Assessment of the Efficacy of the Combination of RNAi of lncRNA DANCR with Chemotherapy to Treat Triple Negative Breast Cancer Using Magnetic Resonance Molecular Imaging.

Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is overexpressed in human triple-negative breast cancer (TNBC) and promotes cell migration and proliferation. TNBC is limited in treatment options relative to hormone-receptor-positive breast cancer and is commonly treated with chemotherapy, which is often compromised by acquired resistance. DANCR has been implicated in the development of chemoresistance across multiple cancer types. Here, we applied magnetic resonance molecular imaging (MRMI) with a targeted contrast agent, MT218, specific to extradomain-B fibronectin (EDB-FN), a marker for epithelial-to-mesenchymal transition, to assess the therapeutic efficacy of the combination of paclitaxel and ZD2-PEG-ECO/siDANCR nanoparticles (ZD2-siDANCR-ELNP) to treat TNBC. The treatment of orthotopic MDA-MB-231 TNBC in mice with paclitaxel significantly suppressed tumor growth but with a significant increase of EDB-FN in the tumor, as revealed by MRMI and immunohistochemistry. Combining ZD2-siDANCR-ELNP with paclitaxel further reduced tumor sizes, along with reduced EDB-FN expression. Interestingly, MT218-MRMI revealed a lower reduction of tumor signal enhancement with the combination treatment than that with the siDANCR treatment alone, which was supported by higher cell density in the tumors treated with the combination therapy, as shown by histochemical analysis. MT218-MRMI clearly revealed the changes of the tumor microenvironment in response to various therapies and is effective to noninvasively assess the response of TNBC tumors to the therapies. Regulating oncogenic lncRNA DANCR is an effective strategy for improving the outcomes of chemotherapy in TNBC.

Intravitreal Delivery of PEGylated-ECO Plasmid DNA Nanoparticles for Gene Therapy of Stargardt Disease.

OBJECTIVE: Current gene therapy of inherited retinal diseases is achieved mainly by subretinal injection, which is invasive with severe adverse effects. Intravitreal injection is a minimally invasive alternative for gene therapy of inherited retinal diseases. This work explores the efficacy of intravitreal delivery of PEGylated ECO (a multifunctional pH-sensitive amphiphilic amino lipid) plasmid DNA (pGRK1-ABCA4-S/MAR) nanoparticles (PEG-ELNP) for gene therapy of Stargardt disease. METHODS: Pigmented Abca4-/- knockout mice received 1 µL of PEG-ELNP solution (200 ng/uL, pDNA concentration) by intravitreal injections at an interval of 1.5 months. The expression of ABCA4 in the retina was determined by RT-PCR and immunohistochemistry at 6 months after the second injection. A2E levels in the treated eyes and untreated controls were determined by HPLC. The safety of treatment was monitored by scanning laser ophthalmoscopy and electroretinogram (ERG). RESULTS: PEG-ELNP resulted in significant ABCA4 expression at both mRNA level and protein level at]6 months after 2 intravitreal injections, and a 40% A2E accumulation reduction compared with non-treated controls. The PEG-ELNP also demonstrated excellent safety as shown by scanning laser ophthalmoscopy, and the eye function evaluation from electroretinogram. CONCLUSIONS: Intravitreal delivery of the PEG-ELNP of pGRK1-ABCA4-S/MAR is a promising approach for gene therapy of Stargardt Disease, which can also be a delivery platform for gene therapy of other inherited retinal diseases.

Targeted Contrast Agents for Magnetic Resonance Molecular Imaging of Cancer.

Magnetic resonance imaging (MRI) is a clinical imaging modality that provides high-resolution images of soft tissues, including cancerous lesions. Stable gadolinium(III) chelates have been used as contrast agents (CA) in MRI to enhance the contrast between the tissues of interest and surrounding tissues for accurate diagnostic imaging. Magnetic resonance molecular imaging (MRMI) of cancer requires targeted CA to specifically elucidate cancer-associated molecular processes and can provide high-resolution delineation and characterization of cancer for precision medicine. The main challenge for MRMI is the lack of sufficient sensitivity to detect the low concentration of the cellular oncogenic markers. In addition, targeted CA must satisfy regulatory safety requirements prior to clinical development. Up to now, there is no FDA-approved targeted CA for MRMI of cancer.In this Account, we discuss the latest developments in the design and development of clinically translatable targeted CA for MRMI of cancer, with an emphasis on our own research. The primary limitation of MRMI can be overcome by designing small molecular targeted CA to target abundant cancer-specific targets found in the tumor microenvironment (TME). For example, aggressive tumors have a unique extracellular matrix (ECM) composed of oncoproteins, which can be used as targetable markers for MRMI. We have designed and prepared small peptide conjugates of clinical contrast agents, including Gd-DTPA and Gd-DOTA, to target fibrin-fibronectin clots in tumors. These small molecular CA have been effective in enhancing MRMI detection of solid tumors and have demonstrated the ability to detect submillimeter cancer micrometastases in mouse tumor models, exceeding the detection limit of current clinical imaging modalities. We have also identified extradomain B fibronectin (EDB-FN), an oncofetal subtype of fibronectin, as a promising TME target to leverage in the design and development of small peptide targeted CA for clinical translation. The expression level of EDB-FN is correlated with invasiveness of cancer cells and poor patient survival of multiple cancer types. ZD2 peptide with a sequence of seven amino acids (TVRTSAD) was identified to specifically bind to the EDB protein fragment. Several ZD2 conjugates of macrocyclic GBCA, including Gd-DOTA and Gd(HP-DO3A), have been synthesized and tested in mouse tumor models. ZD2-N3-Gd(HP-DO3A) (MT218) with a high r1 relaxivity was selected as the lead agent for clinical translation. The physicochemical properties and preclinical assessments of MT218 are summarized in this Account. MRMI of EDB-FN with MT218 can effectively detect invasive tumors of multiple cancers with risk-stratification and monitor tumor response to anticancer therapies in mouse models. Currently, MT218 is in clinical trials for precision cancer MRMI. Herein, we will show that using targeted MRI contrast agents specific to abundant TME biomarkers is a pragmatic solution for effective precision cancer imaging in high spatial resolution. And thus, we illustrate a replicable approach for CA development that is vital for cancer MRMI.

Structure and Function of Cationic and Ionizable Lipids for Nucleic Acid Delivery.

Hereditary genetic diseases, cancer, and infectious diseases are affecting global health and become major health issues, but the treatment development remains challenging. Gene therapies using DNA plasmid, RNAi, miRNA, mRNA, and gene editing hold great promise. Lipid nanoparticle (LNP) delivery technology has been a revolutionary development, which has been granted for clinical applications, including mRNA vaccines against SARS-CoV-2 infections. Due to the success of LNP systems, understanding the structure, formulation, and function relationship of the lipid components in LNP systems is crucial for design more effective LNP. Here, we highlight the key considerations for developing an LNP system. The evolution of structure and function of lipids as well as their LNP formulation from the early-stage simple formulations to multi-components LNP and multifunctional ionizable lipids have been discussed. The flexibility and platform nature of LNP enable efficient intracellular delivery of a variety of therapeutic nucleic acids and provide many novel treatment options for the diseases that are previously untreatable.

Disruption of retinal inflammation and the development of diabetic retinopathy in mice by a CD40-derived peptide or mutation of CD40 in Müller cells.

AIMS/HYPOTHESIS: CD40 expressed in Müller cells is a central driver of diabetic retinopathy. CD40 causes phospholipase Cγ1 (PLCγ1)-dependent ATP release in Müller cells followed by purinergic receptor (P2X7)-dependent production of proinflammatory cytokines in myeloid cells. In the diabetic retina, CD40 and P2X7 upregulate a broad range of inflammatory molecules that promote development of diabetic retinopathy. The molecular event downstream of CD40 that activates the PLCγ1-ATP-P2X7-proinflammatory cytokine cascade and promotes development of diabetic retinopathy is unknown. We hypothesise that disruption of the CD40-driven molecular events that trigger this cascade prevents/treats diabetic retinopathy in mice. METHODS: B6 and transgenic mice with Müller cell-restricted expression of wild-type (WT) CD40 or CD40 with mutations in TNF receptor-associated factor (TRAF) binding sites were made diabetic using streptozotocin. Leucostasis was assessed using FITC-conjugated concanavalin A. Histopathology was examined in the retinal vasculature. Expression of inflammatory molecules and phospho-Tyr783 PLCγ1 (p-PLCγ1) were assessed using real-time PCR, immunoblot and/or immunohistochemistry. Release of ATP and cytokines were measured by ATP bioluminescence and ELISA, respectively. RESULTS: Human Müller cells with CD40 ΔT2,3 (lacks TRAF2,3 binding sites) were unable to phosphorylate PLCγ1 and release ATP in response to CD40 ligation, and could not induce TNF-α/IL-1ß secretion in bystander myeloid cells. CD40-TRAF signalling acted via Src to induce PLCγ1 phosphorylation. Diabetic mice in which WT CD40 in Müller cells was replaced by CD40 ΔT2,3 failed to exhibit phosphorylation of PLCγ1 in these cells and upregulate P2X7 and TNF-α in microglia/macrophages. P2x7 (also known as P2rx7), Tnf-α (also known as Tnf), Il-1ß (also known as Il1b), Nos2, Icam-1 (also known as Icam1) and Ccl2 mRNA were not increased in these mice and the mice did not develop retinal leucostasis and capillary degeneration. Diabetic B6 mice treated intravitreally with a cell-permeable peptide that disrupts CD40-TRAF2,3 signalling did not exhibit either upregulation of P2X7 and inflammatory molecules in the retina or leucostasis. CONCLUSIONS/INTERPRETATION: CD40-TRAF2,3 signalling activated the CD40-PLCγ1-ATP-P2X7-proinflammatory cytokine pathway. Src functioned as a link between CD40-TRAF2,3 and PLCγ1. Replacing WT CD40 with CD40 ΔT2,3 impaired activation of PLCγ1 in Müller cells, upregulation of P2X7 in microglia/macrophages, upregulation of a broad range of inflammatory molecules in the diabetic retina and the development of diabetic retinopathy. Administration of a peptide that disrupts CD40-TRAF2,3 signalling reduced retinal expression of inflammatory molecules and reduced leucostasis in diabetic mice, supporting the therapeutic potential of pharmacological inhibition of CD40-TRAF2,3 in diabetic retinopathy.

Peptide Derivatives of Retinylamine Prevent Retinal Degeneration with Minimal Side Effects on Vision in Mice.

Safe and effective molecular therapeutics for prophylactic treatment of retinal degenerative diseases are greatly needed. Disruptions in the clearance of all-trans-retinal (atRAL) by the visual (retinoid) cycle of the retina can lead to the accumulation of atRAL and its condensation products known to initiate progressive retinal dystrophy. Retinylamine (Ret-NH2) and its analogues are known to be effective in lowering the concentration of atRAL within the eye and thus preventing retinal degeneration in mouse models of human retinopathies. Here, we chemically modified Ret-NH2 with amino acids and peptides to improve the stability and ocular bioavailability of the resulting derivatives and to minimize their side effects. Fourteen Ret-NH2 derivatives were synthesized and tested in vitro and in vivo. These derivatives exhibited structure-dependent therapeutic efficacy in preventing light-induced retinal degeneration in Abca4-/-Rdh8-/- double-knockout mice, with the compounds containing glycine and/or L-valine generally exhibiting greater protective effects than Ret-NH2 or other tested amino acid derivatives of Ret-NH2. Ret-NH2-L-valylglycine amide (RVG) exhibited good stability in storage; and effective uptake and prolonged retention in mouse eyes. RVG readily formed a Schiff base with atRAL and did not inhibit RPE65 enzymatic activity. Administered by oral gavage, this retinoid also provided effective protection against light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Notably, the treatment with RVG had minimal effects on the regeneration of 11-cis-retinal and recovery of retinal function. RVG holds promise as a lead therapy for effective and safe treatment of human retinal degenerative diseases.

Efficacy of Targeted ECO/miR-200c Nanoparticles for Modulating Tumor Microenvironment and Treating Triple Negative Breast Cancer as Non-invasively Monitored by MR Molecular Imaging.

PURPOSE: To investigate the effectiveness of targeted ECO/miR-200c in modulating tumor microenvironment and treating triple negative breast cancer (TNBC) using non-invasive magnetic resonance molecular imaging (MRMI) of extradomain B fibronectin (EDB-FN) with a targeted MRI contrast agent. METHODS: MDA-MB-231 and Hs578T TNBC cells were transfected with RGD-PEG-ECO/miR-200c. Invasive and migratory potential was evaluated using transwell, scratch wound, and spheroid formation assays. Athymic nude mice bearing orthotopic MDA-MB-231 and Hs578T xenografts were treated with weekly i.v. injection of RGD-PEG-ECO/miR-200c nanoparticles at 1.0 mg/kg/week RNA for 6 weeks. MRMI of EDB-FN was performed using a targeted contrast agent MT218 [ZD2-N3-Gd(DO3A)] on a 3 T MRS 3000 scanner. T1-weighted images were acquired following intravenous injection of MT218 at dose of 0.1 mmol/kg using a fast spin echo axial sequence with respiratory gating. RESULTS: Systemic administration of RGD-PEG-ECO/miR-200c nanoparticles in mice bearing orthotopic TNBC xenografts significantly suppressed tumor progression without toxic side-effects. MRMI with MT218 revealed that the treatment significantly suppressed tumor proliferation as compared to the control. MRMI also showed that the miR-200c treatment altered tumor microenvironment by reducing EDB-FN expression, as evidenced by decreased contrast enhancement in both MDA-MB-231 and Hs578T tumors. The reduction of EDB-FN was confirmed by immunohistochemistry. CONCLUSIONS: Targeted delivery of miR-200c with RGD-PEG-ECO/miR-200c nanoparticles effectively modulates tumor microenvironment and suppresses TNBC proliferation in animal models. MRMI of tumor EDB-FN expression is effective to non-invasively monitor tumor response and therapeutic efficacy of RGD-PEG-ECO/miR-200c nanoparticles in TNBC.

Non-viral Gene Therapy for Stargardt Disease with ECO/pRHO-ABCA4 Self-Assembled Nanoparticles.

Stargardt disease (STGD) is an autosomal recessive retinal disorder caused by a monogenic ABCA4 mutation. Currently, there is no effective therapy to cure Stargardt disease. The replacement of mutated ABCA4 with a functional gene remains an attractive strategy. In this study, we have developed a non-viral gene therapy using nanoparticles self-assembled by a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid. The nanoparticles mediated efficient intracellular gene transduction in wild-type (WT) and Abca4-/- mice. Specific ABCA4 expression in the outer segment of photoreceptors was achieved by incorporating a rhodopsin promoter into the plasmids. The ECO/pRHO-ABCA4 nanoparticles induced substantial and specific ABCA4 expression for at least 8 months, 35% reduction in A2E accumulation on average, and a delayed Stargardt disease progression for at least 6 months in Abca4-/- mice. ECO/plasmid nanoparticles constitute a promising non-viral gene therapy platform for Stargardt disease and other visual dystrophies.

Fibronectin in the Tumor Microenvironment.

Fibronectin (FN) is a large glycoprotein that plays a diverse set of biological roles. This chapter discusses the structural biology, the normal biological functions, and the molecular role of FN and its splice variants in cancer cell proliferation, metastasis, and chemoresistance. The potential role of FN in cancer imaging is discussed in detail. The chapter also discusses the future directions of basic and translational research of fibronectin in the context of the tumor microenvironment and its role in tumor biology.

Extradomain-B Fibronectin-Targeted Dextran-Based Chemical Exchange Saturation Transfer Magnetic Resonance Imaging Probe for Detecting Pancreatic Cancer.

A dextran-peptide conjugate was developed for magnetic resonance (MR) molecular imaging of pancreatic ductal adenocarcinoma (PDAC) through its overexpressed microenvironment biomarker, extradomain-B fibronectin (EDB-FN). This new agent consists of diamagnetic and biocompatible dextran and a targeting peptide. Dextrans can be directly detected by chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) without the need for radionuclide or metallic labeling. In addition, large molecular weight dextran, dextran 10 (MW ∼ 10 kDa), provides an approximately 50 times higher sensitivity per molecule than a single glucose unit. The potential of this highly biocompatible diamagnetic probe is demonstrated in a murine syngeneic allograft PDAC tumor model. The biocompatibility and sensitivity of this new agent clearly show potential for a path to clinical translation.

Systemic Delivery of Tumor-Targeting siRNA Nanoparticles against an Oncogenic LncRNA Facilitates Effective Triple-Negative Breast Cancer Therapy.

Long noncoding RNAs (lncRNAs), by virtue of their versatility and multilevel gene regulation, have emerged as attractive pharmacological targets for treating heterogeneous and complex malignancies like triple-negative breast cancer (TNBC). Despite multiple studies on lncRNA functions in tumor pathology, systemic targeting of these "undruggable" macromolecules with conventional approaches remains a challenge. Here, we demonstrate effective TNBC therapy by nanoparticle-mediated RNAi of the oncogenic lncRNA DANCR, which is significantly overexpressed in TNBC. Tumor-targeting RGD-PEG-ECO/siDANCR nanoparticles were formulated via self-assembly of multifunctional amino lipid ECO, cyclic RGD peptide-PEG, and siDANCR for systemic delivery. MDA-MB-231 and BT549 cells treated with the therapeutic RGD-PEG-ECO/siDANCR nanoparticles exhibited 80-90% knockdown in the expression of DANCR for up to 7 days, indicating efficient intracellular siRNA delivery and sustained target silencing. The RGD-PEG-ECO/siDANCR nanoparticles mediated excellent in vitro therapeutic efficacy, reflected by significant reduction in the invasion, migration, survival, tumor spheroid formation, and proliferation of the TNBC cell lines. At the molecular level, functional ablation of DANCR dynamically impacted the oncogenic nexus by downregulating PRC2-mediated H3K27-trimethylation and Wnt/EMT signaling, and altering the phosphorylation profiles of several kinases in the TNBC cells. Furthermore, systemic administration of the RGD-PEG-ECO/siDANCR nanoparticles at a dose of 1 mg/kg siRNA in nude mice bearing TNBC xenografts resulted in robust suppression of TNBC progression with no overt toxic side-effects, underscoring the efficacy and safety of the nanoparticle therapy. These results demonstrate that nanoparticle-mediated modulation of onco-lncRNAs and their molecular targets is a promising approach for developing curative therapies for TNBC and other cancers.

Synthesis and Evaluation of pH-Sensitive Multifunctional Lipids for Efficient Delivery of CRISPR/Cas9 in Gene Editing.

CRISPR/Cas9 system is a promising approach for gene editing in gene therapy. Effective gene editing requires safe and efficient delivery of CRISPR/Cas9 system in target cells. Several new multifunctional pH-sensitive amino lipids were designed and synthesized with modification of the amino head groups for intracellular delivery of CRISPR/Cas9 system. These multifunctional pH-sensitive amino lipids exhibited structurally dependent formulation of stable nanoparticles with the DNA plasmids of CRISPR/Cas9 system with the sizes ranging from 100 to 200 nm. The amino lipid plasmid DNA nanoparticles showed pH-sensitive hemolysis with minimal hemolytic activity at pH 7.4 and increased hemolysis at acidic pH (pH = 5.5, 6.5). The nanoparticles exhibited low cytotoxicity at an N/P ratio of 10. Expression of both Cas9 and sgRNA of the CRISPR/Cas9 system was in the range from 4.4% to 33%, dependent on the lipid structure in NIH3T3-GFP cells. The amino lipids that formed stable nanoparticles with high expression of both Cas9 and sgRNA mediated high gene editing efficiency. ECO and iECO mediated more efficient gene editing than other tested lipids. ECO mediated up to 50% GFP suppression based on observations with confocal microscopy and nearly 80% reduction of GFP mRNA based on RT-PCR measurement in NIH3T3-GFP cells. The multifunctional pH-sensitive amino lipids have the potential for efficient intracellular delivery of CRISPR/Cas9 for effective gene editing.

New GABA modulators protect photoreceptor cells from light-induced degeneration in mouse models.

No clinically approved therapies are currently available that prevent the onset of photoreceptor death in retinal degeneration. Signaling between retinal neurons is regulated by the release and uptake of neurotransmitters, wherein GABA is the main inhibitory neurotransmitter. In this work, novel 3-chloropropiophenone derivatives and the clinical anticonvulsants tiagabine and vigabatrin were tested to modulate GABA signaling and protect against light-induced retinal degeneration. Abca4-/-Rdh8-/- mice, an accelerated model of retinal degeneration, were exposed to intense light after prophylactic injections of one of these compounds. Imaging and functional assessments of the retina indicated that these compounds successfully protected photoreceptor cells from degeneration to maintain a full-visual-field response. Furthermore, these compounds demonstrated a strong safety profile in wild-type mice and did not compromise visual function or damage the retina, despite repeated administration. These results indicate that modulating inhibitory GABA signaling can offer prophylactic protection against light-induced retinal degeneration.-Schur, R. M., Gao, S., Yu, G., Chen, Y., Maeda, A., Palczewski, K., Lu, Z.-R. New GABA modulators protect photoreceptor cells from light-induced degeneration in mouse models.

Magnetic resonance molecular imaging of metastatic breast cancer by targeting extradomain-B fibronectin in the tumor microenvironment.

PURPOSE: Non-invasive early accurate detection of malignant breast cancer is paramount to the clinical management of the life-threatening disease. Here, we aim to test a small peptide targeted MRI contrast agent, ZD2-Gd(HP-DO3A), specific to an oncoprotein, extradomain-B fibronectin (EDB-FN), in the tumor microenvironment for MR molecular imaging of breast cancer. METHOD: EDB-FN expression in 4T1 and MDA-MB-231 cancers was analyzed with quantitative real-time PCR and western blot. Primary and metastatic triple negative breast cancer mouse models were developed using 4T1 and MDA-MB-231 cells. Contrast-enhanced MRI was carried out to evaluate the use of ZD2-Gd(HP-DO3A) in detecting 4T1 and MDA-MB-231 primary and metastatic tumors. RESULTS: EDB-FN was abundantly expressed in the extracellular matrix (ECM) of both the primary and metastatic TNBC tumors. In T1 -weighted MRI, ZD2-Gd(HP-DO3A) generated superior contrast enhancement in primary TNBC tumors than a nonspecific clinical agent Gd(HP-DO3A), during 30 min after contrast injection. ZD2-Gd(HP-DO3A) also produced a significant increase in contrast-to-noise ratio (CNR) of TNBC metastases, enabling sensitive localization and delineation of metastases that occulted in non-contrast-enhanced or Gd(HP-DO3A)-enhanced MRI. CONCLUSIONS: These findings potentiate the use of ZD2-Gd(HP-DO3A) for MR molecular imaging of malignant breast cancers to improve the healthcare of breast cancer patients. Magn Reson Med 79:3135-3143, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Drug Delivery in Cancer Therapy, Quo Vadis?

The treatment of malignancies has undergone dramatic changes in the past few decades. Advances in drug delivery techniques and nanotechnology have allowed for new formulations of old drugs, so as to improve the pharmacokinetics, to enhance accumulation in solid tumors, and to reduce the significant toxic effects of these important therapeutic agents. Here, we review the published clinical data in cancer therapy of several major drug delivery systems, including targeted radionuclide therapy, antibody-drug conjugates, liposomes, polymer-drug conjugates, polymer implants, micelles, and nanoparticles. The clinical outcomes of these delivery systems from various phases of clinical trials are summarized. The success and limitations of the drug delivery strategies are discussed based on the clinical observations. In addition, the challenges in applying drug delivery for efficacious cancer therapy, including physical barriers, tumor heterogeneity, drug resistance, and metastasis, are discussed along with future perspectives of drug delivery in cancer therapy. In doing so, we intend to underscore that efficient delivery of cancer therapeutics to solid malignancies remains a major challenge in cancer therapy, and requires a multidisciplinary approach that integrates knowledge from the diverse fields of chemistry, biology, engineering, and medicine. The overall objective of this review is to improve our understanding of the clinical fate of commonly investigated drug delivery strategies, and to identify the limitations that must be addressed in future drug delivery strategies, toward the pursuit of curative therapies for cancer.

Targeted Contrast Agent Specific to an Oncoprotein in Tumor Microenvironment with the Potential for Detection and Risk Stratification of Prostate Cancer with MRI.

Accurate detection and risk stratification are paramount to the clinical management of prostate cancer. Current diagnostic methods, including prostate specific antigen (PSA) screening, are unable to differentiate high-risk tumors from low-risk tumors, resulting in overdiagnosis and overtreatment. A peptide targeted contrast agent, ZD2-Gd(HP-DO3A), specific to an oncoprotein in tumor microenvironment, EDB-FN, was synthesized for noninvasive detection and characterization of aggressive prostate cancer. EDB-FN, one of the subtypes of oncofetal fibronectin, is involved in tumor epithelial-to-mesenchymal transition (EMT), which is implicated in drug resistance and metastasis. The EDB-FN mRNA level in the metastatic PC3 cells was at least three times higher than that in non-metastatic LNCaP cells. In tumors, EDB-FN protein was highly expressed in PC3 tumor xenografts, but not in LNCaP tumors, as revealed by Western blot analysis. ZD2-Gd(HP-DO3A) produced over two times higher contrast-to-noise ratio in the PC3 tumors than in the LNCaP tumors in contrast-enhanced MRI during 30 min after injection. ZD2-Gd(HP-DO3A) possessed high chelate stability against transmetalation and minimal tissue accumulation. Our results demonstrate that molecular MRI of EDB-FN with ZD2-Gd(HP-DO3A) can potentially be used for noninvasive detection and risk stratification of human prostate cancer. Incorporation of this targeted contrast agent in the existing clinical contrast enhanced MRI procedures has the potential to improve diagnostic accuracy of prostate cancer.

Synthesis and Assessment of Peptide Gd-DOTA Conjugates Targeting Extradomain B Fibronectin for Magnetic Resonance Molecular Imaging of Prostate Cancer.

Contrast enhanced MRI is commonly used in imaging and treatment planning of prostate cancer. However, no tumor targeting contrast agent is commercially available for accurate detection and characterization of prostate cancer with MRI. Extradomain B fibronectin (EDB-FN), an oncoprotein present in aggressive tumors, is a promising molecular target for detection and stratification of high-risk prostate cancer. In this work, we have identified four small peptides (GVK, IGK, SGV, and ZD2) specific to EDB-FN for tumor targeting. In silico simulations of the binding patterns and affinities of peptides to the EDB protein fragment revealed different binding site to different peptide in the ligand-receptor interactions. Tumor specificity and organ distribution of the peptides were assessed using fluorescence imaging in male mice bearing PC-3 human prostate cancer xenografts. Targeted contrast agents were synthesized by conjugating tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to the peptides in the solid phase, followed by complexation with GdCl3. The contrast agents were characterized by MALDI-TOF mass spectrometry and relaxivity measurements. All four peptide Gd-DOTA conjugates resulted in robust tumor contrast enhancement in MR imaging of the PC3 mouse prostate cancer model. The peptide Gd-DOTA conjugates specific to EDB-FN are promising targeted small molecular macrocyclic contrast agents for MR molecular imaging of prostate cancer.