Selective targeting of a laccase from Stachybotrys chartarum covalently linked to a carotenoid‐binding peptide

Abstract: A two‐step targeting strategy was used to identify improved laccases for bleaching carotenoid‐containing stains on fabric. We first applied a modified phage display technique to identify peptide sequences capable of binding specifically to carotenoid stains and not to fabric. Prior deselection on the support on which the carotenoid was localized, increased stringency during the biopanning target selection process, and analysis of the phage peptides’ binding to the target after acid elution and polymerase chain reaction (PCR) postacid elution, were used to isolate phage peptide libraries with increased binding selectivity and affinity. Peptide sequences were selected based on identified consensus motifs. We verified the enhanced carotenoid‐binding properties of the peptide YGYLPSR and subsequently cloned and expressed C‐terminal variants of laccase from Stachybotrys chartarum containing carotenoid‐binding peptides YGYLPSR, IERSAPATAPPP, KASAPAL, CKASAPALC, and SLLNATK. These targeted peptide–laccase fusions demonstrate enhanced catalytic properties on stained fabrics.     

On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters

The integration of pharmaceutical nanocarriers with phage display techniques is emerging as a new paradigm for targeted cancer nanomedicines. We explored the direct use of landscape phage fusion proteins for the self-assembly of phage-derived binding peptides to liposomes for cancer cell targeting. The primary purpose of this study was to elucidate the targeting mechanism with a particular emphasis on the relative contributions of the two motifs that make up the landscape phage fusion protein (a binding peptide and the phage pVIII coat protein) to the targeting efficiency. Using transmission electron microscopy and dynamic light scattering, we confirmed the formation of phage-liposomes. Using FACS analysis, fluorescence microscopy, and fluorescence photospectrometry, we found that liposomes modified with MCF-7-specific phage fusion proteins (MCF-7 binding peptide, DMPGTVLP, fused to the phage PVIII coat protein) provided a strong and specific association with target MCF-7 cancer cells but not with cocultured, nontarget cells including C166-GFP and NIH3T3. The substitution for the binding peptide fused to phage pVIII coat protein abolished the targeting specificity. The addition of free binding peptide, DMPGTVLP, competitively inhibited the interaction of MCF-7-specific phage-liposomes with target MCF-7 cells but showed no reduction of MCF-7-associated plain liposomes. The proteolysis of the binding peptide reduced MCF-7 cell-associated phage-liposomes in a proteinase K (PK) concentration-dependent manner with no effect on the binding of plain liposomes to MCF-7 cells. Overall, only the binding peptide motif was involved in the targeting specificity of phage-liposomes. The presence of phage pVIII coat protein did not interfere with the targeting efficiency.     

Ligand-based targeted therapy for cancer tissue

Background: Limited accessibility of drugs to the tumor tissues, the requirement of high doses, intolerable cytotoxicity, the development of multiple drug resistance and non-specific targeting are obstacles to the clinical use of cancer drugs and cancer therapy. Objective: Drug delivery through carrier systems to cancerous tissue is no longer simply wrapping up cancer drugs in a new formulation for different routes of delivery, rather the focus is on targeted cancer therapy. Methods: This review summarizes the exploitation of drug-loaded nanocarrier conjugates with various targeting moieties for the delivery and targeting of anticancer drugs and describes the current status of and challenges in the field of nanocarrier-aided drug delivery and drug targeting. Conclusion: The discovery of targeting ligand to cancer cells and the development of ligand-targeted therapy will help us to improve therapeutic efficacy and reduce side effects. Unlike other forms of therapy, it will allow us to maintain quality of life for patients, while efficiently attacking the cancer tissue. It indicates that ligands have a pivotal role in cancer cell targeting.     

肺癌特异性结合多肽的体外筛选和鉴定

目的应用噬菌体随机肽库技术筛选出与肺癌细胞特异性结合的多肽。方法以人肺癌细胞NCI-H1299为靶细胞,人胚肺细胞MRC-5为吸附细胞,对噬菌体随机12肽库进行3轮全细胞减性筛选后,随机挑取噬菌体克隆进行ELISA鉴定;对亲和力较高的阳性克隆进行DNA测序并翻译为氨基酸序列;化学合成异硫氰酸荧光素标记的多肽(FITC-ZS-5),采用细胞和组织免疫荧光法鉴定FITC-ZS-5与肺癌细胞的亲和力及特异性。结果通过3轮减性筛选后,与NCI-H1299细胞结合的噬菌体克隆得到有效的富集;ELISA结果显示5号克隆对NCI-H1299细胞亲和力最高,将其命名为Phage ZS-5;测序结果显示Phage ZS-5所表达的多肽序列在国内外均未见报道,细胞及组织免疫荧光实验结果显示FITC-ZS-5对肺癌细胞及组织具有较高的亲和力和特异性。结论应用噬菌体随机肽库技术筛选到肺癌靶向性多肽ZS-5,为肺癌的靶向治疗和诊断奠定基础。     

Identification of oligopeptide binding to colon cancer cells separated from patients using laser capture microdissection

The development of intravascular conjugates that efficiently deliver genes or drugs to tumors is limited by the lack of efficacious targeting ligands. Small targeting peptides, such as those iterated by phage display technology, offer enormous potential for these applications. The majority of reports published to date have focused on the identification of peptides isolated for their ability to bind to human cancer cell lines in vitro, and have failed to account for the loss of polarization and de-differentiation of such cells from their in vivo state. Here, we report a novel approach for the identification of peptides capable of binding specifically to cancer cells derived from clinically resected human colon cancer. In this strategy, laser capture microdissection (LCM) is performed on a surgically resected colon cancer specimen to separate only cancer cells from the specimen. Subsequently, biopanning was performed on the LCM-selected colon cancer cells to identify peptide sequences that bound specifically to them. A peptide containing the SPT motif was selected as the most promising consensus sequence binding specifically to the LCM-selected colon cancer cells. Phage clones displaying the SPT motif demonstrated 9-fold higher binding to colon cancer cells derived from a patient than insertless phage (p < 0.05), while, recovery of the SPT phage from the colon cancer cell lines DLD-1 and HCT-15 was 7-fold higher than that of the control insertless phage (p < 0.05). The binding of SPT phage to colon cancer cells from the patient was confirmed by immunofluorescence. Additionally, a synthesized SPT-containing peptide (SPTKSNS) showed binding activity in the absence of mitogenic effects on colon cancer cells in vitro. In summary, we have introduced LCM into a biopanning procedure and identified a small peptide that binds preferentially to colon cancer cells derived from a clinically resected sample. This procedure could be applicable for the design of customized cancer cell targeting methodologies using clinical biopsy samples from human subjects.     

Display technologies: application for the discovery of drug and gene delivery agents

Recognition of molecular diversity of cell surface proteomes in disease is essential for the development of targeted therapies. Progress in targeted therapeutics requires establishing effective approaches for high-throughput identification of agents specific for clinically relevant cell surface markers. Over the past decade, a number of platform strategies have been developed to screen polypeptide libraries for ligands targeting receptors selectively expressed in the context of various cell surface proteomes. Streamlined procedures for identification of ligand-receptor pairs that could serve as targets in disease diagnosis, profiling, imaging and therapy have relied on the display technologies, in which polypeptides with desired binding profiles can be serially selected, in a process called biopanning, based on their physical linkage with the encoding nucleic acid. These technologies include virus/phage display, cell display, ribosomal display, mRNA display and covalent DNA display (CDT), with phage display being by far the most utilized. The scope of this review is the recent advancements in the display technologies with a particular emphasis on molecular mapping of cell surface proteomes with peptide phage display. Prospective applications of targeted compounds derived from display libraries in the discovery of targeted drugs and gene therapy vectors are discussed.     

Peptide phage display: opportunities for development of personalized anti-cancer strategies

Personalized medicine is critical for cancer patients, because (1) cancer is a highly heterogeneous disease with major molecular differences in the expression and distribution of tumor cell surface markers among patients with the same type and grade of cancer, (2) cellular mutations tend to accumulate as cancer progresses, further increasing tumor heterogeneity, and (3) currently used cancer therapies often are toxic to normal cells, causing severe side effects rarely seen in other diseases. To prevent side effects and to improve effectiveness, cytotoxic therapies should be targeted and each patient should be profiled for the presence of cancer targets before the therapy is administered. Phage display technology utilizes combinatorial libraries of proteins expressed on phage particles that can be selected for specific binding to cancer cells. Such cancer-specific molecules can be used in a variety of applications, including identification of cell-specific targeting molecules; identification of cell surface biomarkers; profiling of specimens obtained from individual cancer patients, and the design of peptide-based anti-cancer therapeutics for personalized treatments. This review is focused on peptide phage display strategies that target cell surfaces because many biomarkers important in cancer are differentially expressed molecules located on the outside of the cell membranes.     

Nanotechnology based platforms for survivin targeted drug discovery

Introduction: Development of an effective, safe and targeted drug delivery system to fight cancer and other diseases is a prime focus in the area of drug discovery. The emerging field of nanotechnology has revolutionised the way cancer therapy and diagnosis is achieved primarily due to the recent advances in material engineering and drug availability. Further, the recognition of the crucial role played by anti-apoptotic proteins such as survivin, has initiated the development of therapeutics that can target this protein as an attempt to develop alternative cancer therapies. However, a key challenge faced in drug development is the efficient delivery of survivin-targeted molecules to specific areas in the body.

Areas covered: This review primarily focuses on the different strategies employing nanotechnology for targeting survivin expressed in human cancers. Different nanomaterials incorporating nucleic molecules or drugs targeted at survivin are discussed and the results obtained from studies are highlighted.

Expert opinion: There are extensive studies reporting different treatment regimens for cancer, however, they still result in systemic toxicity, reduced bioavailability and ineffective delivery. Novel approaches involve the use of biocompatible nanomaterials together with gene or drug molecules to target proteins such as survivin, which is overexpressed in cancerous cells. These nanoformulations allow the benefits of protecting easily degradable molecules, allow controlled release, and enhance targeted delivery and effectiveness. Hence, nanotherapy utilizing survivin targeting can be considered to play a key role in the development of personalized nanomedicine for cancer.     

Is phage display technology on target for developing peptide-based cancer drugs?

New tumor targeting agents are required to advance cancer diagnosis and treatment. Bacteriophage (phage) display technology, a molecular genetic means of combinatorial drug discovery, is an emerging approach to identify and improve peptide molecules as pharmaceuticals. Peptides are thought to have clinically desirable benefits over currently used biomolecules, such as antibodies, because of their rapid blood clearance, increased diffusion and tissue penetration, non-immunogenic nature and ease of synthesis. Using phage display, one can rapidly and simultaneously survey billion-clone peptide libraries, resulting in large numbers of "hits". However, only a few lead compounds resulting from the hits historically reach the drug market. Hence determining which peptide may best translate into a useful drug is of particular importance. Examination of successfully marketed drugs has highlighted key features of a winning agent, including low molecular weight, high affinity, stability, solubility, lipophilicity and conformational rigidity. Although peptide modulators of tumor cell function and cancer targeting agents have been developed, the majority of peptide-based drugs reported thus far are immune and cardiac regulators. In this review, we will highlight how phage display has been employed to isolate peptides that target key steps in cancer progression--from tumor growth to metastasis--and how phage display technology can be harnessed to select a priori peptides with inherent features essential for anti-cancer drug efficacy. In 2003, phage display provided us with several novel peptides not only in clinical trials but approved by the FDA for use as therapeutics in a variety of diseases--suggesting that the future looks bright for phage display in anti-cancer drug development.     

New approaches for pediatric rhabdomyosarcoma drug discovery: targeting combinatorial signaling

Introduction: Rhabdomyosarcomas (RMS) are rare heterogeneous pediatric tumors that are treated by surgery, chemotherapy and irradiation. New therapeutic approaches are needed, especially in the advanced stages to target the pro-oncogenic signals. Exploring the molecular interactions of the regulatory signals and their roles in the developmental aspects of different subtypes of RMS is essential to identify potential targets and develop new therapeutic drugs.

Areas covered: Insights into different drug discovery approaches are discussed with specific emphasis on gene expression profiling, fusion protein, role of small interfering RNA (siRNA)- and microRNA (miRNA)-based discovery approaches, targeting cancer stem cells, and in vitro and in vivo model systems. Targeting some overexpressed signals along with the possibilities of combination therapy of validated drug targets is discussed. Additionally, methods to overcome the limitations of discovery-based research are briefly discussed.

Expert opinion: Due to drug resistance, ineffective therapy in advanced stages and relapse, there is a demand to explore new drug targets and discovery approaches. Implementing miRNA-based profiling would reveal the extent of miR-based regulation, various biomarkers and potential targets in RMS. A suitable combination of innovative techniques and the use of model systems might assist the identification and validation of novel targets and drug discovery methods. Combining specific drugs along with type-specific target inhibition of overexpressed mRNAs through siRNA approaches would enable the development of personalized therapy.     

Drug delivery vectors based on filamentous bacteriophages and phage-mimetic nanoparticles

With the development of nanomedicine, a mass of nanocarriers have been exploited and utilized for targeted drug delivery, including liposomes, polymers, nanoparticles, viruses, and stem cells. Due to huge surface bearing capacity and flexible genetic engineering property, filamentous bacteriophage and phage-mimetic nanoparticles are attracting more and more attentions. As a rod-like bio-nanofiber without tropism to mammalian cells, filamentous phage can be easily loaded with drugs and directly delivered to the lesion location. In particular, chemical drugs can be conjugated on phage surface by chemical modification, and gene drugs can also be inserted into the genome of phage by recombinant DNA technology. Meanwhile, specific peptides/proteins displayed on the phage surface are able to conjugate with nanoparticles which will endow them specific-targeting and huge drug-loading capacity. Additionally, phage peptides/proteins can directly self-assemble into phage-mimetic nanoparticles which may be applied for self-navigating drug delivery nanovehicles. In this review, we summarize the production of phage particles, the identification of targeting peptides, and the recent applications of filamentous bacteriophages as well as their protein/peptide for targeting drug delivery in vitro and in vivo. The improvement of our understanding of filamentous bacteriophage and phage-mimetic nanoparticles will supply new tools for biotechnological approaches.     

An In Vivo Approach to Structure Activity Relationship Analysis of Peptide Ligands

Purpose The goals in this study were several-fold. First, to optimize the in vivo phage display methodology by incorporating phage pharmacokinetic properties, to isolate peptides that target the brain microvasculature, and then to build focused libraries to obtain structure activity relationship information in vivo to identify the optimal targeting motif. Materials and Methods The blood pharmacokinetics of filamentous and T7 phage were evaluated to choose the optimal platform. A randomized peptide library with a motif CX₁₀C was constructed in T7 phage and used for in vivo panning. Focused peptide libraries around each structural element of the brain-specific peptide were constructed to perform kinetic structure activity relationship (kSAR) analysis in vivo. To determine potential function, sepsis was induced in mice by LPS administration and four hours later the effect of GST-peptide on adhesion of rhodamine-labelled lymphocytes or CFDA-labelled platelets to pial microvasculature was observed by intravital microscopy. Results The blood phamacokinetics of T7 was rapid (half-life of 12 min) which aids the clearance of non-specific phage. In vivo panning in brain enriched for isolates expressing the motif CAGALCY. Kinetic analysis of focused libraries built around each structural element of the peptide provided for rapid pharmacophore mapping. The computer modeling data suggested the peptide showed similarities to peptide mimetics of adhesion molecule ligands. GST-CAGALCY but not GST control protein was able to inhibit the rolling and adhesion of labeled platelets to inflamed pial vasculature. GST-CAGALCY had no effect on lymphocyte adhesion. Conclusions Incorporating normal blood phamacokinetics of T7 phage into in vivo phage display improves the ability to recover targeting peptide motifs and allows effective lead optimization by kSAR. This approach led to the isolation of a brain-specific peptide, CAGALCY, which appears to function as an effective antagonist of platelet adhesion to activated pial microvasculature. Targeted Molecules Corp. is now a wholly owned subsidiary of Chromos Molecular Systems Inc, 8081 Lougheed Highway, Burnaby, BC, V5A 1W9, Canada.     

Paradigm shift in bacteriophage-mediated delivery of anticancer drugs: from targeted ‘magic bullets’ to self-navigated ‘magic missiles’

Introduction: New phage-directed nanomedicines have emerged recently as a result of the in-depth study of the genetics and structure of filamentous phage and evolution of phage display and phage nanobiotechnology. This review focuses on the progress made in the development of the cancer-targeted nanomaterials and discusses the trends in using phage as a bioselectable molecular navigation system.

Areas covered: The merging of phage display technologies with nanotechnology in recent years has proved promising in different areas of medicine and technology, such as medical diagnostics, molecular imaging, vaccine development and targeted drug/gene delivery, which is the focus of this review. The authors used data obtained from their research group and sourced using Science Citation Index (Web of Science) and NCBI PubMed search resources.

Expert opinion: First attempts of adapting traditional concepts of direct targeting of tumor using phage-targeted nanomedicines has shown minimal improvements. With discovery and study of biological and technical barriers that prevent anti-tumor drug delivery, a paradigm shift from traditional drug targeting to nanomedicine navigation systems is required. The advanced bacteriophage-driven self-navigation systems are thought to overcome those barriers using more precise, localized phage selection methods, multi-targeting ‘promiscuous’ ligands and advanced multifunctional nanomedicine platforms.     

In vitro optimization of liposomal nanocarriers prepared from breast tumor cell specific phage fusion protein

Fusion proteins created by phage display peptides with tumor cell specificity and the pVIII major coat protein of filamentous phages have been explored recently as a simple and cost-effective means for preparing tumor-targeted liposomes that improve the cytotoxicity of anticancer drugs in vitro. The next step in the development of this approach is the optimization of the liposome composition for the maximum targeting activity and subsequent testing in vivo. This study aimed to investigate the impact of preparation protocols, lipid composition and phage protein content on the targeting efficiency of phage protein-modified liposomes. Analysis of size, zeta potential and morphology was used to investigate the effect of preparation protocols on the stability and homogeneity of the phage liposomes. A previously developed coculture targeting assay and a factorial design approach were used to determine the role of lipid composition of the liposomal membrane on the target cell specificity of the phage liposomes. Western blot combined with proteinase K treatment detected the orientation of targeted phage protein in liposomal membrane. Phage protein, DPPG and PEG_2k-PE showed positive effects on target specificity of phage liposomes. The results served to identify optimal formulation that offer an improved liposomal affinity for target tumor cells over the non-optimized formulation.     

Development of an orphanin FQ (OFQ) receptor antagonist for the treatment of chronic pain

Introduction: Hedgehog (Hh) signaling pathway plays key roles in embryonic development, formation and maintenance of cancer stem cells (CSCs) and acquisition of epithelial-to-mesenchymal transition (EMT). Since CSCs and EMT are important biological factors responsible for cancer cell invasion, metastasis, drug resistance and tumor recurrence, the Hh signaling pathway is believed to be an important target for cancer therapy.

Areas covered: In recent years, small-molecule inhibitors of Hh signaling have been synthesized for cancer treatment. Clinical trials using these inhibitors are being conducted to determine their toxicity profiles and efficacies. In addition, nutraceuticals (such as isoflavones, curcumin, vitamin D, etc) have been shown to inhibit cancer growth through downregulation of Hh signaling.

Expert opinion: Inhibition of Hh signaling is important for suppression of cancer growth, invasion, metastasis and recurrence in cancer therapy. However, targeting only one molecule in Hh signaling may not be sufficient to kill cancer cells because cancers show deregulation of multiple signals. Therefore, utilizing new technologies to determine alterations in Hh and other signals for individuals and designing combination strategies with small-molecule Hh inhibitors, nutraceuticals and other chemotherapeutics in targeted personalized therapy could have a significant effect on improving the overall survival of patients with cancers.     

Integrin targeted drug and gene delivery

Importance of the field: Recently, there has been substantial progress in the development of integrin targeted pharmaceuticals and drug delivery systems. Integrin is an important member in the cell adhesion molecule family, which is involved in regulation of complex biological conditions, from keeping normal physiological activities to causing cellular dysfunction in diseased cells. Hence, it is timely to summarize the recent developments in integrin targeted drug and gene delivery systems to understand better their advantages and limitations.

Areas covered in this review: In this review, advances in the discovery and clinical trials of these integrin antagonists against different integrin subunits are summarized and discussed. Besides using integrin inhibitor as a single therapeutic agent, integrin antagonists that were conjugated to cytotoxic drugs by synthetic chemistry or coupled to biomacromolecules by either DNA recombination technology or fusion protein technology for integrin targeted therapy have been explored. Furthermore, nanoparticles with integrin targeting ligands for both drug and gene delivery, typically for antiangiogenesis and anticancer therapy, are highlighted and evaluated.

What the reader will gain: This review sheds light on the future development of integrin targeted drug and/or gene delivery systems.

Take home message: Although thus far there are still limitations, integrin targeted delivery systems have already shown their potential as important pharmaceuticals in the near future.     

Docking and scoring: applications to drug discovery in the interactomics era

Background: Computational approaches such as docking and scoring are becoming routine in drug discovery as a complement to other more traditional techniques. However, so far, computer drug design methods have been applied to inhibit the function of individual proteins, and there is little available data on the use of these computational techniques to target protein–protein interactions. Objective: To establish a strategy for the use of current computational tools in drug discovery targeting protein–protein interactions. Method: Individual techniques applied to specific cases could be studied to derive a general strategy for targeting protein–protein interactions. Conclusion: Protein docking, interface prediction and hot-spot identification can contribute to the discovery of small molecule inhibitors targeting protein interactions of therapeutic interest, especially when little structural information is available.     

Functional screening of α7 nicotinic receptor ligands

Background: The α7 nicotinic acetylcholine receptor, a ligand-gated ion channel, is an attractive drug discovery target in schizophrenia and Alzheimer's disease. Objective: We have evaluated the various approaches to discovering ligands targeting the α7 nicotinic receptor to define the current paradigm driving drug discovery efforts in this area. Methods: Assays using functional read-outs as a consequence of α7 nicotinic receptor activation have been reviewed. Conclusion: Functional assays using fluorescence-based optical methods in combination with direct electrophysiological recordings of channel function currently provide an integrated approach to the discovery of α7 nicotinic receptor targeted ligands.     

Thermosensitive magnetic liposomes with doxorubicin cell-penetrating peptides conjugate for enhanced and targeted cancer therapy

To specifically deliver cytotoxic drug to tumor cells and enhance cellular uptake is the key for effective cancer therapy. In this paper, we described a novel drug targeting system, which is designed to combine features of biological (cell-penetrating peptides, CPPs) and physical (magnetic) drug targeting for use in the magnetic hyperthermia-triggered release. A doxorubicin–CPPs conjugate (DOX-CPPs) was loaded into thermosensitive magnetic liposomes (TSMLs) (DOX-CPPs/TSMLs), and in vitro DOX-CPPs thermosensitive release activity, anti-proliferation effect, in vivo targeted delivery as well as in vivo antitumor activity were determined. The results demonstrated that the DOX-CPPs/TSMLs showed good physicochemical properties, effective anti-proliferation effect in MCF-7 cells in vitro. Additionally, in vivo study, DOX-CPPs/TSMLs under AC magnetic field displayed superior in vivo targeted delivery efficacy, antitumor efficacy in an MCF-7 xenograft murine model. In conclusion, the application of DOX-CPPs/TSMLs under AC magnetic field may provide a strategy for the selective and efficient delivery of drug.     

Landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency

Tumor-specific cytotoxicity of drugs can be enhanced by targeting them to tumor receptors using tumor-specific ligands. Phage display offers a high-throughput approach to screen for the targeting ligands. We have successfully isolated phage fusion peptides selective and specific for PC3 prostate cancer cells. Also, we have demonstrated a novel approach of targeting liposomes through tumor-specific phage fusion coat proteins, exploiting the intrinsic properties of the phage coat protein as an integral membrane protein. Here we describe the production of Rhodamine-labeled liposomes as well as doxorubicin-loaded long-circulating liposomes targeted to PC3 prostate tumor cells via PC-specific phage peptides, as an extension of our previous studies. Targeting of labeled liposomes was demonstrated using fluorescence microscopy as well as flow cytometry. Targeting of doxorubicin-loaded liposomes enhanced their cytotoxic effect against PC3 cells in vitro, indicating a possible therapeutic advantage. The simplicity of the approach for generating targeted liposomes coupled with the ability to rapidly obtain tumor-specific phage fusion proteins via phage display may contribute to a combinatorial system for the production of targeted liposomal therapeutics for advanced stages of prostate tumor.From the Clinical EditorThis paper demonstrates targeting cytotoxic agents to tumor receptors using tumor-specific ligands. The authors describe the production of Rhodamine-labeled liposomes as well as doxorubicin loaded long circulating liposomes targeted to PC3 prostate tumor cells via PC-specific phage peptides. This approach may be especially relevant for advanced prostate tumors.