Magnetic resonance imaging of intra-pancreatic ductal nanoparticle delivery to islet cells.

BACKGROUND: The absence of reliable drug delivery systems to pancreatic islet cells hampers efficient treatment of type 1 diabetes. Nanoparticle delivery systems equipped with imaging capabilities could enable selective delivery to pancreatic islet cells. Biodistribution of nanoparticles is defined by several factors including the mode of administration, which determines accumulation in various organs. METHODS: In this study, we tested whether intrapancreatic ductal injection of magnetic nanoparticles would result in efficient cellular uptake by pancreatic islet cells. Dextran-coated iron oxide nanoparticles labeled with the near infrared fluorescent dye Cy5.5 were injected into the intrapancreatic ducts of streptozotocin-induced diabetic and healthy mice. To monitor the distribution of the nanoparticles, we performed in vivo magnetic resonance imaging followed by optical imaging and histology. RESULTS: Both imaging modalities demonstrated accumulation of the nanoparticles in the pancreas. However, histology revealed a high accumulation of nanoparticles in the insulin-producing cells in the pancreata of diabetic animals. By contrast, in nondiabetic controls, nanoparticles were mainly restricted to nonendocrine tissues. CONCLUSIONS: Our results demonstrate that pancreatic ductal injection accompanied by image guidance could serve as an alternative pathway for nanoparticle delivery. We expect to utilize this intraductal delivery method for theranostic applications in type 1 diabetes.

Predictive imaging of chemotherapeutic response in a transgenic mouse model of pancreatic cancer.

The underglycosylated mucin 1 tumor antigen (uMUC1) is a biomarker that forecasts the progression of adenocarcinomas. In this study, we evaluated the utility of a dual-modality molecular imaging approach based on targeting uMUC1 for monitoring chemotherapeutic response in a transgenic murine model of pancreatic cancer (KCM triple transgenic mice). An uMUC1-specific contrast agent (MN-EPPT) was synthesized for use with magnetic resonance imaging (MRI) and fluorescence optical imaging. It consisted of dextran-coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1-specific peptide (EPPT). KCM triple transgenic mice were given gemcitabine as chemotherapy while control animals received saline injections following the same schedule. Changes in uMUC1 levels following chemotherapy were monitored using T2-weighted MRI and optical imaging before and 24 hr after injection of the MN-EPPT. uMUC1 expression in tumors from both groups was evaluated by histology and qRT-PCR. We observed that the average delta-T2 in the gemcitabine-treated group was significantly reduced compared to the control group indicating lower accumulation of MN-EPPT, and correspondingly, a lower level of uMUC1 expression. In vivo optical imaging confirmed the MRI findings. Fluorescence microscopy of pancreatic tumor sections showed a lower level of uMUC1 expression in the gemcitabine-treated group compared to the control, which was confirmed by qRT-PCR. Our data proved that changes in uMUC1 expression after gemcitabine chemotherapy could be evaluated using MN-EPPT-enhanced in vivo MR and optical imaging. These results suggest that the uMUC1-targeted imaging approach could provide a useful tool for the predictive assessment of therapeutic response.

Detection of in vivo enzyme activity with CatalyCEST MRI.

PURPOSE: CatalyCEST MRI compares the detection of an enzyme-responsive chemical exchange saturation transfer (CEST) agent with the detection of an unresponsive "control" CEST agent that accounts for other conditions that influence CEST. The purpose of this study was to investigate the feasibility of in vivo catalyCEST MRI. METHODS: CEST agents that were responsive and unresponsive to the activity of urokinase plasminogen activator were shown to have negligible interaction with each other. A CEST-fast imaging with steady state precession (FISP) MRI protocol was used to acquire MR CEST spectroscopic images with a Capan-2 pancreatic tumor model after intravenous injection of the CEST agents. A function of (super)-Lorentzian line shapes was fit to CEST spectra of a region-of-interest that represented the tumor. RESULTS: The CEST effects from each agent showed the same initial uptake into tumor tissues, indicating that both agents had the same pharmacokinetic transport rates. Starting 5 min after injection, CEST from the enzyme-responsive agent disappeared more quickly than CEST from the unresponsive agent, indicating that the enzyme responsive agent was being catalyzed by urokinase plasminogen activator, while both agents also experienced net pharmacokinetic washout from the tumor. CONCLUSION: CatalyCEST MRI demonstrates that dynamic tracking of enzyme-responsive and unresponsive CEST agents during the same in vivo MRI study is feasible.

Detecting enzyme activities with exogenous MRI contrast agents.

This review focuses on exogenous magnetic resonance imaging (MRI) contrast agents that are responsive to enzyme activity. Enzymes can catalyze a change in water access, rotational tumbling time, the proximity of a (19)F-labeled ligand, the aggregation state, the proton chemical-exchange rate between the agent and water, or the chemical shift of (19)F, (31)P, (13)C or a labile (1)H of an agent, all of which can be used to detect enzyme activity. The variety of agents attests to the creativity in developing enzyme-responsive MRI contrast agents.

Targeted imaging of breast tumor progression and therapeutic response in a human uMUC-1 expressing transgenic mouse model.

The ability to monitor breast cancer initiation and progression on the molecular level would provide an effective tool for early diagnosis and therapy. In the present study, we focused on the underglycosylated MUC-1 tumor antigen (uMUC-1), which is directly linked to tumor progression from pre-malignancy to advanced malignancy in breast cancer and has been identified as the independent predictor of local recurrence and tumor response to chemotherapy. We investigated whether changes in uMUC-1 expression during tumor development and therapeutic intervention could be monitored non-invasively using molecular imaging approach with the uMUC-1-specific contrast agent (MN-EPPT) detectable by magnetic resonance and fluorescence optical imaging. This was done in mice that express human uMUC-1 tumor antigen (MMT mice) and develop spontaneous mammary carcinoma in a stage-wise fashion. After the injection of MN-EPPT there was a significant reduction in average T2 relaxation times of the mammary fat pad between pre-malignancy and cancer. In addition, T2 relaxation times were already altered at pre-malignant state in these mice compared to non-tumor bearing mice. This indicated that targeting uMUC-1 could be useful for detecting pre-malignant transformation in the mammary fat pad. We also probed changes in uMUC-1 expression with MN-EPPT during therapy with doxorubicin (Dox). We observed that tumor delta-T2s were significantly reduced by treatment with Dox indicating lower accumulation of MN-EPPT. This correlated with a lower level of MUC-1 expression in the Dox-treated tumors, as confirmed by immunoblotting. Our study could provide a very sensitive molecular imaging approach for monitoring tumor progression and therapeutic response.

Lanthanide-mediated dephosphorylation used for peptide cleavage during solid phase peptide synthesis.

Lanthanide(III) ions can accelerate the hydrolysis of phosphomonoesters and phosphodiesters in neutral aqueous solution. In this paper, lanthanide-mediated dephosphorylation has been applied in aqueous media as an orthogonal cleavage condition that can be employed in conventional solid phase peptide synthesis (SPPS). A phosphorylated polymeric support for SPPS was developed using Boc chemistry. The cleavage of resin-bound phosphates was investigated with the addition of Eu(III), Yb(III), acid or base, a mixture of solvents or different temperatures. To demonstrate the utility of this approach for SPPS, a peptide sequence was synthesized on a phosphorylated polymeric support and quantitatively cleaved with lanthanide ions in neutral aqueous media. The protecting groups for side chains were retained during peptide cleavage using lanthanide ions. This new methodology provides a mild orthogonal cleavage condition of phosphoester as a linker during SPPS.

Clinical Applications of Short Non-Coding RNA-Based Therapies in the Era of Precision Medicine.

Traditional targeted therapeutic agents have relied on small synthetic molecules or large proteins, such as monoclonal antibodies. These agents leave a lot of therapeutic targets undruggable because of the lack or inaccessibility of active sites and/or pockets in their three-dimensional structure that can be chemically engaged. RNA presents an attractive, transformative opportunity to reach any genetic target with therapeutic intent. RNA therapeutic design is amenable to modularity and tunability and is based on a computational blueprint presented by the genetic code. Here, we will focus on short non-coding RNAs (sncRNAs) as a promising therapeutic modality because of their potency and versatility. We review recent progress towards clinical application of small interfering RNAs (siRNAs) for single-target therapy and microRNA (miRNA) activity modulators for multi-target therapy. siRNAs derive their potency from the fact that the underlying RNA interference (RNAi) mechanism is catalytic and reliant on post-transcriptional mRNA degradation. Therapeutic siRNAs can be designed against virtually any mRNA sequence in the transcriptome and specifically target a disease-causing mRNA variant. Two main classes of microRNA activity modulators exist to increase (miRNA mimics) or decrease (anti-miRNA inhibitors) the function of a specific microRNA. Since a single microRNA regulates the expression of multiple target genes, a miRNA activity modulator can have a more profound effect on global gene expression and protein output than siRNAs do. Both types of sncRNA-based drugs have been investigated in clinical trials and some siRNAs have already been granted FDA approval for the treatment of genetic, cardiometabolic, and infectious diseases. Here, we detail clinical results using siRNA and miRNA therapeutics and present an outlook for the potential of these sncRNAs in medicine.

Case report: MicroRNA-10b as a therapeutic target in feline metastatic mammary carcinoma and its implications for human clinical trials.

Ninety percent of deaths from cancer are caused by metastasis. miRNAs are critical players in biological processes such as proliferation, metastasis, apoptosis, and self-renewal. We and others have previously demonstrated that miRNA-10b promotes metastatic cell migration and invasion. Importantly, we also showed that miR-10b is a critical driver of metastatic cell viability and proliferation. To treat established metastases by inhibiting miR-10b, we utilized a therapeutic, termed MN-anti-miR10b, composed of anti-miR-10b antagomirs, conjugated to iron oxide nanoparticles, that serve as delivery vehicles to tumor cells in vivo and a magnetic resonance imaging (MRI) reporter. In our previous studies using murine models of metastatic breast cancer, we demonstrated the effectiveness of MN-anti-miR10b in preventing and eliminating existing metastases. With an outlook toward clinical translation of our therapeutic, here we report studies in large animals (companion cats) with spontaneous feline mammary carcinoma (FMC). We first investigated the expression and tissue localization of miR-10b in feline tumors and metastases and showed remarkable similarity to these features in humans. Next, in the first case study involving this therapeutic we intravenously dosed an FMC patient with MN-anti-miR10b and demonstrated its delivery to the metastatic lesions using MRI. We also showed the initial safety profile of the therapeutic and demonstrated significant change in miR-10b expression and its target HOXD10 after dosing. Our results provide support for using companion animals for further MN-anti-miR10b development as a therapy and serve as a guide for future clinical trials in human patients.

MiRNA10b-directed nanotherapy effectively targets brain metastases from breast cancer.

RNA interference represents one of the most appealing therapeutic modalities for cancer because of its potency, versatility, and modularity. Because the mechanism is catalytic and affects the expression of disease-causing antigens at the post-transcriptional level, only small amounts of therapeutic need to be delivered to the target in order to exert a robust therapeutic effect. RNA interference is also advantageous over other treatment modalities, such as monoclonal antibodies or small molecules, because it has a much broader array of druggable targets. Finally, the complementarity of the genetic code gives us the opportunity to design RNAi therapeutics using computational, rational approaches. Previously, we developed and tested an RNAi-targeted therapeutic, termed MN-anti-miR10b, which was designed to inhibit the critical driver of metastasis and metastatic colonization, miRNA-10b. We showed in animal models of metastatic breast cancer that MN-anti-miR10b accumulated into tumors and metastases in the lymph nodes, lungs, and bone, following simple intravenous injection. We also found that treatment incorporating MN-anti-miR10b was effective at inhibiting the emergence of metastases and could regress already established metastases in the lymph nodes, lungs, and bone. In the present study, we extend the application of MN-anti-miR10b to a model of breast cancer metastatic to the brain. We demonstrate delivery to the metastatic lesions and obtain evidence of a therapeutic effect manifested as inhibition of metastatic progression. This investigation represents an additional step towards translating similar RNAi-targeted therapeutics for the systemic treatment of metastatic disease.

Radiolabeling and PET-MRI microdosing of the experimental cancer therapeutic, MN-anti-miR10b, demonstrates delivery to metastatic lesions in a murine model of metastatic breast cancer.

BACKGROUND: In our earlier work, we identified microRNA-10b (miR10b) as a master regulator of the viability of metastatic tumor cells. This knowledge allowed us to design a miR10b-targeted therapeutic consisting of anti-miR10b and ultrasmall iron oxide magnetic nanoparticles (MN), termed MN-anti-miR10b. In mouse models of breast cancer, we demonstrated that MN-anti-miR10b caused durable regressions of established metastases with no evidence of systemic toxicity. As a first step towards translating MN-anti-miR10b for the treatment of metastatic breast cancer, we needed to determine if MN-anti-miR10b, which is so effective in mice, will also accumulate in human metastases. RESULTS: In this study, we devised a method to efficiently radiolabel MN-anti-miR10b with Cu-64 (64Cu) and evaluated the pharmacokinetics and biodistribution of the radiolabeled product at two different doses: a therapeutic dose, referred to as macrodose, corresponding to 64Cu-MN-anti-miR10b co-injected with non-labeled MN-anti-miR10b, and a tracer level dose of 64Cu-MN-anti-miR10b, referred to as microdose. In addition, we evaluated the uptake of 64Cu-MN-anti-miR10b by metastatic lesions using both in vivo and ex vivo positron emission tomography-magnetic resonance imaging (PET-MRI). A comparable distribution of the therapeutic was observed after administration of a microdose or macrodose. Uptake of the therapeutic by metastatic lymph nodes, lungs, and bone was also demonstrated by PET-MRI with a significantly higher PET signal than in the same organs devoid of metastatic lesions. CONCLUSION: Our results demonstrate that PET-MRI following a microdose injection of the agent will accurately reflect the innate biodistribution of the therapeutic. The tools developed in the present study lay the groundwork for the clinical testing of MN-anti-miR10b and other similar therapeutics in patients with cancer.

Screening of potential miRNA therapeutics for the prevention of multi-drug resistance in cancer cells.

Chemotherapy, a major cancer treatment approach, suffers seriously from multidrug resistance (MDR), generally caused by innate DNA repair proteins that reverse the DNA modification by anti-cancer therapeutics or trans-membrane efflux proteins that pump anti-cancer therapeutics out of the cytosol. This project focused on finding microRNAs that can regulate MDR proteins by managing corresponding mRNA levels through post-transcriptional regulation based on nucleotide sequence matching. Screening was done with bioinformatics databases for unpublished/unexplored microRNAs with high nucleotide sequence correspondence to two representative MDR proteins, MGMT (a DNA repair protein) and ABCB1 (an efflux protein), revealing microRNA-4539 and microRNA-4261 respectively. To investigate the enhancement of chemotherapeutics in cancer cells, high MGMT expressing glioblastoma (T98G) and a high ABCB1 expressing triple-negative breast cancer cell line (MDA-MB-231-luc) were treated with varying concentrations of chemotherapeutics and corresponding miRNAs. Newly identified MDR-related miRNAs (MDRmiRs) enhanced the response to anti-cancer therapeutics and resulted in effective cell death. In this study, we demonstrated that therapeutic miRNAs could be identified based on the nucleotide sequence matching of miRNAs to targeted mRNA and the same approach could be employed for the screening of therapeutic candidates to regulate specific target proteins in diverse diseases.

PARACEST MRI with improved temporal resolution.

PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) is a novel contrast mechanism for MRI. A PARACEST MRI methodology with high temporal resolution is highly desired for in vivo MRI applications of molecular imaging. To address this need, a strategy has been developed that includes a long selective saturation period before each repetition of a Rapid Acquisition with Relaxation Enhancement (RARE) pulse sequence. This strategy is suitable for the application of PARACEST contrast agents to environments with long T1 relaxation times. An alternative strategy uses short selective saturation periods before the acquisition of each k-space trajectory to maintain steady state conditions, which can be implemented with a Fast Low Angle Shot (FLASH) pulse sequence. These short saturation periods lengthen the total scan time as compared to the first approach but compensate for the loss in PARACEST contrast related to T1 relaxation. Both approaches have been demonstrated in vitro and in vivo with significantly improved temporal resolutions as compared to a conventional gradient-echo PARACEST method without sacrificing CNR efficiency. These demonstrations also adopted a strategy for measuring the PARACEST effect that only requires selective saturation at a single MR frequency, which further improves temporal resolution for PARACEST detection.

Improvement in survival among HIV-infected individuals in the Republic of Korea: need for an early HIV diagnosis.

BACKGROUND: There is little information describing survival in HIV-infected patients after primary diagnosis in Korea, and changes in survival over time. This study investigated survival times, survival characteristics, and changes in survival after initial HIV diagnosis. Survival was characterized by evaluation of the immune status at primary HIV diagnosis nationwide. METHODS: A total of 5,323 HIV-infected individuals were registered with the government and followed until the end of 2007. Survival following HIV diagnosis was estimated based on epidemiological characteristics. We examined 3,369 individuals with available initial CD4+ T-cell counts within 6 months of HIV diagnosis to estimate survival based on immune status at diagnosis. The association between epidemiological variables and survival times was analyzed with univariate and multivariate Cox's proportional hazards model. RESULTS: Individuals died during the study period (n = 980), and 45% of the individuals died within 6 months of HIV diagnosis. The median survival following HIV diagnosis was 16.7 years. Survival were longer in women, in younger persons, in individuals diagnosed at blood centers, and in individuals diagnosed later in the study period. Survival were shortest in individuals with CD4+ T-cell counts <200 cells/mm3 at HIV diagnosis. These results suggest that early HIV diagnosis in Korea is imperative to increase survival and to promote the quality of life for HIV-infected individuals with governmental support. CONCLUSION: The median survival time of HIV-infected individuals following HIV diagnosis was 16.7 years in Korea. The survival was significantly lower in individuals with CD4+ T-cell counts <200 cells/mm3 at HIV diagnosis and higher by introduction of drugs and development of therapy.

An amine-derivatized, DOTA-loaded polymeric support for Fmoc Solid Phase Peptide Synthesis.

An amine-derivatized DOTA has been used to modify the surface of a polymeric support for conventional Solid Phase Peptide Synthesis (SPPS) following standard Fmoc chemistry methods. This methodology was used to synthesize a peptide-DOTA conjugate that was demonstrated to be a PARACEST MRI contrast agent. Therefore, this synthesis methodology can facilitate Fmoc SPPS of molecular imaging contrast agents.

MicroRNA-710 regulates multiple pathways of carcinogenesis in murine metastatic breast cancer.

Prior research has shown that critical differences between non-metastatic and metastatic tumor cells are at the level of microRNA. Consequently, harnessing these molecules for the treatment of metastatic cancer could have significant clinical impact. In the present study, we set out to identify metastasis-specific microRNAs which drive metastatic colonization of distant organs. Using a murine model of metastatic breast cancer, we employed a directed approach in which we screened for microRNAs that are differentially expressed between the primary tumors and metastatic lesions but concordantly expressed in all of the metastatic lesions irrespective of the tissue that is colonized. Of the identified targets, we focused on miR-710, which was consistently and significantly downregulated in the metastatic lesions relative to the primary tumors. The level of downregulation was independent of the distant organ that is involved, suggesting that miR-710 plays a fundamental role in metastatic colonization. Computational target prediction suggested a pleiotropic role for miR-710 in apoptosis, migration and invasion, and stemness. Using a previously validated oligonucleotide delivery system, we introduced miR-710 mimics into 4T1 metastatic breast adenocarcinoma cells and assessed the resultant phenotypic effects. We demonstrated significant inhibition of cell viability, migration, and invasion. We also showed that the treatment profoundly enhanced cell senescence, reduced stemness, and influenced markers of epithelial to mesenchymal transition, as evidenced by enhanced E-cadherin and reduced vimentin expression. This knowledge represents a first step towards harnessing a similar approach to discover novel microRNA targets with therapeutic potential in metastasis.

RNAi-Mediated PD-L1 Inhibition for Pancreatic Cancer Immunotherapy.

The recent past has seen impressive progress in the treatment of various malignancies using immunotherapy. One of the most promising approaches involves immune checkpoint inhibitors. However, the clinical results with these agents have demonstrated variability in the response. Pancreatic cancer, in particular, has proven resistant to initial immunotherapy approaches. Here, we describe an alternative strategy that relies on combining gemcitabine and a novel programmed death-ligand 1 (PD-L1) inhibitor, termed MN-siPDL1. MN-siPDL1 incorporates small interfering RNA against PD-L1 (siPDL1) conjugated to a magnetic nanocarrier (MN). We show that noninvasive magnetic resonance imaging (MRI) could be used to monitor therapeutic response. Combination therapy consisting of gemcitabine and MN-siPDL1 in a syngeneic murine pancreatic cancer model resulted in a significant reduction in tumor growth and an increase in survival. Following optimization, a 90% reduction in tumor volume was achieved 2 weeks after the beginning of treatment. Whereas 100% of the control animals had succumbed to their tumors by week 6 after the beginning of treatment, there was no mortality in the experimental group by week 5, and 67% of the experimental animals survived for 12 weeks. This method could provide therapeutic benefit against an intractable disease for which there are no effective treatments and which is characterized by a mere 1% 5-year survival.

An overview of responsive MRI contrast agents for molecular imaging.

This review focuses on MR contrast agents that are responsive to a change in physiological environment. The "response" mechanisms are dependent on 6 physicochemical phenomena, including the accessibility of water to the agent, rotational tumbling time, proton exchange rate, electron spin state, MR frequency, or local field inhomogenieties caused by the agent. These phenomena can be affected by the physiological environment, including changes in concentrations or activities of proteins, enzymes, nucleic acids, metabolites, oxygen and metal ions, and changes in pH and temperature. A total of 52 examples are presented, which demonstrate the variety and creativity of different approaches used to create responsive MRI contrast agents.

New Directions in the Study and Treatment of Metastatic Cancer.

Traditional cancer therapy has relied on a strictly cytotoxic approach that views non-metastatic and metastatic tumor cells as identical in terms of molecular biology and sensitivity to therapeutic intervention. Mounting evidence suggests that, in fact, non-metastatic and metastatic tumor cells differ in key characteristics that could explain the capacity of the metastatic cells to not only escape the primary organ but also to survive while in the circulation and to colonize a distant organ. Here, we lay out a framework for a new multi-pronged therapeutic approach. This approach involves modifying the local microenvironment of the primary tumor to inhibit the formation and release of metastatic cells; normalizing the microenvironment of the metastatic organ to limit the capacity of metastatic tumor cells to invade and colonize the organ; remediating the immune response to tumor neoantigens; and targeting metastatic tumor cells on a systemic level by restoring critical and unique aspects of the cell's phenotype, such as anchorage dependence. Given the limited progress against metastatic cancer using traditional therapeutic strategies, the outlined paradigm could provide a more rational alternative to patients with metastatic cancer.

Potent and selective effect of the mir-10b inhibitor MN-anti-mir10b in human cancer cells of diverse primary disease origin.

Since microRNAs (miRNAs, miRs) have been implicated in oncogenesis, many of them have been identified as therapeutic targets. Previously we have demonstrated that miRNA-10b acts as a master regulator of the viability of metastatic tumor cells and represents a target for therapeutic intervention. We designed and synthesized an inhibitor of miR-10b, termed MN-anti-miR10b. We showed that treatment with MN-anti-miR10b led to durable regression/elimination of established metastases in murine models of metastatic breast cancer. Since miRNA-10b has been associated with various metastatic and non-metastatic cancers, in the present study, we investigated the effect of MN-anti-miR10b in a panel of over 600 cell lines derived from a variety of human malignancies. We observed an effect on the viability of multiple cell lines within each cancer type and a mostly dichotomous response with cell lines either strongly responsive to MN-anti-miR10b or not at all even at maximum dose tested, suggesting a very high specificity of the effect. Genomic modeling of the drug response showed enrichment of genes associated with the proto-oncogene, c-Jun.

Guidelines for Rational Cancer Therapeutics.

Traditionally, cancer therapy has relied on surgery, radiation therapy, and chemotherapy. In recent years, these interventions have become increasingly replaced or complemented by more targeted approaches that are informed by a deeper understanding of the underlying biology. Still, the implementation of fully rational patient-specific drug design appears to be years away. Here, we present a vision of rational drug design for cancer that is defined by two major components: modularity and image guidance. We suggest that modularity can be achieved by combining a nanocarrier and an oligonucleotide component into the therapeutic. Image guidance can be incorporated into the nanocarrier component by labeling with a specific imaging reporter, such as a radionuclide or contrast agent for magnetic resonance imaging. While limited by the need for additional technological advancement in the areas of cancer biology, nanotechnology, and imaging, this vision for the future of cancer therapy can be used as a guide to future research endeavors.