Blocking the adhesion cascade at the premetastatic niche for prevention of breast cancer metastasis.

Shear-resistant adhesion and extravasation of disseminated cancer cells at the target organ is a crucial step in hematogenous metastasis. We found that the vascular adhesion molecule E-selectin preferentially promoted the shear-resistant adhesion and transendothelial migration of the estrogen receptor (ER)(-)/CD44(+) hormone-independent breast cancer cells, but not of the ER(+)/CD44(-/low) hormone-dependent breast cancer cells. Coincidentally, CD44(+) breast cancer cells were abundant in metastatic lung and brain lesions in ER(-) breast cancer, suggesting that E-selectin supports hematogenous metastasis of ER(-)/CD44(+) breast cancer. In an attempt to prevent hematogenous metastasis through the inhibition of a shear-resistant adhesion of CD44(+) cancer cells to E-selectin-expressing blood vessels on the premetastatic niche, an E-selectin targeted aptamer (ESTA) was developed. We demonstrated that a single intravenous injection of ESTA reduced metastases to a baseline level in both syngeneic and xenogeneic forced breast cancer metastasis models without relocating the site of metastasis. The effect of ESTA was absent in E-selectin knockout mice, suggesting that E-selectin is a molecular target of ESTA. Our data highlight the potential application of an E-selectin antagonist for the prevention of hematogenous metastasis of ER(-)/CD44(+) breast cancer.

Etchable plasmonic nanoparticle probes to image and quantify cellular internalization.

There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, as there is no methodology to turn particles 'off'. We have developed a simple technique to rapidly remove silver nanoparticles outside living cells, leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate-thiosulphate redox-based destain solution. In demonstration of the technique we present a class of multicoloured plasmonic nanoprobes comprising dye-labelled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice, and that show tumour uptake in vivo.

Safety evaluation of intravenously administered mono-thioated aptamer against E-selectin in mice.

The medical applications of aptamers have recently emerged. We developed an antagonistic thioaptamer (ESTA) against E-selectin. Previously, we showed that a single injection of ESTA at a dose of 100µg inhibits breast cancer metastasis in mice through the functional blockade of E-selectin. In the present study, we evaluated the safety of different doses of intravenously administered ESTA in single-dose acute and repeat-dose subacute studies in ICR mice. Our data indicated that intravenous administration of up to 500µg ESTA did not result in hematologic abnormality in either study. Additionally, intravenous injection of ESTA did not affect the levels of plasma cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, GM-CSF, IFN-γ, and TNF-α) or complement split products (C3a and C5a) in either study. However, repeated injections of ESTA slightly increased plasma ALT and AST activities, in accordance with the appearance of small necrotic areas in the liver. In conclusion, our data demonstrated that intravenous administration of ESTA does not cause overt hematologic, organs, and immunologic responses under the experimental conditions.

Nanomedicine in cancer therapy: innovative trends and prospects.

Cancer is a leading cause of morbidity and mortality worldwide, with recent advancements resulting in modest impacts on patient survival. Nanomedicine represents an innovative field with immense potential for improving cancer treatment, having ushered in several established drug delivery platforms. Nanoconstructs such as liposomes are widely used in clinics, while polymer micelles are in advanced phases of clinical trials in several countries. Currently, the field of nanomedicine is generating a new wave of nanoscale drug delivery strategies, embracing trends that involve the functionalization of these constructs with moieties that enhance site-specific delivery and tailored release. Herein, we discuss several advancements in established nanoparticle technologies such as liposomes, polymer micelles, and dendrimers regarding tumor targeting and controlled release strategies, which are being incorporated into their design with the hope of generating a more robust and efficacious nanotherapeutic modality. We also highlight a novel strategy known as multistage drug delivery; a rationally designed nanocarrier aimed at overcoming numerous biological barriers involved in drug delivery through the decoupling of various tasks that comprise the journey from the moment of systemic administration to arrival at the tumor site.

Immune-mediated ECM depletion improves tumour perfusion and payload delivery.

High extracellular matrix (ECM) content in solid cancers impairs tumour perfusion and thus access of imaging and therapeutic agents. We have devised a new approach to degrade tumour ECM, which improves uptake of circulating compounds. We target the immune-modulating cytokine, tumour necrosis factor alpha (TNFα), to tumours using a newly discovered peptide ligand referred to as CSG. This peptide binds to laminin-nidogen complexes in the ECM of mouse and human carcinomas with little or no peptide detected in normal tissues, and it selectively delivers a recombinant TNFα-CSG fusion protein to tumour ECM in tumour-bearing mice. Intravenously injected TNFα-CSG triggered robust immune cell infiltration in mouse tumours, particularly in the ECM-rich zones. The immune cell influx was accompanied by extensive ECM degradation, reduction in tumour stiffness, dilation of tumour blood vessels, improved perfusion and greater intratumoral uptake of the contrast agents gadoteridol and iron oxide nanoparticles. Suppressed tumour growth and prolonged survival of tumour-bearing mice were observed. These effects were attainable without the usually severe toxic side effects of TNFα.

Increased expression of tissue transglutaminase in pancreatic ductal adenocarcinoma and its implications in drug resistance and metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplastic diseases and is virtually incurable. The molecular mechanisms that contribute to the intrinsic resistance of PDAC to various anticancer therapies are not well understood. Recently, we have observed that several drug-resistant and metastatic tumors and tumor cell lines expressed elevated levels of tissue transglutaminase (TG2). Because PDAC exhibits inherent resistance to various drugs, we determined the constitutive expression of TG2 in 75 PDAC and 12 PDAC cell lines. Our results showed that 42 of 75 (56%) PDAC tumor samples expressed higher basal levels of TG2 compared with the normal pancreatic ducts [odds ratio (OR), 2.439; P = 0.012]. The increased expression of TG2 in PDAC was strongly associated with nodal metastasis (OR, 3.400; P = 0.017) and lymphovascular invasion (OR, 3.055; P = 0.045). Increased expression of TG2 was also evident in all 12 cell lines examined. The elevated expression of TG2 in PDAC cell lines was associated with gemcitabine resistance and increased invasive potential. Overexpression of catalytically active or inactive (C(277)S mutant) TG2 induced focal adhesion kinase (FAK) activation and augmented invasive functions in the BxPC-3 cell line. Conversely, down-regulation of TG2 by small interfering RNA attenuated FAK phosphorylation. Immunoprecipitation and confocal microscopy data revealed that TG2 was associated with FAK protein in PDAC cells. The activated FAK colocalized with TG2 at focal adhesion points. These results show for the first time that elevated expression of TG2 can induce constitutive activation of FAK and thus may contribute to the development of drug resistance and invasive phenotypes in PDAC.

Publisher Correction: Identification of a peptide recognizing cerebrovascular changes in mouse models of Alzheimer's disease.

The original version of the Supplementary Information associated with this Article inadvertently omitted Supplementary Table 1. The HTML has now been updated to include a corrected version of the Supplementary Information.

Antibiotic-loaded nanoparticles targeted to the site of infection enhance antibacterial efficacy.

Bacterial resistance to antibiotics has made it necessary to resort to antibiotics that have considerable toxicities. Here, we show that the cyclic 9-amino acid peptide CARGGLKSC (CARG), identified via phage display on Staphylococcus aureus (S. aureus) bacteria and through in vivo screening in mice with S. aureus-induced lung infections, increases the antibacterial activity of CARG-conjugated vancomycin-loaded nanoparticles in S. aureus-infected tissues and reduces the needed overall systemic dose, minimizing side effects. CARG binds specifically to S. aureus bacteria but not Pseudomonas bacteria in vitro, selectively accumulates in S. aureus-infected lungs and skin of mice but not in non-infected tissue and Pseudomonas-infected tissue, and significantly enhances the accumulation of intravenously injected vancomycin-loaded porous silicon nanoparticles bearing the peptide in S. aureus-infected mouse lung tissue. The targeted nanoparticles more effectively suppress staphylococcal infections in vivo relative to equivalent doses of untargeted vancomycin nanoparticles or of free vancomycin. The therapeutic delivery of antibiotic-carrying nanoparticles bearing peptides targeting infected tissue may help combat difficult-to-treat infections.

Vascular changes in tumors resistant to a vascular disrupting nanoparticle treatment.

Anti-angiogenic and vascular disrupting therapies rely on the dependence of tumors on new blood vessels to sustain tumor growth. We previously reported a potent vascular disrupting agent, a theranostic nanosystem consisting of a tumor vasculature-homing peptide (CGKRK) fused to a pro-apoptotic peptide [D(KLAKLAK)2] coated on iron oxide nanoparticles. This nanosystem showed promising therapeutic efficacy in glioblastoma (GBM) and breast cancer models. However, complete control of the tumors was not achieved, and some tumors became non-responsive to the treatment. Here we examined the non-responder phenomenon in an aggressive MCF10-CA1a breast tumor model. In the treatment-resistant tumors we noted the emergence of CD31-negative patent neovessels and a concomitant loss of tumor homing of the nanosystem. In vivo phage library screening in mice bearing non-responder tumors showed that compared to untreated and treatment-sensitive tumors, treatment sensitive tumors yield a distinct landscape of vascular homing peptides characterized by over-representation of peptides that target αv integrins. Our approach may be generally applicable to the development of targeted therapies for tumors that have failed treatment.

Identification of a peptide recognizing cerebrovascular changes in mouse models of Alzheimer's disease.

Cerebrovascular changes occur in Alzheimer's disease (AD). Using in vivo phage display, we searched for molecular markers of the neurovascular unit, including endothelial cells and astrocytes, in mouse models of AD. We identified a cyclic peptide, CDAGRKQKC (DAG), that accumulates in the hippocampus of hAPP-J20 mice at different ages. Intravenously injected DAG peptide homes to neurovascular unit endothelial cells and to reactive astrocytes in mouse models of AD. We identified connective tissue growth factor (CTGF), a matricellular protein that is highly expressed in the brain of individuals with AD and in mouse models, as the target of the DAG peptide. We also showed that exogenously delivered DAG homes to the brain in mouse models of glioblastoma, traumatic brain injury, and Parkinson's disease. DAG may potentially be used as a tool to enhance delivery of therapeutics and imaging agents to sites of vascular changes and astrogliosis in diseases associated with neuroinflammation.

Porous silicon-graphene oxide core-shell nanoparticles for targeted delivery of siRNA to the injured brain.

We report the synthesis, characterization, and assessment of a nanoparticle-based RNAi delivery platform that protects siRNA payloads against nuclease-induced degradation and efficiently delivers them to target cells. The nanocarrier is based on biodegradable mesoporous silicon nanoparticles (pSiNPs), where the voids of the nanoparticles are loaded with siRNA and the nanoparticles are encapsulated with graphene oxide nanosheets (GO-pSiNPs). The graphene oxide encapsulant delays release of the oligonucleotide payloads in vitro by a factor of 3. When conjugated to a targeting peptide derived from the rabies virus glycoprotein (RVG), the nanoparticles show 2-fold greater cellular uptake and gene silencing. Intravenous administration of the nanoparticles into brain-injured mice results in substantial accumulation specifically at the site of injury.

A peptide for targeted, systemic delivery of imaging and therapeutic compounds into acute brain injuries.

Traumatic brain injury (TBI) is a major health and socio-economic problem, but no pharmacological agent is currently approved for the treatment of acute TBI. Thus, there is a great need for advances in this field. Here, we describe a short peptide (sequence CAQK) identified by in vivo phage display screening in mice with acute brain injury. The CAQK peptide selectively binds to injured mouse and human brain, and systemically injected CAQK specifically homes to sites of brain injury in mouse models. The CAQK target is a proteoglycan complex upregulated in brain injuries. Coupling to CAQK increased injury site accumulation of systemically administered molecules ranging from a drug-sized molecule to nanoparticles. CAQK-coated nanoparticles containing silencing oligonucleotides provided the first evidence of gene silencing in injured brain parenchyma by systemically administered siRNA. These findings present an effective targeting strategy for the delivery of therapeutics in clinical management of acute brain injuries.

Thioaptamer conjugated liposomes for tumor vasculature targeting.

Recent developments in multi-functional nanoparticles offer a great potential for targeted delivery of therapeutic compounds and imaging contrast agents to specific cell types, in turn, enhancing therapeutic effect and minimizing side effects. Despite the promise, site specific delivery carriers have not been translated into clinical reality. In this study, we have developed long circulating liposomes with the outer surface decorated with thioated oligonucleotide aptamer (thioaptamer) against E-selectin (ESTA) and evaluated the targeting efficacy and PK parameters. In vitro targeting studies using Human Umbilical Cord Vein Endothelial Cell (HUVEC) demonstrated efficient and rapid uptake of the ESTA conjugated liposomes (ESTA-lip). In vivo, the intravenous administration of ESTA-lip resulted in their accumulation at the tumor vasculature of breast tumor xenografts without shortening the circulation half-life. The study presented here represents an exemplary use of thioaptamer and liposome and opens the door to testing various combinations of thioaptamer and nanocarriers that can be constructed to target multiple cancer types and tumor components for delivery of both therapeutics and imaging agent.

Identification of thioaptamer ligand against E-selectin: potential application for inflamed vasculature targeting.

Active targeting of a drug carrier to a specific target site is crucial to provide a safe and efficient delivery of therapeutics and imaging contrast agents. E-selectin expression is induced on the endothelial cell surface of vessels in response to inflammatory stimuli but is absent in the normal vessels. Thus, E-selectin is an attractive molecular target, and high affinity ligands for E-selectin could be powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. In this study, we identified a thiophosphate modified aptamer (thioaptamer, TA) against E-selectin (ESTA-1) by employing a two-step selection strategy: a recombinant protein-based TA binding selection from a combinatorial library followed by a cell-based TA binding selection using E-selectin expressing human microvascular endothelial cells. ESTA-1 selectively bound to E-selectin with nanomolar binding affinity (K(D) = 47 nM) while exhibiting minimal cross reactivity to P- and L-selectin. Furthermore, ESTA-1 binding to E-selectin on the endothelial cells markedly antagonized the adhesion (over 75% inhibition) of sLe(x) positive HL-60 cells at nanomolar concentration. ESTA-1 also bound specifically to the inflamed tumor-associated vasculature of human carcinomas derived from breast, ovarian, and skin but not to normal organs, and this binding was highly associated with the E-selectin expression level. Similarly, intravenously injected ESTA-1 demonstrated distinct binding to the tumor vasculature in a breast cancer xenograft model. Together, our data substantiates the discovery of a thioaptamer (ESTA-1) that binds to E-selectin with high affinity and specificity, thereby highlighting the potential application of ESTA-1 for E-selectin targeted delivery.

Sustained small interfering RNA delivery by mesoporous silicon particles.

RNA interference (RNAi) is a powerful approach for silencing genes associated with a variety of pathologic conditions; however, in vivo RNAi delivery has remained a major challenge due to lack of safe, efficient, and sustained systemic delivery. Here, we report on a novel approach to overcome these limitations using a multistage vector composed of mesoporous silicon particles (stage 1 microparticles, S1MP) loaded with neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC) containing small interfering RNA (siRNA) targeted against the EphA2 oncoprotein, which is overexpressed in most cancers, including ovarian. Our delivery methods resulted in sustained EphA2 gene silencing for at least 3 weeks in two independent orthotopic mouse models of ovarian cancer following a single i.v. administration of S1MP loaded with EphA2-siRNA-DOPC. Furthermore, a single administration of S1MP loaded with-EphA2-siRNA-DOPC substantially reduced tumor burden, angiogenesis, and cell proliferation compared with a noncoding control siRNA alone (SKOV3ip1, 54%; HeyA8, 57%), with no significant changes in serum chemistries or in proinflammatory cytokines. In summary, we have provided the first in vivo therapeutic validation of a novel, multistage siRNA delivery system for sustained gene silencing with broad applicability to pathologies beyond ovarian neoplasms.

Overexpression of tissue transglutaminase leads to constitutive activation of nuclear factor-kappaB in cancer cells: delineation of a novel pathway.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays an important role in regulating cell growth, apoptosis, and metastatic functions. Constitutive activation of NF-kappaB has been observed in various cancers; however, molecular mechanisms resulting in such activation remain elusive. Based on our previous results showing that drug-resistant and metastatic cancer cells have high levels of tissue transglutaminase (TG2) expression and that this expression can confer chemoresistance to certain types of cancer cells, we hypothesized that TG2 contributes to constitutive activation of NF-kappaB. Numerous lines of evidence showed that overexpression of TG2 is linked with constitutive activation of NF-kappaB. Tumor cells with overexpression of TG2 exhibited increased levels of constitutively active NF-kappaB. Activation of TG2 led to activation of NF-kappaB; conversely, inhibition of TG2 activity inhibited activation of NF-kappaB. Similarly, ectopic expression of TG2 caused activation of NF-kappaB, and inhibition of expression of TG2 by small interfering RNA abolished the activation of NF-kappaB. Our results further indicated that constitutive NF-kappaB reporter activity in pancreatic cancer cells is not affected by dominant-negative I kappaB alpha. Additionally, coimmunoprecipitation and confocal microscopy showed that I kappaB alpha is physically associated with TG2. Lastly, immunohistochemical analysis of pancreatic ductal carcinoma samples obtained from 61 patients further supported a strong correlation between TG2 expression and NF-kappaB activation/overexpression (P = 0.0098, Fisher's exact test). We conclude that TG2 induces constitutive activation of NF-kappaB in tumor cells via a novel pathway that is most likely independent of I kappaB alpha kinase. Therefore, TG2 may be an attractive alternate target for inhibiting constitutive NF-kappaB activation and rendering cancer cells sensitive to anticancer therapies.