Tumor-targeting Ligand Research Articles

Self-Assembled Lipid Nanomedicines for siRNA Tumor Targeting

Lipid-based nanoparticle technology has developed from chemical drug carrier into an efficient multifunctional siRNA tumor targeting delivery system. In this review, we start with an overview of the lipid-based nanomedicine history and the two classes of lipidic vectors for DNA or siRNA delivery. Then we discuss the features of lipid-based nanomedicine that lead to effective tumor targeting and the principles behind. We also discuss nanoparticle surface modification, classes of tumor targeting ligands, and other state-of-the-art strategies for enhancing endosome release primarily focused on lipid-based systems. At the end, we show that multifunctional self-assembled lipid-based nanoparticles could also be versatile delivery vehicles for cancer molecular imaging probes.

Oncolytic virus therapy for pancreatic cancer using the adenovirus library displaying random peptides on the fiber knob

A conditionally replicative adenovirus is a novel anticancer agent designed to replicate selectively in tumor cells. However, a leak of the virus into systemic circulation from the tumors often causes ectopic infection of various organs. Therefore, suppression of naive viral tropism and addition of tumor-targeting potential are necessary to secure patient safety and increase the therapeutic effect of an oncolytic adenovirus in the clinical setting. We have recently developed a direct selection method of targeted vector from a random peptide library displayed on an adenoviral fiber knob to overcome the limitation that many cell type-specific ligands for targeted adenovirus vectors are not known. Here we examined whether the addition of a tumor-targeting ligand to a replication-competent adenovirus ablated for naive tropism enhances its therapeutic index. First, a peptide-display adenovirus library was screened on a pancreatic cancer cell line (AsPC-1), and particular peptide sequences were selected. The replication-competent adenovirus displaying the selected ligand (AdDeltaCAR-SYE) showed higher oncolytic potency in several other pancreatic cancer cell lines as well as AsPC-1 compared with the untargeted adenovirus (AdDeltaCAR). An intratumoral injection of AdDeltaCAR-SYE significantly suppressed the growth of AsPC-1 subcutaneous tumors, and an analysis of adenovirus titer in the tumors revealed an effective replication of the virus in the tumors. Ectopic liver gene transduction following the intratumoral injection of AdDeltaCAR-SYE was not increased compared with the AdDeltaCAR. The results showed that a tumor-targeting strategy using an adenovirus library is promising for optimizing the safety and efficacy of oncolytic adenovirus therapy.

Cyclodextrin-covered gold nanoparticles for targeted delivery of an anti-cancer drug

We report on the therapeutic ability of a novel cyclodextrin-covered gold nanoparticle (AuNP) carrier for noncovalent encapsulation of an anti-cancer drug. The surface of the AuNPs was functionalized with cyclodextrin as a drug pocket, anti-epidermal growth factor receptor (anti-EGFR) antibody as a targeting moiety, and poly(ethyleneglycol) (PEG) as an anti-fouling shell. β-Lapachone, an anti-cancer drug, was efficiently encapsulated into the hydrophobic cavity of cyclodextrin on the surface of the AuNP carriers (AuNP-1). The glutathione-mediated release of β-lapachone from the surface of AuNP-1 was demonstrated by an experiment with MCF-7 (low glutathione concentration) and A549 cells (high glutathione concentration). We also show that the introduction of an anti-EGFR antibody onto the AuNP carriers (AuNP-2) increased the intracellular uptake of AuNP carriers as compared with AuNP-1, which does not contain a targeting ligand. In the in vitro cytotoxicity study, AuNP-2 with β-lapachone exhibited a higher apoptosis effect than that caused by AuNP-1 with β-lapachone. This work suggests that AuNPs covered with cyclodextrin and tumor-targeting ligands may find useful applications for the development of nanoparticles with therapeutic and diagnostic modalities.

Development of PEGylated Adenovirus Vector for Cancer Gene Therapy

Adenovirus vectors (Ad) have been frequently used for cancer gene therapy research because of their high gene transduction efficiency. However, systemic administration of conventional Ad can lead to the acute accumulation of virus particles and transgene expression in the liver, which may cause severe hepatotoxicity. For these reasons, clinical application of Ad for systemic administration has been limited, although intratumor administration of Ad has shown marked antitumor effects. Therefore, to promote the application of Ad in systemic cancer gene therapy, especially against the distant metastatic cancer, a novel Ad with marked accumulation in tumors and minimal hepatic distribution is needed. From this perspective, bioconjugation with polyethylene glycol (PEGylation) to Ad surface is a promising strategy, and we are trying to develop cancer targeted Ad by PEGylation approach. Through our study, we particularly clarified that PEGylated Ad (PEG-Ad) with optimized PEG modification ratio exhibited the enhanced distribution and gene expression in tumor tissue via systemic injection, which was based on the enhanced permeability and retention (EPR) effect. Moreover, PEG-Ad encoding therapeutic gene demonstrated not only stronger tumor-suppressive activity but also fewer hepatotoxic side effects compared with conventional Ad. In addition, we further attempted the active targeting using targeting ligand on the tip of PEG. We revealed that PEG-Ad with transferrin as a tumor targeting ligand could transduce more efficiently into tumor cells, which express transferrin receptor, compared with conventional PEG-Ad. In this symposium, I will present our approach for development of cancer targeted Ad by PEGylation.

Folate Functionalized Boron Nitride Nanotubes and their Selective Uptake by Glioblastoma Multiforme Cells: Implications for their Use as Boron Carriers in Clinical Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is increasingly being used in the treatment of several aggressive cancers, including cerebral glioblastoma multiforme. The main requirement for this therapy is selective targeting of tumor cells by sufficient quantities of10B atoms required for their capture/irradiation with low-energy thermal neutrons. The low content of boron targeting species in glioblastoma multiforme accounts for the difficulty in selective targeting of this very malignant cerebral tumor by this radiation modality. In the present study, we have used for the first time boron nitride nanotubes as carriers of boron atoms to overcome this problem and enhance the selective targeting and ablative efficacy of BNCT for these tumors. Following their dispersion in aqueous solution by noncovalent coating with biocompatible poly-l-lysine solutions, boron nitride nanotubes were functionalized with a fluorescent probe (quantum dots) to enable their tracking and with folic acid as selective tumor targeting ligand. Initial in vitro studies have confirmed substantive and selective uptake of these nanovectors by glioblastoma multiforme cells, an observation which confirms their potential clinical application for BNCT therapy for these malignant cerebral tumors.

Single-step surface functionalization of polymeric nanoparticles for targeted drug delivery

Targeted drug delivery using nanocarriers is achieved by functionalizing the carrier surface with a tissue-recognition ligand. Current surface modification methods require tedious and inefficient synthesis and purification steps, and are not easily amenable to incorporating multiple functionalities on a single surface. In this report, we describe a versatile, single-step surface functionalizing technique for polymeric nanoparticles. The technique utilizes the fact that when a diblock copolymer like polylactide–polyethylene glycol (PLA–PEG) is introduced in the oil/water emulsion used in polymeric nanoparticle formulation, the PLA block partitions into the polymer containing organic phase and PEG block partitions into the aqueous phase. Removal of the organic solvent results in the formation of nanoparticles with PEG on the surface. When a PLA–PEG–ligand conjugate is used instead of PLA–PEG copolymer, this technique permits a ‘one-pot’ fabrication of ligand-functionalized nanoparticles. In the current study, the IAASF approach facilitated the simultaneous incorporation of biotin and folic acid, known tumor-targeting ligands, on drug-loaded nanoparticles in a single step. Incorporation of the ligands on nanoparticles was confirmed by using NMR, surface plasmon resonance, transmission electron microscopy and tumor cell uptake studies. Simultaneous functionalization with both ligands significantly enhanced nanoparticle accumulation in tumors in vivo, and resulted in greatly improved efficacy of paclitaxel-loaded nanoparticles in a mouse xenograft tumor model. This new surface functionalization approach will enable the development of targeting strategies based on the use of multiple ligands on a single surface to target a tissue of interest.

Targeted magnetic iron oxide nanoparticles for tumor imaging and therapy

Magnetic iron oxide (IO) nanoparticles with a long blood retention time, biodegradability and low toxicity have emerged as one of the primary nanomaterials for biomedical applications in vitro and in vivo. IO nanoparticles have a large surface area and can be engineered to provide a large number of functional groups for cross-linking to tumor-targeting ligands such as monoclonal antibodies, peptides, or small molecules for diagnostic imaging or delivery of therapeutic agents. IO nanoparticles possess unique paramagnetic properties, which generate significant susceptibility effects resulting in strong T2 and T*2 contrast, as well as T1 effects at very low concentrations for magnetic resonance imaging (MRI), which is widely used for clinical oncology imaging. We review recent advances in the development of targeted IO nanoparticles for tumor imaging and therapy.

Does a targeting ligand influence nanoparticle tumor localization or uptake?

Inclusion of a tumor-targeting molecule in nanosized delivery systems increases their in vivo efficacy. However, the biodistribution and pharmacokinetics of the uptake of such particles have not yet been well addressed. Several recent papers have suggested that tumor-targeting ligands function primarily to increase intracellular uptake of the nanocomplex and do not influence tumor localization. However, other reports indicate that they do play a role in the accumulation in the tumor. One difference might be the presence or absence of poly-[ethylene glycol] (PEG) in the complex and its impact on the enhanced permeability and retention (EPR) effect. Further studies are clearly needed to more fully elucidate the influence of composition on tumor-targeted, systemic delivery of nanoparticles.

Folate-encoded and Fe3O4-loaded polymeric micelles for dual targeting of cancer cells

Diblock copolymers of poly(ethylene glycol) (PEG) and poly(ɛ-caprolactone) (PCL) bearing a tumor-targeting ligand, folate, were self-assembled into micelles. Superparamagnetic iron oxide (SPIO) nanoparticles and an anticancer drug doxorubicin (DOX) were coencapsulated within the micelles less than 100nm in diameters. These SPIO–DOX-loaded micelles were superparamagnetic at room temperature, but turned ferrimagnetic at 10K, consistent with magnetic properties of primary SPIO nanoparticles. Cell culture experiments demonstrated the potential of these polymeric micelles as an effective dual targeting nanoplatform for the delivery of anticancer drugs. Folate attachment to micelles resulted in the recognition of the micelles by tumor cells over-expressing folate receptors, leading to facilitation in cellular uptake of micelles, and the transport efficiency of the SPIO-loaded and folate-functionalized micelles into the tumor cells can be further enhanced by applying an external magnetic field to the cells.

PH-Sensitive microgels functionalized with folic acid

One of the most promising strategies in anticancer therapies is the targeted delivery through malignancy-associated cellular markers. The design of new synthetic devices with enhanced stimuli-responsive sensitivity and targeting ligands is a promising field for the development of cancer-specific delivery systems. One of the pathways to achieve this aim is the chemical functionalization of nanodevices such as microgels. The p-nitrophenyl acrylate (NPA) is an active ester molecule with a group that can be easily cleavaged by the nucleophilic attack of species such as amines. This modification consists of an easy chemical reaction that leads to several types of functionalized microgels, which are originally made up of NPA as one of their constituent monomers.Here is reported the chemical functionalization of NPA-based microgels by incorporating pH-sensitive functional groups and folic acid as a tumor targeting ligand into the same initial polymer network. For this purpose, microgels of p-nitrophenyl acrylate (NPA)-co-methacrylamide (MeAM) synthesized by precipitation polymerization, were modified with two different pyridine derivatives: 2-aminomethylpyridine (2-AMP) and 2-aminopyridine (2-AP), thus pH-sensitive microgels with acid pH swelling capacity were obtained. The equilibrium swelling behaviour was studied as a function of pH, ionic strength, copolymer composition and type of pyridine derivative. In addition, the microgels were derivatized with ethylene diamine, to obtain amino-functionalized microgels to which the folic acid was subsequently attached as the targeting ligand. As final step, pH-sensitive groups and folic acid were equimolarly attached to the polymer chains to obtain the fully functionalized microgels.

In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags

We describe biocompatible and nontoxic nanoparticles for in vivo tumor targeting and detection based on pegylated gold nanoparticles and surface-enhanced Raman scattering (SERS). Colloidal gold has been safely used to treat rheumatoid arthritis for 50 years, and has recently been found to amplify the efficiency of Raman scattering by 14-15 orders of magnitude. Here we show that large optical enhancements can be achieved under in vivo conditions for tumor detection in live animals. An important finding is that small-molecule Raman reporters such as organic dyes were not displaced but were stabilized by thiol-modified polyethylene glycols. These pegylated SERS nanoparticles were considerably brighter than semiconductor quantum dots with light emission in the near-infrared window. When conjugated to tumor-targeting ligands such as single-chain variable fragment (ScFv) antibodies, the conjugated nanoparticles were able to target tumor biomarkers such as epidermal growth factor receptors on human cancer cells and in xenograft tumor models.

Nanotechnology Applications in Cancer

Cancer nanotechnology is an interdisciplinary area of research in science, engineering, and medicine with broad applications for molecular imaging, molecular diagnosis, and targeted therapy. The basic rationale is that nanometer-sized particles, such as semiconductor quantum dots and iron oxide nanocrystals, have optical, magnetic, or structural properties that are not available from molecules or bulk solids. When linked with tumor targeting ligands such as monoclonal antibodies, peptides, or small molecules, these nanoparticles can be used to target tumor antigens (biomarkers) as well as tumor vasculatures with high affinity and specificity. In the mesoscopic size range of 5-100 nm diameter, nanoparticles also have large surface areas and functional groups for conjugating to multiple diagnostic (e.g., optical, radioisotopic, or magnetic) and therapeutic (e.g., anticancer) agents. Recent advances have led to bioaffinity nanoparticle probes for molecular and cellular imaging, targeted nanoparticle drugs for cancer therapy, and integrated nanodevices for early cancer detection and screening. These developments raise exciting opportunities for personalized oncology in which genetic and protein biomarkers are used to diagnose and treat cancer based on the molecular profiles of individual patients.

Molecular profiling of single cancer cells and clinical tissue specimens with semiconductor quantum dots

Semiconductor quantum dots (QDs) are a new class of fluorescent labels with broad applications in biomedical imaging, disease diagnostics, and molecular and cell biology. In comparison with organic dyes and fluorescent proteins, quantum dots have unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to multifunctional nanoparticle probes that are highly bright and stable under complex in vitro and in vivo conditions. New designs involve encapsulating luminescent QDs with amphiphilic block copolymers, and linking the polymer coating to tumor-targeting ligands and drug-delivery functionalities. These improved QDs have opened new possibilities for real-time imaging and tracking of molecular targets in living cells, for multiplexed analysis of biomolecular markers in clinical tissue specimens, and for ultrasensitive imaging of malignant tumors in living animal models. In this article, we briefly discuss recent developments in bioaffinity QD probes and their applications in molecular profiling of individual cancer cells and clinical tissue specimens.

Selection of Tumor-binding Ligands in Cancer Patients with Phage Display Libraries

Phage display has been used extensively in vitro and in animal models to generate ligands and to identify cancer-relevant targets. We report here the use of phage-display libraries in cancer patients to identify tumor-targeting ligands. Eight patients with stage IV cancer, including breast, melanoma, and pancreas, had phage-displayed random peptide or scFv library (1.6 x 10(8)-1 x 10(11) transducing units/kg) administered i.v.; tumors were excised after 30 minutes; and tumor-homing phage were recovered. In three patients, repeat panning was possible using phage recovered and amplified from that same patient's tumor. No serious side effects, including allergic reactions, were observed with up to three infusions. Patients developed antiphage antibodies that reached a submaximal level within the 10-day protocol window for serial phage administration. Tumor phage were recoverable from all the patients. Using a filter-based ELISA, several clones from a subset of the patients were identified that bound to a tumor from the same patient in which clones were recovered. The clone-binding to tumor was confirmed by immunostaining, bioassay, and real-time PCR-based methods. Binding studies with noncancer and cancer cell lines of the same histology showed specificity of the tumor-binding clones. Analysis of insert sequences of tumor-homing peptide clones showed several motifs, indicating nonrandom accumulation of clones in human tumors. This is the first reported series of cancer patients to receive phage library for serial panning of tumor targeting ligands. The lack of toxicity and the ability to recover clones with favorable characteristics are a first step for further research with this technology in cancer patients.

Quantum Dots and Multifunctional Nanoparticles: New Contrast Agents for Tumor Imaging

Nanometer-sized particles, such as semiconductor quantum dots and iron oxide nanocrystals, have novel optical, electronic, magnetic or structural properties that are not available from either molecules or bulk solids. When linked with tumor-targeting ligands, such as monoclonal antibodies, peptide fragments of tumor-specific proteins or small molecules, these nanoparticles can be used to target tumor antigens (biomarkers) and tumor vasculatures with high affinity and specificity. In the mesoscopic size range of 5-100 nm diameter, quantum dots and related nanoparticles have large surface areas and functional groups that can be linked to multiple diagnostic (e.g., optical, radioisotopic or magnetic) and therapeutic (e.g., anticancer) agents. In this review, recent advances in the development and applications of bioconjugated quantum dots and multifunctional nanoparticles for in vivo tumor imaging and targeting are discussed.

Enhanced baculovirus-mediated transduction of human cancer cells by tumor-homing peptides.

Tumor cells and vasculature offer specific targets for the selective delivery of therapeutic genes. To achieve tumor-specific gene transfer, baculovirus tropism was manipulated by viral envelope modification using baculovirus display technology. LyP-1, F3, and CGKRK tumor-homing peptides, originally identified by in vivo screening of phage display libraries, were fused to the transmembrane anchor of vesicular stomatitis virus G protein and displayed on the baculoviral surface. The fusion proteins were successfully incorporated into budded virions, which showed two- to fivefold-improved binding to human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells. The LyP-1 peptide inhibited viral binding to MDA-MB-435 cells with a greater magnitude and specificity than the CGKRK and F3 peptides. Maximal 7- and 24-fold increases in transduction, determined by transgene expression level, were achieved for the MDA-MB-435 and HepG2 cells, respectively. The internalization of each virus was inhibited by ammonium chloride treatment, suggesting the use of a similar endocytic entry route. The LyP-1 and F3 peptides showed an apparent inhibitory effect in transduction of HepG2 cells with the corresponding display viruses. Together, these results imply that the efficiency of baculovirus-mediated gene delivery can be significantly enhanced in vitro when tumor-targeting ligands are used and therefore highlight the potential of baculovirus vectors in cancer gene therapy.

165. Enhanced Baculovirus-Mediated Transduction of Human Cancer Cells by Tumor- Homing Peptides

Tumor cells and tumor vasculature both offer specific molecular targets that can be utilized for site-directed delivery of therapeutic genes. In order to achieve tumor-specific gene delivery, the tropism of baculovirus can be manipulated by modification of the virus envelope using baculovirus display technology. We genetically engineered a series of recombinant display viruses by fusing various tumor-homing peptides to the transmembrane anchor of vesicular stomatitis virus G-protein to establish more efficient and selective baculovirus vectors for targeted gene delivery into human cancer cells. The viruses were further equipped with a luciferase expression cassette, allowing transduction monitoring in mammalian cells by measuring luciferase activity. The fusion proteins were successfully incorporated into budded virions and these modified viruses showed significantly improved binding to both human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells as measured by flow cytometry. Binding of the tumor-homing peptide displaying viruses to target cells was reduced to the level of the control virus by preincubating the cells with the corresponding soluble peptides. Moreover, a maximum of 7- and 24-fold increase in transgene expression was achieved for these cell lines, respectively. The enhanced binding and transduction strongly suggest that the displayed peptides dictated this behavior. Together, these results imply that the specificity and efficiency of baculovirus-mediated gene delivery can be notably enhanced in vitro when tumor-targeting ligands are used and therefore also highlight the potential of baculovirus vectors in cancer gene therapy in vivo. Tumor cells and tumor vasculature both offer specific molecular targets that can be utilized for site-directed delivery of therapeutic genes. In order to achieve tumor-specific gene delivery, the tropism of baculovirus can be manipulated by modification of the virus envelope using baculovirus display technology. We genetically engineered a series of recombinant display viruses by fusing various tumor-homing peptides to the transmembrane anchor of vesicular stomatitis virus G-protein to establish more efficient and selective baculovirus vectors for targeted gene delivery into human cancer cells. The viruses were further equipped with a luciferase expression cassette, allowing transduction monitoring in mammalian cells by measuring luciferase activity. The fusion proteins were successfully incorporated into budded virions and these modified viruses showed significantly improved binding to both human breast carcinoma (MDA-MB-435) and hepatocarcinoma (HepG2) cells as measured by flow cytometry. Binding of the tumor-homing peptide displaying viruses to target cells was reduced to the level of the control virus by preincubating the cells with the corresponding soluble peptides. Moreover, a maximum of 7- and 24-fold increase in transgene expression was achieved for these cell lines, respectively. The enhanced binding and transduction strongly suggest that the displayed peptides dictated this behavior. Together, these results imply that the specificity and efficiency of baculovirus-mediated gene delivery can be notably enhanced in vitro when tumor-targeting ligands are used and therefore also highlight the potential of baculovirus vectors in cancer gene therapy in vivo.

Protein toxins: intracellular trafficking for targeted therapy

The immunotoxin approach is based on the use of tumor-targeting ligands or antibodies that are linked to the catalytic (toxic) moieties of bacterial or plant protein toxins. In this review, we first discuss the current state of clinical development of immunotoxin approaches describing the results obtained with the two toxins most frequently used: diphtheria and Pseudomonas toxin-derived proteins. In the second part of the review, a novel concept will be presented in which the roles are inverted: nontoxic receptor-binding toxin moieties are used for the targeting of therapeutic and diagnostic compounds to cancer or immune cells. The cell biological basis of these novel types of toxin-based therapeutics will be discussed, and we will summarize ongoing preclinical and clinical testing.

Specific Targets in Tumor Tissue for the Delivery of Therapeutic Genes

Gene therapy is part of a growing field in molecular medicine, which will gain importance in the treatment of human diseases. Until now, almost two thirds of all clinical trials performed in gene therapy are directed against Cancer As solid tumors exceeding a certain size rely on blood supply, the administration of particulate gene delivery vectors via the bloodstream is a promising concept. Tumor cells and the tumor vasculature both offer specific molecular targets, which can be utilized for the site directed delivery of therapeutic genes. Passive targeting of macromolecular drugs including gene delivery vectors to tumors can be achieved by the so called enhanced permeability and retention (EPR) effect. The specificity can be markedly enhanced when tumor targeting ligands are used. Viral vectors, which usually do not have a natural tropism for tumor tissue, were generated to carry tumor targeting molecules on their surface. Synthetic gene delivery vectors, based on cationic lipids or cationic polymers were biochemically modified to incorporate ligands specific for tumor cells or tumor vasculature. For systemic application, these delivery systems have to fulfill certain conditions. The delivery vector should not induce any immunogenic and inflammatory responses. Several studies were conducted to reduce the immunogenicity of viral vectors; surface modification of non-viral gene delivery systems reduced their non-specific interaction with blood components. On the genetic level, tumor specific promoters add additional layers of specificity restricting the transgene expression to the tumor tissue. This review will cover the systemic application of particulate gene transfer vectors targeted to tumors and will give an overview of therapeutic concepts for cancer gene therapy.

In vivo molecular and cellular imaging with quantum dots

Quantum dots (QDs), tiny light-emitting particles on the nanometer scale, are emerging as a new class of fluorescent probe for in vivo biomolecular and cellular imaging. In comparison with organic dyes and fluorescent proteins, QDs have unique optical and electronic properties: size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to the development of multifunctional nanoparticle probes that are very bright and stable under complex in vivo conditions. A new structural design involves encapsulating luminescent QDs with amphiphilic block copolymers and linking the polymer coating to tumor-targeting ligands and drug delivery functionalities. Polymer-encapsulated QDs are essentially nontoxic to cells and animals, but their long-term in vivo toxicity and degradation need more careful study. Bioconjugated QDs have raised new possibilities for ultrasensitive and multiplexed imaging of molecular targets in living cells, animal models and possibly in humans.