Non-targeted Counterparts Research Articles

LHRH-peptide conjugated dextran nanoparticles for targeted delivery of cisplatin to breast cancer.

Cisplatin is one of the most common anticancer agents for treating different kinds of solid tumors today. However, its broader therapeutic applications are limited by the severe side effects and nonspecific biodistribution. In this study, luteinizing hormone-releasing hormone (LHRH)-targeted polysaccharide nanoparticles for tumor-targeted delivery and controlled release of cisplatin were developed. This nanoparticle delivery system possessed the following unique properties: (1) as the degradation products of the carrier, both dextran and succinic acid have been proved by the United States Food and Drug Administration for parenteral use, indicating good safety and great application potential; (2) both the drug loading and LHRH conjugation procedures were carried out with efficiency in aqueous medium without the use of organic solvents, thus representing a green chemistry approach; and (3) the design followed the principle of drug encapsulation first and subsequent targeting ligand modification, guaranteeing that the targeting molecules were conjugated on the surface of nanoparticles. As compared to free cisplatin, both the non-targeted and targeted nanoparticles displayed sustained drug release, prolonged blood circulation and reduced systemic toxicity. Foremost, the LHRH-targeted nanoparticles led to significant higher cellular internalization in MCF-7 tumor cells in vitro and enhanced accumulation in MCF-7 xenograft tumors in vivo, compared with the non-targeted counterparts. Systemic delivery of the targeted nanoparticles carrying cisplatin via intravenous injection showed enhanced tumor suppression in MCF-7 tumor bearing mice compared to the non-targeted nanoparticles and free CDDP. Collectively, the LHRH-mediated polysaccharide nanoparticles appeared to be a promising nanomedicine drug delivery system for tumor-targeted delivery of cisplatin.

An NIR-responsive and sugar-targeted polypeptide composite nanomedicine for intracellular cancer therapy.

The upconversion nanoparticle (UCNP)-loaded polypeptide composite nanoparticles that present fast NIR-sensitivity and tunable sugar-targeting properties are fabricated, opening a new avenue for on-demand and targeted cancer therapy. The half maximal inhibitory concentration (IC50) of the nanoparticles dropped 4.7-fold or 3.1-fold compared to non-targeted or non-irradiated counterparts.

Convection-Enhanced Delivery of Targeted Quantum Dot–Immunoliposome Hybrid Nanoparticles to Intracranial Brain Tumor Models

The aim of this work is to evaluate combining targeting strategy and convection-enhanced delivery in brain tumor models by imaging quantum dot-immunoliposome hybrid nanoparticles. An EGF receptor-targeted, quantum dot-immunoliposome hybrid nanoparticle (QD-IL) was synthesized. In vitro uptake was measured by flow cytometry and intracellular localization was imaged by confocal microscopy. In the in vivo study, QD-ILs were delivered to intracranial xenografts via convection-enhanced delivery and fluorescence was monitored noninvasively in real-time. QD-ILs exhibited specific and efficient uptake in vitro and exhibited approximately 1.3- to 5.0-fold higher total fluorescence compared with nontargeted counterpart in intracranial brain tumor xenografts in vivo. QD-ILs serve as an effective imaging agent in vitro and in vivo, and the data suggest that ligand-directed liposomal nanoparticles in conjunction with convection-enhanced delivery may offer therapeutic benefits for glioblastoma treatment as a result of specific and efficient uptake by malignant cells.

Augmented Anticancer Activity of a Targeted, Intracellularly Activatable, Theranostic Nanomedicine Based on Fluorescent and Radiolabeled, Methotrexate-Folic Acid-Multiwalled Carbon Nanotube Conjugate

The present study reports the design, synthesis, and biological evaluation of a novel, intravenously injectable, theranostic prodrug based on multiwalled carbon nanotubes (MWCNTs) concomitantly decorated with a fluorochrome (Alexa-fluor, AF488/647), radionucleide (Technitium-99m), tumor-targeting module (folic acid, FA), and anticancer agent (methotrexate, MTX). Specifically, MTX was conjugated to MWCNTs via a serum-stable yet intracellularly hydrolyzable ester linkage to ensure minimum drug loss in circulation. Cell uptake studies corroborated the selective internalization of AF-FA-MTX-MWCNTs (1) by folate receptor (FR) positive human lung (A549) and breast (MCF 7) cancer cells through FR mediated endocytosis. Lysosomal trafficking of 1 enabled the conjugate to exert higher anticancer activity as compared to its nontargeted counterpart that was mainly restricted to cytoplasm. Tumor-specific accumulation of 1 in Ehlrich Ascites Tumor (EAT) xenografted mice was almost 19 and 8.6 times higher than free MTX and FA-deprived MWCNTs. Subsequently, the conjugate 1 was shown to arrest tumor growth more effectively in chemically breast tumor induced rats, when compared to either free MTX or nontargeted controls. Interestingly, the anticancer activities of the ester-linked CNT-MTX conjugates (including the one deprived of FA) were significantly higher than their amide-linked counterpart, suggesting that cleavability of linkers between drug and multifunctional nanotubes critically influence their therapeutic performance. The results were also supported by in silico docking and ligand similarity analysis. Toxicity studies in mice confirmed that all CNT-MTX conjugates were devoid of any perceivable hepatotoxicity, cardiotoxicity, and nephrotoxicity. Overall, the delivery property of MWCNTs, high tumor binding avidity of FA, optical detectability of AF fluorochromes, and radio-traceability of (99m)Tc could be successfully integrated and partitioned on a single CNT-platform to augment the therapeutic efficacy of MTX against FR overexpressing cancer cells while allowing a real-time monitoring of treatment response through multimodal imaging.

Abstract 4521: A novel targeted triggered release nanoparticle against cancer cells of diverse histological origin.

Abstract The paradigm of cancer treatment has changed substantially over the last decade. The discovery of new therapeutic targets within the tumor cell and the recognition of the interdependence with the tumor microenvironment have been the basis of such changes. However, no common ground was yet found for the treatment of cancer within its diverse histological origin. Conventional therapy against human canc¬er, relying on the use of drugs that do not differentiate between cancer and normal cells, is progressively giving room to novel therapeutic strategies. Specificity became mandatory for treatment effectiveness and target selection one of the main priorities. In this scenario, delivery systems rise as promising approaches, for the reason they can improve pharmacokinetic features of anticancer drugs and efficiently address a particular payload to a selected target, namely through exploitation of high specificity and affinity between cell-surface receptors and targeting ligands coupled at their surface. We evidence the overexpression of a target cell surface receptor (nucleolin) in several cancer cell lines of diverse histological origins: breast (hormone-dependent and triple negative subtypes), colon, prostate and small cell and non-small cell lung cancer. We demonstrate the ability of the nucleolin receptor to specifically internalize a F3-targeted sterically stabilized lipid-based formulation. Moreover, we show that the unique combination of targeting specificity and intracellular triggered release of a therapeutic agent, doxorubicin, results in enhanced cytotoxic activity in those specific cell lines, when compared to the non-targeted counterpart. Active accumulation of the targeted nanoparticle into human orthotopic tumors, implanted in the mammary fat pad of nude mice, was registered for a time-point as short as 4 h, reaching 48% of the injected dose/g of tissue. Twenty-four hours post-injection accumulation in the tumor tissue was 33-fold higher than the non-targeted counterpart. In mice treated with the targeted nanoparticle containing doxorubicin, significant decrease of the tumor viable rim area and microvascular density, as well as limited invasion to surrounding healthy tissues were observed (as opposed to other tested controls). Moreover, the clinical potential of such strategy was demonstrated by the successful specific cellular association by breast cancer cells harvested from tumors of patients submitted to mastectomy. Overall, we provide evidence of a nanoparticle with specificity to cancer cells of diverse histological origins and proven therapeutic efficacy in an animal model of breast cancer, which may be a breakthrough in the development of anticancer therapies, especially when considering cancer cell heterogeneity. Citation Format: Vera Moura, Manuela Lacerda, Paulo Figueiredo, Maria L. Corvo, Maria M E Cruz, Raquel Soares, Sérgio Simões, João Moreira. A novel targeted triggered release nanoparticle against cancer cells of diverse histological origin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4521. doi:10.1158/1538-7445.AM2013-4521

Policy resistance undermines superspreader vaccination strategies for influenza.

Theoretical models of infection spread on networks predict that targeting vaccination at individuals with a very large number of contacts (superspreaders) can reduce infection incidence by a significant margin. These models generally assume that superspreaders will always agree to be vaccinated. Hence, they cannot capture unintended consequences such as policy resistance, where the behavioral response induced by a new vaccine policy tends to reduce the expected benefits of the policy. Here, we couple a model of influenza transmission on an empirically-based contact network with a psychologically structured model of influenza vaccinating behavior, where individual vaccinating decisions depend on social learning and past experiences of perceived infections, vaccine complications and vaccine failures. We find that policy resistance almost completely undermines the effectiveness of superspreader strategies: the most commonly explored approaches that target a randomly chosen neighbor of an individual, or that preferentially choose neighbors with many contacts, provide at best a relative improvement over their non-targeted counterpart as compared to when behavioral feedbacks are ignored. Increased vaccine coverage in super spreaders is offset by decreased coverage in non-superspreaders, and superspreaders also have a higher rate of perceived vaccine failures on account of being infected more often. Including incentives for vaccination provides modest improvements in outcomes. We conclude that the design of influenza vaccine strategies involving widespread incentive use and/or targeting of superspreaders should account for policy resistance, and mitigate it whenever possible.

Toward a siRNA-containing nanoparticle targeted to breast cancer cells and the tumor microenvironment

The present work aimed at designing a lipid-based nanocarrier for siRNA delivery toward two cell sub-populations within breast tumors, the cancer and the endothelial cells from angiogenic tumor blood vessels. To achieve such goal, the F3 peptide, which is specifically internalized by nucleolin overexpressed on both those sub-populations, was used as a targeting moiety.The developed F3-targeted stable nucleic acid lipid particles presented adequate features for systemic administration. In addition, the attachment of the F3 peptide onto the liposomal surface enabled an internalization by both cancer and endothelial cells from angiogenic blood vessels that was significantly higher than the one observed with non-cancer cells. Sequence-specific downregulation of enhanced green fluorescent protein (eGFP) in eGFP-overexpressing human cancer cell lines, both at the protein and mRNA levels, was further observed upon delivery of anti-eGFP siRNA by F3-targeted liposomes, in contrast with the non-targeted counterpart. This effect was highly dependent on the content of poly(ethylene glycol) (PEG), as evidenced by the co-localization studies between the siRNA and the lysosomes.Overall, the present work represents an important contribution toward a nanoparticle with multi-targeting capabilities in breast cancer, both at the cellular and molecular level.

Preparation, Characterization of 2-Deoxy-D-Glucose Functionalized Dimercaptosuccinic Acid-Coated Maghemite Nanoparticles for Targeting Tumor Cells

To report a modified preparation and to systematically study the structure, magnetic and other properties of γ-Fe(2)O(3)-DMSA-DG NPs (2-deoxy-D-glucose (2-DG) conjugated meso-2,3-dimercaptosuccinic acid coated γ-Fe(2)O(3) nanoparticles) and test its ability to improve Hela tumor cells targeting in vitro compared to the γ-Fe(2)O(3)-DMSA NPs. The conjugation of 2-DG on the surface of γ-Fe(2)O(3)-DMSA NPs was performed by esterification reaction and characterized. Acute toxicity was evaluated using MTT assay. Cellular uptake was investigated by Prussian blue staining and UV colorimetric assay. DG was successfully functionalized onto the surface of γ-Fe(2)O(3)-DMSA NPs; binding efficiency was ~60%. The mean diameter of single core of γ-Fe(2)O(3)-DMSA-DG NPs was 10nm. Particle size and polydispersity index of its aggregates were 156.2nm and 0.162, respectively. 2-DG-conjugated nanoparticles caused little cytotoxic effects on Hela cells at the concentration range of 0-600μg/mL. When 2-DG-conjuated and non-conjugated nanoparticles were incubated with Hela cells for 4, 8 and 12h, the 2-DG-conjugated nanoparticle showed significant amount of uptake in cells compared to their non-targeted counterparts. γ-Fe(2)O(3)-DMSA-DG NPs could be developed as a tumor-targeted probe for cervical cancer imaging and therapy.

Abstract C233: Limiting tumor invasion with multifunctional nanoparticle targeting the tumor microenvironment.

Abstract Combining multi-targeted strategies with in vivo stable drug retention and increased intracellular delivery is a promising strategy to treat solid tumors. We have evaluated the therapeutic impact of targeting two different populations within the tumor microenvironment (endothelial and cancer cells), using the same F3 peptide-targeted sterically-stabilized pH-sensitive liposome containing doxorubicin. We have demonstrated that it targets the nucleolin receptor on a cell- and ligand-specific manner and has the ability to promote intracellular and triggered drug release into breast cancer cells (hormone-dependent and triple negative subtypes). We have further evidenced that the overexpression of nucleolin in several cancer cell lines of diverse histological origins. We have demonstrated that the unique combination of targeting specificity and intracellular triggered release of a therapeutic agent, doxorubicin, has resulted in enhanced cytotoxic activity in those specific cell lines, when compared to the non-targeted counterpart. Active tumor accumulation of the radioactive lipid and drug tracers of targeted pH-sensitive liposomes, performed on Balb/c nude female mice bearing breast tumors implanted orthotopically, was registered for a time-point as short as 4 h and reaching 48% of the injected dose/g of tumor. In compliance to these data was the doxorubicin tumor-to-heart ratio of 80.8 for F3-targeted pH-sensitive liposomes vs 3.3 and 12.2 for the non-targeted and targeted non-pH-sensitive liposomes, respectively, 24 h post-injection. These data suggested a decrease on the potential dose-cumulative toxicity to the heart, namely relative to the non-targeted non-pH-sensitive counterpart. In mice treated with the F3-targeted pH-sensitive nanoparticle, significant decrease of the viable rim area (50%, accompanied by a strong necrosis) and microvascular density (80%) was observed. Moreover, suppression of invasion to surrounding healthy tissues was depicted, which may increase the probability of tumors falling in the category of “negative margins”, with reduced risk of relapse. The clinical potential of such strategy was reinforced by the successful ex-vivo targeting studies, where specific cellular association was observed in tumor cells harvested from tumors of patients submitted to mastectomy and overexpressing the nucleolin receptor, independent of age, proliferation index, oestrogen, progesterone and HER2 receptor expression. Overall, this work has demonstrated that the combination of tumor and vascular targeting with intracellular triggered release of the encapsulated drug is crucial for the successful therapeutic outcome of a nanotechnology-based strategy against a solid tumor, and should be considered for clinical evaluation. In addition, we have provided evidence of specific targeting to tumor cells of diverse histological origins with the same nanotechnology-based platform, which may be a breakthrough in the development of anticancer therapies, especially when considering tumor cell heterogeneity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C233.

Abstract 4444: Targeted delivery of siRNAs to tumor cells and the tumor microenvironment

Abstract The limited effectiveness of conventional treatment strategies has generated considerable interest on the development of new types of anticancer agents, with improved molecular target specificity. In this context, gene silencing technology constitutes such new class of anticancer agents. The identification of activated oncogenes, as fundamental genetic differences relative to normal cells has made it possible to consider such genes as targets for antitumor therapy. A tumor is an organ, not a single cell type, which encompasses multiple cell types, each one contributing to the overall tumor aggressiveness. It is now recognized that novel therapeutic strategies should envisage the simultaneous inhibition of multiple targets or pathways, at different cell levels within a tumor, that regulate processes promoting tumor development. The main goal of this work was to design a novel ligand-mediated targeted lipid-based nanocarrier, containing a nucleic acid (siRNA), aiming at targeting, simultaneously, human breast cancer and endothelial cells from angiogenic vessels. We have been able to develop a novel ligand-targeted sterically stabilized lipid-based nanoparticle with adequate features for systemic administration: it is characterized by high siRNA encapsulation efficiency, efficient protection of siRNA, average size around 200 nm, and charge close to neutrality. Our results have shown that the covalent attachment of a specific ligand at the extremity of poly(ethylene glycol) chains, brings a major advantage as it significantly improves the internalization of the lipid-based nanoparticle by both human cancer cells (MDA-MB-435 and MDA-MB-231) and endothelial cells from angiogenic blood vessels (HMEC-1). Moreover, it was not observed a significant internalization by the non-transformed cell line, BJ fibroblasts, indicating the cellular specificity of the developed targeted nanoparticle. In order to evaluate the potential of the developed targeted nanoparticle to silence a target gene, the green fluorescent protein (GFP)-overexpressing MDA-MB-435 cells were used. A specific downregulation of GFP, both at the protein and mRNA levels, was further observed with the targeted nanoparticle when compared with the non-targeted counterpart. As the developed nanoparticle is adequate for the encapsulation and delivery of any siRNA sequence, studies with a siRNA targeting a validated molecular target are now ongoing. 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 4444. doi:10.1158/1538-7445.AM2011-4444

Folate receptor targeted, carboxymethyl chitosan functionalized iron oxide nanoparticles: a novel ultradispersed nanoconjugates for bimodal imaging

This article delineates the design and synthesis of a novel, bio-functionalized, magneto-fluorescent multifunctional nanoparticles suitable for cancer-specific targeting, detection and imaging. Biocompatible, hydrophilic, magneto-fluorescent nanoparticles with surface-pendant amine, carboxyl and aldehyde groups were designed using o-carboxymethyl chitosan (OCMC). The free amine groups of OCMC stabilized magnetite nanoparticles on the surface allow for the covalent attachment of a fluorescent dye such as rhodamine isothiocyanate (RITC) with the aim to develop a magneto-fluorescent nanoprobe for optical imaging. In order to impart specific cancer cell targeting properties, folic acid and its aminated derivative was conjugated onto these magneto-fluorescent nanoparticles using different pendant groups (-NH(2), -COOH, -CHO). These newly synthesized iron-oxide folate nanoconjugates (FA-RITC-OCMC-SPIONs) showed excellent dispersibility, biocompatibility and good hydrodynamic sizes under physiological conditions which were extensively studied by a variety of complementary techniques. The cellular internalization efficacy of these folate-targeted and its non-targeted counterparts were studied using a folate-overexpressed (HeLa) and a normal (L929 fibroblast) cells by fluorescence microscopy and magnetically activated cell sorting (MACS). Cell-uptake behaviors of nanoparticles clearly demonstrate that cancer cells over-expressing the human folate receptor internalized a higher level of these nanoparticle-folate conjugates than normal cells. These folate targeted nanoparticles possess specific magnetic properties in the presence of an external magnetic field and the potential of these nanoconjugates as T(2)-weighted negative contrast MR imaging agent were evaluated in folate-overexpressed HeLa and normal L929 fibroblast cells.

Polyethylenimine-grafted copolymer of poly( l-lysine) and poly(ethylene glycol) for gene delivery

A major challenge in gene therapy is the development of effective gene delivery vectors with low toxicity. In the present study, linear poly(ethylenimine) (lPEI) with low molecular weight was grafted onto the block copolymer (PPL) of poly( l-lysine) (PLL) and poly(ethylene glycol)(PEG), yielding a ternary copolymer PEG- b-PLL- g-lPEI (PPI) for gene delivery. In such molecular design, PLL, lPEI and PEG blocks were expected to render the vector biodegradability, proton buffering capacity, low cationic toxicity and potentially long circulation in vivo, respectively. Given proper control of molecular composition, the copolymers demonstrated lower cytotoxicity, proton buffering capacity, ability to condense pDNA and mediate effective gene transfection in various cell lines. With folate as an exemplary targeting ligand, the FA-PPI/pDNA complex showed much higher transgene activity than its nontargeting counterpart for both reporter and therapeutic genes in folate receptor(FR)-positive cells. FA-PPI mediated effective transfection of the TNF-related apoptosis-inducing ligand gene (TRAIL) in human hepatoma Bel 7402 cells, leading to cell apoptosis and great suppression of cell viability. Our results indicate that the copolymers might be a promising vector combining low cytotoxicity, biodegradability, and high gene transfection efficiency.

PECAM‐targeted delivery of SOD inhibits endothelial inflammatory response

Elevated generation of reactive oxygen species (ROS) by endothelial enzymes, including NADPH-oxidase, is implicated in vascular oxidative stress and endothelial proinflammatory activation involving exposure of vascular cell adhesion molecule-1 (VCAM-1). Catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet/endothelial cell adhesion molecule 1 (PECAM-1) bind specifically to endothelium and inhibit effects of corresponding ROS, H(2)O(2), and superoxide anion. In this study, anti-PECAM/SOD, but not anti-PECAM/catalase or nontargeted enzymes, including polyethylene glycol (PEG)-SOD, inhibited 2- to 3-fold VCAM expression caused by tumor necrosis factor (TNF), interleukin-1β, and lipopolysaccharide. Anti- PECAM/SOD, but not nontargeted counterparts, accumulated in vascular endothelium after intravenous injection, localized in endothelial endosomes, and inhibited by 70% lipopolysaccharide-caused VCAM-1 expression in mice. Anti-PECAM/SOD colocalized with EEA-1-positive endothelial vesicles and quenched ROS produced in response to TNF. Inhibitors of NADPH oxidase and anion channel ClC3 blocked TNF-induced VCAM expression, affirming that superoxide produced and transported by these proteins, respectively, mediates inflammatory signaling. Anti-PECAM/SOD abolished VCAM expression caused by poly(I:C)-induced activation of toll-like receptor 3 localized in intracellular vesicles. These results directly implicate endosomal influx of superoxide in endothelial inflammatory response and suggest that site-specific interception of this signal attained by targeted delivery of anti-PECAM/SOD into endothelial endosomes may have anti-inflammatory effects.

Targeting non-viral vectors to tumor cells and the tumor microenvironment

Targeted therapy towards tumor cells has been regarded as a promising strategy, since it offers the possibility of directing and concentrating the therapeutic agent only at the desired target site, increasing therapeutic efficacy through increased tumor cell death and decreased incidence of side effects in healthy tissues. Nonetheless, choosing only one cellular target within the tumor microenvironment is an approach with low probabilities to succeed. In this respect, we aim at designing a nonviral vector that targets, simultaneously, tumor cells and endothelial cells from tumor blood vessels. It is hypothesized that such nanoparticle, with this dual targeting ability, will present an active tumor accumulation. Specific intracellular release of the encapsulated payload will strongly contribute to improve the therapeutic outcome. Our results show a substantial increase in the levels of association for ligand-targeted nanoparticles, on a ligandand cell-specific manner, both in vitro and ex vivo (in breast tumor cells removed from patients, after mastectomy or tumorectomy). Cytotoxicity studies show that targeted nanoparticles containing doxorubicin are more cytotoxic than the non-targeted formulation (4 to 180-fold), against tumor and endothelial cells, indicating that internalization of the former is contributing to a more efficient delivery of the payload to the target cells. Experiments with tumor-bearing mice report a higher tumor accumulation of the radiolabeled targeted nanoparticle over the non-targeted counterpart. Overall, these results represent a novel and a valuable contribution for delivery strategies targeting tumor cells and the tumor microenvironment. Author details Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal. Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal. Portuguese Institute of Oncology Francisco Gentil, Coimbra, Portugal. Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal. Department of Life Sciences, University of Coimbra, Coimbra, Portugal.

Targeted gene silencing for cancer treatment

Cancer arises in the twenty-first century as one of the leading causes for mortality in the western civilization. In the last decades, several genes were identified as important players in the transformation of a normal cell into a tumor cell. Therefore, modulation of those genes is a promising strategy for cancer treatment. Gene downregulation can be mediated by small-interfering RNA (siRNA), 21-23 nucleotides long double strand of RNA, which has the potential to inhibit the expression of a target gene through specific cleavage of perfectly complementary mRNA. However, the clinical use of these molecules has been impaired by their unfavourable pharmacokinetics profile and low intracellular accumulation. In order to address this issue, we have developed a novel targeted sterically stabilized lipid-based nanoparticle characterized by high siRNA encapsulation efficiency, efficient protection of siRNA, average size around 200 nm, and charge close to neutrality. Overall, these are nanoparticles that present adequate features for systemic administration. Our results have shown that the targeted nanoparticles were specifically internalized by human cancer cells (MDA-MB-435 and MDA-MB-231) and endothelial cells (HMEC-1). In experiments performed with green fluorescent protein (GFP)-overexpressing human cancer cell lines, specific downregulation of GFP, both at the protein and mRNA levels, was further observed with the targeted nanoparticle but not with the non-targeted counterpart. As the developed nanoparticle is adequate for the encapsulation and delivery of any siRNA sequence, studies with a siRNA against a therapeutic molecular target are now ongoing.

Targeting EGFR-overexpressing tumor cells using Cetuximab-immunomicelles loaded with doxorubicin and superparamagnetic iron oxide

Epidermal growth factor receptor (EGFR), a cellular transmembrane receptor, plays a key role in cell proliferation and is linked to a poor prognosis in various human cancers. In this study, we constructed Cetuximab-immunomicelles in which the anti-EGFR monoclonal antibody was linked to poly(ethylene glycol)-block-poly(ɛ-caprolactone) (PEG-PCL) nanomicelles that were loaded with doxorubicin (DOX) and superparamagnetic iron oxide (SPIO). The specific interactions between EGFR-overexpressing tumor cells (A431) and immunomicelles were observed using confocal laser scanning microscopy (CLSM) and flow cytometry. Furthermore, the capacity of transporting SPIO into tumor cells using these immunomicelles was evaluated with a 1.5 T clinical magnetic resonance imaging (MRI) scanner. It was found that the acquired MRI T2 signal intensity of A431 cells that were treated with the SPIO-loaded and antibody-functionalized micelles decreased significantly. Using the thiazolyl blue tetrazolium bromide (MTT) assay, we also demonstrated that the immunomicelles inhibited cell proliferation more effectively than their nontargeting counterparts. Our results suggest that Cetuximab-immunomicelles are a useful delivery vehicle for DOX and SPIO to EGFR-overexpressing tumor cells in vitro and that Cetuximab-immunomicelles can serve as a MRI-visible and targeted drug delivery agent for better tumor imaging and therapy.

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

Importance of the field: Targeted liposomal drugs represent the next evolution of liposomal drug delivery in cancer treatment. In various preclinical cancer models, antibody-targeted PEGylated liposomal drugs have demonstrated superior therapeutic effects over their non-targeted counterparts. Single chain Fv (scFv) has gained popularity in recent years as the targeting agent of choice over traditional targeting agents such as monoclonal antibodies (mAb) and antibody fragments (e.g., Fab′).Areas covered in this review: This review is focused mainly on advances in scFv-targeted liposomal drug delivery for the treatment of cancers, based on a survey of the recent literature, and on experiments done in a murine model of human B-lymphoma, using anti-CD19 targeted liposomes targeted with whole mAb, Fab′ fragments and scFv fragments.What the reader will gain: This review examines the recent advances in PEGylated immunoliposomal drug delivery, focusing on scFv fragments as targeting agents, in comparison with Fab′ and mAb.Take home message: For clinical development, scFv are potentially preferred targeting agents for PEGylated liposomes over mAb and Fab′, owing to factors such as decreased immunogenicity, and pharmacokinetics/biodistribution profiles that are similar to non-targeted PEGylated (Stealth®) liposomes.

Targeting of a Mutant Plasminogen Activator to Circulating Red Blood Cells for Prophylactic Fibrinolysis

Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl(3) models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.

Experimental Study on Targeted Methotrexate Delivery to the Rabbit Brain via Magnetic Resonance Imaging-Guided Focused Ultrasound

The purpose of this study was to investigate the effects of targeted and reversible disruption of the blood-brain barrier (BBB) by magnetic resonance imaging (MRI)-guided focused ultrasound (FUS) and delivery of methotrexate (MTX) to the rabbit brain. The brains of 20 rabbits were sonicated by MRI-guided FUS at different exposure times, and then Evans blue extravasation, contrast-enhanced MRI, and histologic examination were performed to determine the optimal exposure time for reversible BBB disruption with minimal damage. Five rabbits were sonicated at the optimal exposure time after MTX was injected intravenously (IV); the targeted locations were included in the sonicated group, and the nontargeted contralateral counterparts were included in the IV control group. Five other rabbits were not subjected to sonication and were administered internal carotid artery (ICA) injections of MTX; the specimens of the counterpart brain tissue were harvested as the ICA group. The MTX concentration in all of the specimens was determined by high-performance liquid chromatography. The MTX concentration in the sonicated group (mean +/- SD, 7.412 +/- 1.471 microg/g of tissue) was notably higher than that in both the IV control group (0.544 +/- 0.084 microg/g) and ICA group (1.984 +/- 0.65 microg/g; P <.01). Magnetic resonance imaging-guided FUS can disrupt the BBB reversibly and deliver IV administered MTX to targeted brain locations; it brings about a greater than 10-fold increase in the drug level and is much more effective (approximately 3.7-fold) than drug delivery through the ICA without sonication. This may facilitate the development of improved treatment methods for central nervous system disorders.

Characterization and Subcellular Targeting of GCaMP-Type Genetically-Encoded Calcium Indicators

Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca2+] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator—GCaMP2—in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17±10% ΔF/F [mean±SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302±50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca2+ accumulations evoked by activation of synaptic NMDA receptors. We observed robust ΔF/F responses (range: 37%–264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.