function of a polycationic peptide is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a cleavable linker present between the polycationic cell-penetrating peptide and polyanionic peptide affords dissociation of both domains and enables the activated cell-penetrating peptide to enter cells. Here, we aimed to develop an ACPP sensitive to matrix metalloproteinase-2 and -9 (MMP-2/9) for nuclear imaging purposes. MMP-2/9 ACPPs and nonactivatable cell-penetrating peptides (non-ACPP) were prepared by 9-fluorenylmethyloxycarbonyl solid-phase peptide synthesis and labeled with (177)Lu or (177)Lu/(125)I for dual-isotope studies. The in vivo biodistribution of these probes was assessed in MMP-2/9-positive tumor-bearing mice (n = 6) and healthy mice (n = 4) using γ-counting. Furthermore, a radiolabeled cell-penetrating peptide serving as a positive control was evaluated in tumor-bearing mice (n = 6). Biodistribution studies showed a 5-fold-higher retention of ACPP in tumor than in muscle (P < 0.01) and a 6-fold-higher tumor retention relative to non-ACPP (P < 0.01), supporting earlier studies on fluorescently labeled ACPPs proposing activation by tumor-associated MMP-2/9. Surprisingly, however, the uptake of ACPP was significantly higher than that of non-ACPP in almost all tissues (P < 0.01). To unravel the activation process of ACPP in vivo, we developed dual-isotope ACPP analogs (dACPPs) that allowed us to discriminate between uncleaved dACPP and activated dACPP. In vivo biodistribution of dACPP indicated that the tissue-associated counts originated from activated dACPP. Interestingly, dACPP administration to healthy mice, compared with MMP-2/9-positive tumor-bearing mice, resulted in a similar dACPP biodistribution. Furthermore, a radiolabeled cell-penetrating peptide showed tumor-to-tissue ratios equal to those found for ACPP (P > 0.05). This study demonstrates that the tumor targeting of radiolabeled MMP-2/9 ACPPs is most likely caused by the activation in the vascular compartment rather than tumor-specific activation, as suggested earlier. The results in the present paper indicate that different and more tissue-specific enzyme-ACPP combinations are needed to unleash the full potential of the elegant ACPP concept in living animals.

Design, synthesis, and imaging of an activatable photoacoustic probe.

Photoacoustic tomography is a rapidly growing imaging modality that can provide images of high spatial resolution and high contrast at depths up to 5 cm. We report here the design, synthesis, and evaluation of an activatable probe that shows great promise for enabling detection of the cleaved probe in the presence of high levels of nonactivated, uncleaved probe, a difficult task to attain in absorbance-based modality. Before the cleavage by its target, proteolytic enzyme MMP-2, the probe, an activatable cell-penetrating peptide, Ceeee[Ahx]PLGLAGrrrrrK, labeled with two chromophores, BHQ3 and Alexa750, shows photoacoustic signals of similar intensity at the two wavelengths corresponding to the absorption maxima of the chromophores, 675 and 750 nm. Subtraction of the images taken at these two wavelengths makes the probe effectively photoacoustically silent, as the signals at these two wavelengths essentially cancel out. After the cleavage, the dye associated with the cell-penetrating part of the probe, BHQ3, accumulates in the cells, while the other dye diffuses away, resulting in photoacoustic signal seen at only one of the wavelengths, 675 nm. Subtraction of the photoacoustic images at two wavelengths reveals the location of the cleaved (activated) probe. In the search for the chromophores that are best suited for photoacoustic imaging, we have investigated the photoacoustic signals of five chromophores absorbing in the near-infrared region. We have found that the photoacoustic signal did not correlate with the absorbance and fluorescence of the molecules, as the highest photoacoustic signal arose from the least absorbing quenchers, BHQ3 and QXL 680.

Parallel in Vivo and in Vitro Selection Using Phage Display Identifies Protease-dependent Tumor-targeting Peptides

We recently developed activatable cell-penetrating peptides (ACPPs) that target contrast agents to in vivo sites of matrix metalloproteinase activity, such as tumors. Here we use parallel in vivo and in vitro selection with phage display to identify novel tumor-homing ACPPs with no bias for primary sequence or target protease. Specifically, phage displaying a library of ACPPs were either injected into tumor-bearing mice, followed by isolation of cleaved phage from dissected tumor, or isolated based on selective cleavage by extracts of tumor versus normal tissue. Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts. The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast. This uniquely identified ACPP was not cleaved by matrix metalloproteinases or various coagulation factors but was efficiently cleaved by plasmin and elastases, both of which have been shown to be aberrantly overexpressed in tumors. The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.