Recent progress in LyP-1-based strategies for targeted imaging and therapy

The identification of markers expressed by pathological cells or their microenvironment would help to distinguish such cells from the normal tissues. The strategies derived from this theory can be a promising modality for imaging and treating diseases. LyP-1, a tumor homing peptide, can selectively bind to its receptor p32 protein overexpressed in various tumor-associated cells and atherosclerotic plaque macrophages. During recent decades, multiple types of LyP-1-based imaging probes and drug delivery systems have been designed and developed for diagnostic and therapeutic applications. This review first introduces LyP-1 and its receptor p32, as well as its homing, internalization and proapoptotic properties. Next, we highlight recent studies focusing on the applications of LyP-1-based strategies in the diagnosis and treatment of tumors, metastatic lesions, and atherosclerotic plaques. Finally, several limitations in the clinical translation of LyP-1-based bioconjugates are summarized.

Related papers:

title: Tumour-homing peptides: tools for targeting, imaging and destruction.
abstract: Each normal organ and pathological condition contains organ- or disease-specific molecular tags on its vasculature that constitute a vascular 'zip code' system. Tissue-selective tumour metastasis may also depend on vascular addresses. We have used phage display peptide libraries to map disease-specific differences in the vasculature. By using this technology, we have isolated several peptides which are targeted specifically to tumour blood vessels, lymphatic vessels and/or tumour cells. Some of the tumour-homing peptides recognize common angiogenesis markers and are capable of binding to several types of tumour, whereas other peptides recognize tumour-type-specific differences. We have also shown that the vasculature of a pre-malignant lesion differs from that of a full-blown tumour and also from the vasculature of the corresponding normal organ. Our peptides have revealed molecules that act as novel biomarkers of this vascular heterogeneity. Interestingly, some of our homing peptides are able to penetrate the target cells. This internalization differs from that of the Tat, penetratins and other related peptides in that our peptides enter the cell in a cell-type-specific manner. These peptides appear to be able to concentrate in the target tissue, making them particularly efficient delivery vectors for the targeting of drugs, other therapeutic moieties and imaging agents.

title: Peptide ligand‐modified nanomedicines for targeting cells at the tumor microenvironment☆
abstract: Since their initial discovery more than 30 years ago, tumor‐homing peptides have become an increasingly useful tool for targeted delivery of therapeutic and diagnostic agents into tumors. Today, it is well accepted that cells at the tumor microenvironment (TME) contribute in many ways to cancer development and progression. Tumor‐homing peptide‐decorated nanomedicines can interact specifically with surface receptors expressed on cells in the TME, improve cellular uptake of nanomedicines by target cells, and impair tumor growth and progression. Moreover, peptide ligand‐modified nanomedicines can potentially accumulate in the target tissue at higher concentrations than would small conjugates, thus increasing overall target tissue exposure to the therapeutic agent, enhance therapeutic efficacy and reduce side effects. This review describes the most studied peptide ligands aimed at targeting cells in the TME, discusses major obstacles and principles in the design of ligands for drug targeting and provides an overview of homing peptides in ligand‐targeted nanomedicines that are currently in development for cancer therapy and diagnosis.

title: Advances in phage display technology for drug discovery
abstract: Over the past decade, several library-based methods have been developed to discover ligands with strong binding affinities for their targets. These methods mimic the natural evolution for screening and identifying ligand–target interactions with specific functional properties. Phage display technology is a well-established method that has been applied to many technological challenges including novel drug discovery.  This review describes the recent advances in the use of phage display technology for discovering novel bioactive compounds. Furthermore, it discusses the application of this technology to produce proteins and peptides as well as minimize the use of antibodies, such as antigen-binding fragment, single-chain fragment variable or single-domain antibody fragments like VHHs.  Advances in screening, manufacturing and humanization technologies demonstrate that phage display derived products can play a significant role in the diagnosis and treatment of disease. The effects of this technology are inevitable in the development pipeline for bringing therapeutics into the market, and this number is expected to rise significantly in the future as new advances continue to take place in display methods. Furthermore, a widespread application of this methodology is predicted in different medical technological areas, including biosensing, monitoring, molecular imaging, gene therapy, vaccine development and nanotechnology.

title: Antibody-Drug Conjugates and Small Molecule-Drug Conjugates: Opportunities and Challenges for the Development of Selective Anticancer Cytotoxic Agents.
abstract: Conventional cancer chemotherapy heavily relies on the use of cytotoxic agents, which typically do not preferentially localize at the tumor site and cause toxicity to normal organs, preventing dose escalation to therapeutically active regimens. In principle, antibodies and other ligands could be used for the selective pharmacodelivery of cytotoxic agents to the neoplastic mass. For many years, the availability of ligands, capable of selective internalization into tumor cells, has been considered to be an essential requirement for the development of targeted cytotoxics. This assumption, however, has recently been challenged on the basis of therapeutic data obtained with noninternalizing drug conjugates. Moreover, quantitative evaluations of the tumor targeting properties of antibodies and of small organic ligands have provided new insights for the implementation of optimal strategies for the development of targeted cytotoxics. In this article, we highlight opportunities and challenges associated with the clinical and industrial development of antibody-drug conjugates and small molecule-drug conjugates for cancer therapy.

title: Tumor target amplification: Implications for nano drug delivery systems
abstract: &NA; Tumor cells overexpress surface markers which are absent from normal cells. These tumor‐restricted antigenic signatures are a fundamental basis for distinguishing on‐target from off‐target cells for ligand‐directed targeting of cancer cells. Unfortunately, tumor heterogeneity impedes the establishment of a solid expression pattern for a given target marker, leading to drastic changes in quality (availability) and quantity (number) of the target. Consequently, a subset of cancer cells remains untargeted during the course of treatment, which subsequently promotes drug‐resistance and cancer relapse. Since target inefficiency is only problematic for cancer treatment and not for treatment of other pathological conditions such as viral/bacterial infections, target amplification or the generation of novel targets is key to providing eligible antigenic markers for effective targeted therapy. This review summarizes the limitations of current ligand‐directed targeting strategies and provides a comprehensive overview of tumor target amplification strategies, including self‐amplifying systems, dual targeting, artificial markers and peptide modification. We also discuss the therapeutic and diagnostic potential of these approaches, the underlying mechanism(s) and established methodologies, mostly in the context of different nanodelivery systems, to facilitate more effective ligand‐directed cancer cell monitoring and targeting.

title: Combination therapy: opportunities and challenges for polymer-drug conjugates as anticancer nanomedicines.
abstract: The discovery of new molecular targets and the subsequent development of novel anticancer agents are opening new possibilities for drug combination therapy as anticancer treatment. Polymer-drug conjugates are well established for the delivery of a single therapeutic agent, but only in very recent years their use has been extended to the delivery of multi-agent therapy. These early studies revealed the therapeutic potential of this application but raised new challenges (namely, drug loading and drugs ratio, characterisation, and development of suitable carriers) that need to be addressed for a successful optimisation of the system towards clinical applications.

title: Engineering folate-drug conjugates to target cancer: from chemistry to clinic.
abstract: The folate receptor (FR) is a potentially useful biological target for the management of many human cancers. This membrane protein binds extracellular folates with very high affinity and, through an endocytic process, physically delivers them inside the cell for biological consumption. There are now many examples of how this physiological system can be exploited for the targeted delivery of biologically active molecules to cancer. In fact, strong preclinical as well as emerging clinical evidence exists showing how FR-positive cancers can be (i) anatomically identified using folate conjugates of radiodiagnostic imaging agents and (ii) effectively treated with companion folate-targeted chemotherapies. While the biological results are compelling, it is of equal importance to understand the conjugation chemistries that were developed to produce these active molecules. Therefore, this review will focus on the methods utilized to construct folate-based small-molecule drug conjugates (SMDCs), with particular attention focused on modular design, hydrophilic spacers, and self-immolative linkers.

title: Imaging tumor-induced sentinel lymph node lymphangiogenesis with LyP-1 peptide
abstract: Lymphangiogenesis in tumor-draining lymph nodes (LNs) starts before the onset of metastasis and is associated with metastasis to distant LNs and organs. In this study, we aimed to visualize tumor-induced lymphangiogenesis with a tumor lymphatics-specific peptide LyP-1. The LyP-1 peptide was labeled with a near-infrared fluorophore (Cy5.5) for optical imaging. At days 3, 7, 14 and 21 after subcutaneous 4T1 tumor inoculation, Cy5.5-LyP-1 was administered through the middle phalanges of the upper extremities of the tumor-bearing mice. At 45 min and 24 h postinjection, brachial LN fluorescence imaging was performed. Ex vivo fluorescence images were acquired for quantitative analysis of the fluorescence intensity. Tumor-induced lymphangiogenesis was confirmed by LYVE-1 immunostaining and increased size of tumor side brachial LNs. Cy5.5-LyP-1 staining in LNs co-localized with LYVE-1, suggesting lymphatics-specific binding of LyP-1 peptide. The brachial LNs were clearly visualized by optical imaging at both time points. The tumor side LNs showed significantly higher fluorescence intensities than the contralateral brachial LNs at days 7, 14, and 21, but not day 3 after tumor inoculation. At day 21 after tumor inoculation, the average signal of tumor-draining LNs was 78.0 ± 2.44, 24.3 ± 5.43, 25.6 ± 0.25 (×103 photon/cm2/s) using Cy5.5-LyP-1, Cy5.5-LyP-1 with blocking, and Cy5.5 only, respectively. Tumor-draining brachial LNs showed extensive growth of lymphatic sinuses throughout the cortex and medulla. Use of LyP-1 based imaging probes with optical imaging offers a useful tool for the study of tumor-induced lymphangiogenesis. LyP-1 may serve as a marker of lymphangiogenesis useful in detecting “high risk” LNs before tumor metastasis and after micro-metastasis, as well as for screening potential anti-lymphatic therapies.

title: A tumor-homing peptide with a targeting specificity related to lymphatic vessels
abstract: Blood vessels of tumors carry specific markers that are usually angiogenesis-related. We previously used phage-displayed peptide libraries in vivo to identify peptides that home to tumors through the circulation and that specifically bind to the endothelia of tumor blood vessels. Here we devised a phage screening procedure that would favor tumor-homing to targets that are accessible to circulating phage, but are not blood vessels. Screening on MDA-MB-435 breast carcinoma xenografts yielded multiple copies of a phage that displays a cyclic 9-amino-acid peptide, LyP-1. Homing and binding to tumor-derived cell suspensions indicated that LyP-1 also recognizes an osteosarcoma xenograft, and spontaneous prostate and breast cancers in transgenic mice, but not two other tumor xenografts. Fluorescein-labeled LyP-1 peptide was detected in tumor structures that were positive for three lymphatic endothelial markers and negative for three blood vessel markers. LyP-1 accumulated in the nuclei of the putative lymphatic cells, and in the nuclei of tumor cells. These results suggest that tumor lymphatics carry specific markers and that it may be possible to specifically target therapies into tumor lymphatics.

title: Macrophages: promising targets for the treatment of atherosclerosis.
abstract: Atherosclerosis is now recognised as a chronic inflammatory disease occurring within the artery wall and ultimately responsible for myocardial infarction, stroke and peripheral vascular disease. A crucial step in atherogenesis is the infiltration of monocytes into the subendothelial space of large arteries where they differentiate into macrophages and become functionally active. Macrophage accumulation within plaques is a hallmark of all stages of atherosclerosis, indeed recent studies have shown their presence has the potential to act as a non-invasive marker of disease activity and plaque stability. Activated macrophages are major players in all stages of lesion development. They not only accumulate lipids but also express effector molecules that are pro-inflammatory, cytotoxic and chemotactic. Furthermore, they secrete enzymes that degrade extracellular matrix leading to plaque destabilisation and increased risk of rupture. However, macrophages are heterogeneous and when appropriately activated they have the potential to drive tissue remodelling and ultimately vascular repair. Pharmacological modulation of macrophage activities therefore represents an important strategy for the prevention and treatment of atherosclerosis and other inflammatory diseases. The aim of this review is to give a brief overview of our current understanding of macrophage activation, distribution and function within inflamed tissue. This will provide the basis for highlighting already available and future methods to exploit specifically activated macrophages as diagnostic and therapeutic targets for atherosclerosis.

title: Protein cage nanoparticles bearing the LyP-1 peptide for enhanced imaging of macrophage-rich vascular lesions.
abstract: Cage-like protein nanoparticles are promising platforms for cell- and tissue-specific targeted delivery of imaging and therapeutic agents. Here, we have successfully modified the 12 nm small heat shock protein from Methanococcus jannaschii (MjHsp) to detect atherosclerotic plaque lesions in a mouse model system. As macrophages are centrally involved in the initiation and progression of atherosclerosis, targeted imaging of macrophages is valuable to assess the biologic status of the blood vessel wall. LyP-1, a nine residue peptide, has been shown to target tumor-associated macrophages. Thus, LyP-1 was genetically incorporated onto the exterior surface of MjHsp, while a fluorescent molecule (Cy5.5) was conjugated on the interior cavity. This bioengineered protein cage, LyP-Hsp, exhibited enhanced affinity to macrophage in vitro. Furthermore, in vivo injection of LyP-Hsp allowed visualization of macrophage-rich murine carotid lesions by in situ and ex vivo fluorescence imaging. These results demonstrate the potential of LyP-1-conjugated protein cages as nanoscale platforms for delivery of imaging agents for the diagnosis of atherosclerosis.

title: Mitochondrial/cell-surface protein p32/gC1qR as a molecular target in tumor cells and tumor stroma.
abstract: A tumor homing peptide, LyP-1, selectively binds to tumor-associated lymphatic vessels and tumor cells in certain tumors and exhibits an antitumor effect. Here, we show that the protein known as p32 or gC1q receptor is the receptor for LyP-1. Various human tumor cell lines were positive for p32 expression in culture, and the expression was increased in xenograft tumors grown from the positive cell lines. Fluorescence-activated cell sorting analyses with anti-p32 antibodies showed that p32-positive cell lines expressed p32 at the cell surface. These cells bound and internalized LyP-1 peptide in proportion to the cell-surface expression level, which correlated with malignancy rather than total p32 expression in the cells. Like the LyP-1 peptide, p32 antibodies highlighted hypoxic areas in tumors, where they bound to both tumor cells and cells that expressed macrophage/myeloid cell markers and often seemed to be incorporated into the walls of tumor lymphatics. Significant p32 expression was common in human cancers and the p32 levels were often greatly elevated compared with the corresponding normal tissue. These results establish p32, particularly its cell-surface-expressed form, as a new marker of tumor cells and tumor-associated macrophages/myeloid cells in hypoxic/metabolically deprived areas of tumors. Its unique localization in tumors and its relative tumor specificity may make p32 a useful target in tumor diagnosis and therapy.

title: Antitumor activity of a homing peptide that targets tumor lymphatics and tumor cells.
abstract: LyP-1 is a peptide selected from a phage-displayed peptide library that specifically binds to tumor and endothelial cells of tumor lymphatics in certain tumors. Fluorescein-conjugated LyP-1 and a related peptide, LyP-1b, strongly accumulated in primary MDA-MB-435 breast cancer xenografts and their metastases from i.v. peptide injections, allowing visualization of orthotopic tumors in intact mice. The LyP peptide accumulation coincided with hypoxic areas in tumors. LyP-1 induced cell death in cultured human breast carcinoma cells that bind and internalize the peptide. Melanoma cells that do not bind LyP-1 were unaffected. Systemic LyP-1 peptide treatment of mice with xenografted tumors induced with the breast cancer cells inhibited tumor growth. The treated tumors contained foci of apoptotic cells and were essentially devoid of lymphatics. These results reveal an unexpected antitumor effect by the LyP-1 peptide that seems to be dependent on a proapoptotic/cytotoxic activity of the peptide. As LyP-1 affects the poorly vascularized tumor compartment, it may complement treatments directed at tumor blood vessels.

title: Techniques for physicochemical characterization of nanomaterials.
abstract: Advances in nanotechnology have opened up a new era of diagnosis, prevention and treatment of diseases and traumatic injuries. Nanomaterials, including those with potential for clinical applications, possess novel physicochemical properties that have an impact on their physiological interactions, from the molecular level to the systemic level. There is a lack of standardized methodologies or regulatory protocols for detection or characterization of nanomaterials. This review summarizes the techniques that are commonly used to study the size, shape, surface properties, composition, purity and stability of nanomaterials, along with their advantages and disadvantages. At present there are no FDA guidelines that have been developed specifically for nanomaterial based formulations for diagnostic or therapeutic use. There is an urgent need for standardized protocols and procedures for the characterization of nanoparticles, especially those that are intended for use as theranostics.

title: Magnetic mesoporous nanospheres anchored with LyP-1 as an efficient pancreatic cancer probe.
abstract: Immobilization of a ligand that selectively interacts with cancer cells to nanomaterials can enhance their diagnostic and therapeutic efficiency. In this study, we firstly demonstrate the high expression of receptor for cyclic nine-amino acid peptide LyP-1 (Cys-Gly-Asn-Lys-Arg-Thr-Arg-Gly-Cys) in both mouse and human pancreatic cancer. Based on these findings, sub-50 nm multifunctional superparamagnetic mesoporous nanospheres with surface modified with LyP-1 are rationally designed. Theses nanospheres have a core of silica-protected magnetite nanoparticle and a shell of FITC-labeled mesoporous silica, and they are able to specifically recognize and conjugate with the pancreatic cancer cell in vitro, as verified by the combined techniques of fluorescent imaging and T2 weight magnetic resonance imaging. After systematic administration, these LyP-1 immobilized nanospheres are found to actively target to mouse orthotopic xenograft of pancreatic cancer, which opens up the door for applications in early probing and diagnosis of pancreatic cancer by the multimodal imaging.

title: Design and In Vitro Evaluation of Bispecific Complexes and Drug Conjugates of Anticancer Peptide, LyP-1 in Human Breast Cancer
abstract: PurposeLyP-1, a nine-amino-acid tumor homing peptide, selectively binds to its cognate receptor, p32. Overexpression of p32 in certain tumors should allow use of LyP-1 as a targeting agent for the delivery of therapeutic or diagnostic agents. Peptide conjugates are developed for enhanced pre-targeting of MDA-MB-231 breast cancer cells with peptide-antibody bispecific complexes and targeting with multiple-drug/-fluorophore-conjugated nano-polymers.MethodsLyP-1-anti-DTPA bispecific antibody complexes (LyP-1-bsAbCx) were generated by conjugation of anti-DTPA antibody and LyP-1. LyP-1–doxorubicin (Dox), Dox-DTPA-succinyl-polylysine (Dox-DSPL), Dox-DSPL-LyP-1, DTPA-Dox-poly glutamic acid (D-Dox-PGA) or DTPA-rhodamine conjugated polylysine (DSPL-RITC) were prepared. In vitro therapeutic efficacy and targeting by immunofluorescence in MDA-MB-231 breast cancer cells were assessed with Dox-LyP-1. Immunofluorescence visualization of cancer cells was evaluated after pretargeting with LyP-1-bsAbCx and targeting with DSPL-RITC.ResultsCytotoxicity of Dox-LyP-1 conjugates was significantly greater than free doxorubicin (p < 0.0001). For fluorescent-labeled LyP-1, internalization occurred in 30 min in tumor cells. Fluorescence intensity of two-step targeted cells showed that pretargeting with LyP-1-bsAbC, followed by targeting with DSPL-RITC was greater than non-pretargeted DSPL-RITC (p < 0.05).ConclusionsPeptide-conjugates are effective targeting agents for MDA-MB-231 breast cancer cells in culture. LyP-1-bsAbCx and Dox-LyP-1 conjugates may allow development of novel targeted cancer therapy and diagnosis.

title: Increasing Tumor Accessibility with Conjugatable Disulfide-Bridged Tumor-Penetrating Peptides for Cancer Diagnosis and Treatment
abstract: Tumor-homing peptides with tissue-penetrating properties increase the efficacy of targeted cancer therapy by delivering an anticancer agent to the tumor interior. LyP-1 (CGNKRTRGC) and iRGD (CRGDKGPDC) are founding members of this class of peptides. The presence of the cysteines forming the cyclizing disulfide bond complicates conjugation of these peptides with other molecules, such as drugs. Here, we report the synthesis of conjugatable disulfide-bridged peptides and their conjugation to biologically important molecules. We have synthesized the LyP-1, iRGD, and CRGDC (GACRGDCLGA) peptides with a cysteine or maleimidohexanoic acid added externally at N-terminus of the sequences. Subsequent conjugation to payloads yielded stable compounds in which the tumor-homing properties of the peptide and the biological activity of the payload were retained.

title: Molecular dynamics, thermodynamic, and mutational binding studies for tumor-specific LyP-1 in complex with p32
abstract: Recent studies in tumor homing peptides have shown the specificity of LyP-1 (CGNKRTRGC) to tumor lymphatics. In this present work, we evaluated the possible interactions between cyclic LyP-1 and its receptor, p32, with molecular dynamics and docking studies in order to lead the design of novel LyP-1 derivatives, which could bind to p32 more effectively and perform enhanced antitumor effect. The total binding enthalpy energies have been obtained by MM-PBSA thermodynamic computations and the favorability of p32.LyP-1 complex in water has been shown by explicit water MD computations. The last 30 ns of molecular dynamics trajectory have shown the strong interaction of LyP-1 with the inner surface chains of p32, especially with chains B and C. ALA-SCAN mutagenesis studies have indicated the considerable influence of Asn3, Lys4, Arg5, and Arg7 amino acid residues on the specific binding of LyP-1. Within the knowledge of the critical role of p32 receptor in cancer cell metabolism, this study can lead to further developments in anticancer therapy by targeting p32 with LyP-1 derivatives as active targeting moiety. This data can also be applied for the development of new drug delivery systems in which LyP-1 can be used for its targeting and anticancer properties.

title: Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein.
abstract: Human p32 (also known as SF2-associated p32, p32/TAP, and gC1qR) is a conserved eukaryotic protein that localizes predominantly in the mitochondrial matrix. It is thought to be involved in mitochondrial oxidative phosphorylation and in nucleus-mitochondrion interactions. We report the crystal structure of p32 determined at 2.25 A resolution. The structure reveals that p32 adopts a novel fold with seven consecutive antiparallel beta-strands flanked by one N-terminal and two C-terminal alpha-helices. Three monomers form a doughnut-shaped quaternary structure with an unusually asymmetric charge distribution on the surface. The implications of the structure on previously proposed functions of p32 are discussed and new specific functional properties are suggested.

title: Molecular Cloning of Human Fibroblast Hyaluronic Acid-binding Protein Confirms Its Identity with P-32, a Protein Co-purified with Splicing Factor SF2
abstract: The purification of a 68-kDa hyaluronic acid-binding protein (HA-binding protein), a homodimer of 34 kDa that binds specifically to hyaluronic acid, has been reported earlier by us (Gupta, S., Batchu, R. B., and Datta, K. (1991) ). Here, we report the isolation of a partial cDNA clone from a gt cDNA expression library of human skin fibroblast by immunoscreening with HA-binding protein antiserum. The internal polypeptide sequence (83 residues) of the purified hyaluronic acid-binding protein is identical to the predicted protein sequence derived from hyaluronic acid-binding protein cDNA, suggesting the authenticity of the clone. Interestingly, this hyaluronic acid-binding protein cDNA sequence has complete homology with the cDNA sequence of a protein P-32, co-purified with the human pre-mRNA splicing factor SF2 (Krainer, A. R., 5eda, A., Kozak, D., and Binns, G.(1991) Cell 66, 383-394). Furthermore, the data on the N-terminal sequence of hyaluronic acid-binding protein and the predicted polypeptide of P-32 revealed the identical coding sequence of 209 amino acids for both the proteins. As the identity and functional characterization of P-32 have not yet been reported, P-32 cDNA was expressed in Escherichia coli, and the recombinant P-32 protein was purified by hyaluronic acid-Sepharose affinity chromatography. The recombinant P-32 protein showed immunocross-reactivity with the polyclonal antibodies raised against HA-binding protein. The predicted amino acid sequence of the protein fulfilled the minimal criteria for binding to hyaluronic acid, i.e. two basic amino acids flanking a seven-amino acid stretch, as reported for other hyaluronic acid-binding proteins. Furthermore, the hyaluronic acid affinity of the recombinant P-32 protein was confirmed by biotinylated hyaluronic acid binding assay. The binding of recombinant P-32 protein to biotinylated hyaluronic acid can be competed only with excess unlabeled hyaluronic acid, confirming its specificity toward hyaluronic acid. All these results suggest that both P-32, co-purified with the human pre-mRNA splicing factor SF2, and 34-kDa hyaluronic acid-binding protein reported by us are the same protein and that it is a new member of the hyaluronic acid- binding protein family, the “hyaladherins.”

title: The multiligand-binding protein gC1qR, putative C1q receptor, is a mitochondrial protein.
abstract: A protein of 33 kDa (p33) that tightly binds to the globular domains of the first complement component, C1q, is thought to serve as the major C1q receptor (gC1qR) on B cells, neutrophils, and mast cells. However, the cellular routing and the subcellular localization of p33/gC1qR are unknown. We have performed confocal laser-scanning microscopy and found that p33/gC1qR is present in intracellular compartments, where it colocalizes with the mitochondrial marker protein, pyruvate dehydrogenase. No surface staining for p33/gC1qR on endothelial EA.hy926 cells was observed. A fusion protein of the p33/gC1qR presequence with green fluorescent protein translocated to the mitochondria of transfected COS-7 cells. Concomitantly, a 6-kDa portion of the fusion protein was proteolytically removed. The 33 amino-terminal residues of the presequence proved sufficient to direct reporter constructs to mitochondria. Association of p33/gC1qR with mitoplasts indicated that the mature protein of 209 residues resides in the matrix and/or the inner membrane of mitochondria. Immunocytochemistry of fetal mice tissues revealed a ubiquitous expression of p33/gC1qR, most prominently in tissues that are rich in mitochondria. Thus, the candidate complement receptor p33/gC1qR of intact cells cannot interact with plasma C1q due to mutually exclusive localizations of the components. The functional role of p33/gC1qR needs to be reconsidered.

title: Localization of P32 protein (gC1q-R) in mitochondria and at specific extramitochondrial locations in normal tissues
abstract: P32 protein, also known as the gC1q receptor for complement component C1q, is a binding protein for nuclear pre-mRNA splicing factor SF2/ASF and numerous other nuclear and cell surface proteins, yet is targeted to the mitochondrial matrix compartment where these proteins are not present. In the present study, we use immunogold electron microscopy to evaluate the subcellular distribution of P32 protein (gC1q-R) in cultured cell lines and in rat tissues embedded in the acrylic resin LR Gold. Immunogold labeling of Raji lymphoma, CHO, human fibroblasts, HeLa and B-SC-1 cells shows reactivity primarily within mitochondria. Highly specific labeling of mitochondria is also obtained in rat tissues, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen, testes and thyroid. However, strong P32 (gC1q-R) reactivity is also present in (i) zymogen granules, condensing vacuoles, endoplasmic reticulum, and on the cell surface of pancreatic acinar cells, (ii) on the cell surface of microvascular endothelial cells in pancreas and kidney, (iii) on the cell surface and in nuclei of splenic lymphocytes, and (iv) in the acrosome of developing spermatids in testes. Western immunoblots show that the polyclonal antibody to P32 (gC1q-R) used in this study reacts specifically with a 32-kDa protein in both purified pancreatic zymogen granules and in mitochondria, and no other proteins are reactive. These results provide evidence that P32 (gC1q-R) is a mitochondrial protein that also localizes outside mitochondria in certain cells and tissues under normal physiological conditions.

title: Protein kinase C [micro] is regulated by the multifunctional chaperon protein p32.
abstract: We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKCalpha, PKCzeta, PKCdelta, and PKC mu. We have analyzed the interaction of PKC mu with p32 in detail, and we show here in vivo association of PKC mu, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC mu is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKC mu in a compartment-specific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKC mu, it does not serve as a substrate. Interestingly, PKC mu-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKC mu, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKC mu kinase activity.

title: p32 Protein, a Splicing Factor 2-associated Protein, Is Localized in Mitochondrial Matrix and Is Functionally Important in Maintaining Oxidative Phosphorylation*
abstract: Human p32, originally cloned as a splicing factor 2-associated protein, has been reported to interact with a variety of molecules including human immunodeficiency virus Tat and complement 1q (C1q). p32 protein is supposed to be in the nucleus and on the plasma membrane for the association with human immunodeficiency virus Tat and C1q, respectively. None of the interactions, however, is proven to have a physiological role. To investigate the physiological function of p32, we determined the intracellular localization of p32. The fractionation of cells, fluorescent immunocytochemistry, and electron microscopic immunostaining show that p32 is exclusively localized in the mitochondrial matrix. We cloned a Saccharomyces cerevisiaehomologue of human p32 gene, referred to yeast p30 gene. The yeast p30 protein is also localized in the mitochondrial matrix. The disruption of the p30 gene caused the growth retardation of yeast cells in a glycerol medium but not in a glucose medium, i.e. the impairment of the mitochondrial ATP synthesis. The growth impairment was restored by the introduction of the human p32 cDNA, indicating that p30 is a functional yeast counterpart of human p32. Taken together, both p32 and p30 reside in mitochondrial matrix and play an important role in maintaining mitochondrial oxidative phosphorylation.

title: Truncated variants of hyaluronan-binding protein 1 bind hyaluronan and induce identical morphological aberrations in COS-1 cells.
abstract: Hyaluronan (HA)-binding protein 1 (HABP1) is multifunctional in nature and exists as a trimer through coiled-coil interaction between alpha-helices at its N- and C-termini. To investigate the importance of trimeric assemblage and HA-binding ability of HABP1, we generated and overexpressed variants of HABP1 by truncating the alpha-helices at its termini. Subsequently, these variants were transiently expressed in COS-1 cells to examine the influence of these structural variations on normal cell morphology, as compared with those imparted by HABP1. Substantiating the centrality of coiled-coil interaction for maintaining the trimeric assembly of HABP1, we demonstrate that disruption of trimerization does not alter the affinity of variants towards its ligand HA. Transient expression of HABP1 altered the morphology of COS-1 cells by generating numerous cytoplasmic vacuoles along with disruption of the f-actin network. Interestingly, the truncated variants also imparted identical morphological changes. Characterization of the cytoplasmic vacuoles revealed that most of these vacuoles were autophagic in nature, resembling those generated under stress conditions. The identical morphological changes manifested in COS-1 cells on transient expression of HABP1 or its variants is attributed to their comparable HA-binding ability, which in concert with endogenous HABP1, may deplete the cellular HA pool. Such quenching of HA below a threshold level in the cellular milieu could generate a stress condition, manifested through cytoplasmic vacuoles and a disassembly of the f-actin network.

title: Structure Reconstruction of LyP-1: Lc(LyP-1) Coupling by Amide Bond Inspires the Brain Metastatic Tumor Targeted Drug Delivery.
abstract: The stability and binding affinity of targeting ligands are very important in active targeting drug delivery. Herein we used LyP-1 peptide as a model peptide to investigate chemical-biology-based strategies in the design of peptide ligands for active targeting. LyP-1 is a short peptide cyclized with a disulfide bond. It can specifically bind to tumor cells and tumor lymphatics through the interaction with cell-surface protein p32/gC1qR. Lc(LyP-1), with a same sequence of LyP-1, is coupled by amide bond. It showed better cellular uptake and stability in blood in our previous research. Further, usually d-peptide demonstrates higher stability than l-peptide, and it may contribute to better active targeting ability in vivo. Herein, we designed a retro-inverso isomer of Lc(LyP-1), termed Dc(LyP-1), expecting to inspire brain metastatic tumor targeted drug delivery. However, although Lc(LyP-1) showed lower stability than Dc(LyP-1) in fresh rat bold serum, both the 4T1 cellular uptake capacity (89.20%) and p32 protein binding affinity (7.39 × 10-6) were significantly higher than those (33.41%, 1.37 × 10-5) of Dc(LyP-1). Further, Lc(LyP-1) modified PEG-PLA micelles displayed much higher in vivo distribution in brain metastatic tumor than Dc(LyP-1). All results suggested that Lc(LyP-1) had a better performance than Dc(LyP-1) in brain metastatic tumor-targeted drug delivery.

title: A novel small molecule inhibitor of p32 mitochondrial protein overexpressed in glioma
abstract: BackgroundThe mitochondrial protein p32 is a validated therapeutic target of cancer overexpressed in glioma. Therapeutic targeting of p32 with monoclonal antibody or p32-binding LyP-1 tumor-homing peptide can limit tumor growth. However, these agents do not specifically target mitochondrial-localized p32 and would not readily cross the blood–brain barrier to target p32-overexpressing gliomas. Identifying small molecule inhibitors of p32 overexpressed in cancer is a more rational therapeutic strategy. Thus, in this study we employed a pharmacophore modeling strategy to identify small molecules that could bind and inhibit mitochondrial p32.MethodsA pharmacophore model of C1q and LyP-1 peptide association with p32 was used to screen a virtual compound library. A primary screening assay for inhibitors of p32 was developed to identify compounds that could rescue p32-dependent glutamine-addicted glioma cells from glutamine withdrawal. Inhibitors from this screen were analyzed for direct binding to p32 by fluorescence polarization assay and protein thermal shift. Affect of the p32 inhibitor on glioma cell proliferation was assessed by Alamar Blue assay, and affect on metabolism was examined by measuring lactate secretion.ResultsIdentification of a hit compound (M36) validates the pharmacophore model. M36 binds directly to p32 and inhibits LyP-1 tumor homing peptide association with p32 in vitro. M36 effectively inhibits the growth of p32 overexpressing glioma cells, and sensitizes the cells to glucose depletion.ConclusionsThis study demonstrates a novel screening strategy to identify potential inhibitors of mitochondrial p32 protein overexpressed in glioma. High throughput screening employing this strategy has potential to identify highly selective, potent, brain-penetrant small molecules amenable for further drug development.

title: C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration
abstract: Screening of phage libraries expressing random peptides for binding to prostate cancer cells primarily yielded peptides that had a C-terminal arginine (or rarely lysine) residue, usually in a consensus context R/KXXR/K. Phage expressing these sequences and synthetic nanoparticles coated with them bound to and were internalized into cells. The C-terminal arginine (or lysine) was essential to the activity; adding another amino acid, or even blocking the free carboxyl group of this arginine residue by amidation, eliminated the binding and internalizing activity. An internal R/KXXR/K can be exposed and switched on by a cleavage by a protease. The strict requirement for C-terminal exposure of the motif prompted us to term the phenomenon the C-end rule (CendR). Affinity chromatography showed that the CendR peptides bind to neuropilin-1 (NRP-1) on the target cells. NRP-1 is a cell-surface receptor that plays an essential role in angiogenesis, regulation of vascular permeability, and the development of the nervous system. VEGF-A165 and other ligands of NRP-1 possess a C-terminal CendR sequence that interacts with the b1 domain of NRP-1 and causes cellular internalization and vascular leakage. Our CendR peptides have similar effects, particularly when made multivalent through coupling to a particle. We also noted a unique and important activity of these peptides: penetration and transportation through tissues. The peptides were able to take payloads up to the nanoparticle size scale deep into extravascular tissue. Our observations have implications in drug delivery and penetration of tissue barriers and tumors.

title: Coadministration of a Tumor-Penetrating Peptide Enhances the Efficacy of Cancer Drugs
abstract: Penetrating Attack on Tumors While considerable research effort in oncology is focused on the design of new cancer drugs, an important but relatively understudied research area is the development of methods that optimize the delivery and tumor penetration of existing cancer drugs. Previous work has characterized a peptide (iRGD) that selectively targets and penetrates tumor tissue by virtue of its specific interaction with tumor blood vessels. Now, studying mouse models, Sugahara et al. (p. 1031, see the cover) show that coinjection of the iRGD peptide increases the tumor penetration and antitumor activity of several cancer drugs, including the cytotoxic agent doxorubicin and the therapeutic antibody trastuzumab (Herceptin), without increasing their harmful effects on healthy tissue. Importantly, these effects did not require chemical conjugation of the cancer drugs to the peptide. Anticancer drugs are more effective in mice when they are injected with a peptide that helps the drugs penetrate the tumor. Poor penetration of anticancer drugs into tumors can be an important factor limiting their efficacy. We studied mouse tumor models to show that a previously characterized tumor-penetrating peptide, iRGD, increased vascular and tissue permeability in a tumor-specific and neuropilin-1–dependent manner, allowing coadministered drugs to penetrate into extravascular tumor tissue. Importantly, this effect did not require the drugs to be chemically conjugated to the peptide. Systemic injection with iRGD improved the therapeutic index of drugs of various compositions, including a small molecule (doxorubicin), nanoparticles (nab-paclitaxel and doxorubicin liposomes), and a monoclonal antibody (trastuzumab). Thus, coadministration of iRGD may be a valuable way to enhance the efficacy of anticancer drugs while reducing their side effects, a primary goal of cancer therapy research.

title: Transtumoral targeting enabled by a novel neuropilin-binding peptide
abstract: We have recently described a class of peptides that improve drug delivery by increasing penetration of drugs into solid tumors. These peptides contain a C-terminal C-end Rule (CendR) sequence motif (R/K)XX(R/K), which is responsible for cell internalization and tissue-penetration activity. Tumor-specific CendR peptides contain both a tumor-homing motif and a cryptic CendR motif that is proteolytically unmasked in tumor tissue. A previously described cyclic tumor-homing peptide, LyP-1 (sequence: CGNKRTRGC), contains a CendR element and is capable of tissue penetration. We use here the truncated form of LyP-1, in which the CendR motif is exposed (CGNKRTR; ), and show that both LyP-1 and tLyP-1 internalize into cells through the neuropilin-1-dependent CendR internalization pathway. Moreover, we show that neuropilin-2 also binds tLyP-1 and that this binding equally activates the CendR pathway. Fluorescein-labeled tLyP-1 peptide and tLyP-1-conjugated nanoparticles show robust and selective homing to tumors, penetrating from the blood vessels into the tumor parenchyma. The truncated peptide is more potent in this regard than the parent peptide LyP-1. tLyP-1 furthermore improves extravasation of a co-injected nanoparticle into the tumor tissue. These properties make tLyP-1 a promising tool for targeted delivery of therapeutic and diagnostic agents to breast cancers and perhaps other types of tumors.

title: Specific penetration and accumulation of a homing peptide within atherosclerotic plaques of apolipoprotein E-deficient mice
abstract: The ability to selectively deliver compounds into atherosclerotic plaques would greatly benefit the detection and treatment of atherosclerotic disease. We describe such a delivery system based on a 9-amino acid cyclic peptide, LyP-1. LyP-1 was originally identified as a tumor-homing peptide that specifically recognizes tumor cells, tumor lymphatics, and tumor-associated macrophages. As the receptor for LyP-1, p32, is expressed in atherosclerotic plaques, we tested the ability of LyP-1 to home to plaques. Fluorescein-labeled LyP-1 was intravenously injected into apolipoprotein E (ApoE)-null mice that had been maintained on a high-fat diet to induce atherosclerosis. LyP-1 accumulated in the plaque interior, predominantly in macrophages. More than 60% of cells released from plaques were positive for LyP-1 fluorescence. Another plaque-homing peptide, CREKA, which binds to fibrin-fibronectin clots and accumulates at the surface of plaques, yielded fewer positive cells. Tissues that did not contain plaque yielded only traces of LyP-1+ cells. LyP-1 was capable of delivering intravenously injected nanoparticles to plaques; we observed abundant accumulation of LyP-1–coated superparamagnetic iron oxide nanoparticles in the plaque interior, whereas CREKA-nanoworms remained at the surface of the plaques. Intravenous injection of 4-[18F]fluorobenzoic acid ([18F]FBA)-conjugated LyP-1 showed a four- to sixfold increase in peak PET activity in aortas containing plaques (0.31% ID/g) compared with aortas from normal mice injected with [18F]FBA-LyP-1(0.08% ID/g, P < 0.01) or aortas from atherosclerotic ApoE mice injected with [18F]FBA-labeled control peptide (0.05% ID/g, P < 0.001). These results indicate that LyP-1 is a promising agent for the targeting of atherosclerotic lesions.

title: Plaque-penetrating peptide inhibits development of hypoxic atherosclerotic plaque.
abstract: Atherosclerosis treatments are generally aimed at altering systemic lipid metabolism such that atherogenesis, the formation of plaque, is curtailed. The plaques themselves offer some potential therapeutic targets. For example, selective depletion of macrophages, which play a key role in atherogenesis, inhibits plaque formation. However, it has not been possible to take advantage of these targets because the drugs that have been tested have not been sufficiently selective. We have developed a peptide, LyP-1, which specifically targets atherosclerotic plaques, penetrates into plaque interior, and accumulates in plaque macrophages. In tumors, LyP-1 can cause apoptosis in cells that take up the peptide. Here we show, using three different atherosclerosis models in ApoE null mice that prolonged systemic treatment with LyP-1 triggers apoptosis of plaque macrophages and reduces plaque in advanced hypoxic plaques, and that it does so without increasing necrotic core of plaques or causing detectable side effects. We also show that LyP-1 recognizes human plaque. These findings suggest that LyP-1 could serve as a lead compound for the development of a new class of anti-atherosclerosis drugs.

title: Mitochondrial p32 Protein Is a Critical Regulator of Tumor Metabolism via Maintenance of Oxidative Phosphorylation
abstract: HABP1 is a mitochondrial/cell surface protein overexpressed in certain cancer cells. Here we show that knocking down p32 expression in human cancer cells strongly shifts their metabolism from oxidative phosphorylation (OXPHOS) to glycolysis. The p32 knockdown cells exhibited reduced synthesis of the mitochondrial-DNA-encoded OXPHOS polypeptides and were less tumorigenic in vivo. Expression of exogenous p32 in the knockdown cells restored the wild-type cellular phenotype and tumorigenicity. Increased glucose consumption and lactate production, known as the Warburg effect, are almost universal hallmarks of solid tumors and are thought to favor tumor growth. However, here we show that a protein regularly overexpressed in some cancers is capable of promoting OXPHOS. Our results indicate that high levels of glycolysis, in the absence of adequate OXPHOS, may not be as beneficial for tumor growth as generally thought and suggest that tumor cells use p32 to regulate the balance between OXPHOS and glycolysis.

title: Peptide Targeting of Tumor Lymph Vessels
abstract: In addition to having a blood vasculature, most normal organs and pathologic conditions involve a second vascular system, the lymphatic vasculature, and many tumors induce the growth of new lymphatic vessels. However, compared to the blood vasculature, very little is known about the lymphatic vessels in tumors. We have used the in vivo phage display technology to map tumor‐specific differences in the lymphatic vasculature, and identified peptides that specifically home to tumor lymphatics. Each of these peptides recognizes lymphatic vessels in a different set of tumors, and some of them also recognize tumor cells. Furthermore, these peptides can differentiate lymphatic vasculature of a premalignant lesion from that of a full‐blown tumor, indicating tumor stage–specific differences in the lymphatic vessels. None of the lymphatic homing peptides recognizes blood endothelial cells, nor do they home to any normal organ. Of interest, some of our homing peptides are able to penetrate the target cells in a cell type–specific manner. These peptides appear to be able to concentrate in the target tissue, making them particularly efficient delivery vectors for the targeting of therapeutic moieties and imaging agents. Conjugation of the lymphatic homing peptides to drugs provides an opportunity to specifically deliver therapeutic agents into tumors using a route not previously exploited. The surprising degree of selectivity of these lymphatic homing peptides suggests extensive molecular specialization of tumor lymphatic vessels, positing the existence of a molecular lymphatic “zip code” system, as has been previously demonstrated for the tumor blood vasculature.

title: Effective tumor targeting and enhanced anti-tumor effect of liposomes engrafted with peptides specific for tumor lymphatics and vasculature.
abstract: The use of liposomes to target drugs to tumors represents an attractive therapeutic strategy, especially when used with convenient targeting moieties such as peptides. Here we explored several peptides for their ability to target liposomes to tumors. The metal chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) was incorporated into liposomes to enable the engraftment of His-tagged peptides containing targeting motifs specific for tumor vasculature markers VEGFR-1 (p39-Flt-1) and neuropilin-1 (p24-NRP-1), or a motif known to accumulate in hypoxic areas of tumors (p47-LyP-1). Peptide-engrafted liposomes were examined for their biodistribution and anti-tumor effects after i.v. administration. Our results show that radiolabelled liposomes engrafted with either p24-NRP-1 or p47-LyP-1 and then injected into mice bearing subcutaneous B16-F1 tumors, show increased accumulation in the tumor. For p24-NRP-1-liposomes, tumor targeting was significantly increased when the stabilizing lipid phosphatidylethanolamine polyethylene glycol-750 (PE-PEG(750)) was used instead of PE-PEG(2000) in the liposome lipid mixture. Importantly, compared to the controls, p24-NRP-1 liposomes containing 10 mol% PE-PEG(750) and loaded with doxorubicin significantly inhibited the rate of tumor growth in the tumor-bearing mice. Our findings demonstrate that the use of drug-containing liposomes incorporating NTA(3)-DTDA and engrafted with NRP-1 targeting peptide is a convenient strategy to enhance the therapeutic effect of non-targeted doxorubicin.

title: LyP-1-conjugated PEGylated liposomes: a carrier system for targeted therapy of lymphatic metastatic tumor.
abstract: The application of liposomes in targeted therapy of lymphatic metastatic tumors has been hampered by the low uptake rate of liposome by metastatic lymph nodes. In this report, LyP-1, a peptide that can specifically bind tumor cells, tumor lymphatics and tumor-associated macrophages, was conjugated to liposomes for targeting and treating lymphatic metastatic tumors. Firstly, LyP-1-conjugated PEGylated liposomes loaded with fluorescein or doxorubicin (DOX) were prepared and showed satisfactory vesicle size and size distribution. The in vitro cellular uptake and in vivo near-infrared fluorescence imaging results showed that LyP-1 modification increased liposome uptake by tumor cells and metastatic lymph nodes, but did not increase uptake by normal lymph nodes. The immunofluorescence analysis evidenced that LyP-1-conjugated liposomes were distributed adjacent to tumor lymphatics and tumor-associated macrophages in metastatic lymph nodes. The pharmacodynamic study suggested that compared with unmodified liposomes, LyP-1-conjugated DOX-loaded liposomes exhibited enhanced inhibition effect on tumor cells in vitro and lymphatic metastatic tumors in vivo. Pathological examination showed that liposomal DOX caused reduced tissue damage to injection site compared with DOX solution. In summary, LyP-1-conjugated PEGylated liposomes could be targeted to metastatic lymph nodes based on their specific binding to tumor cells, tumor lymphatics and tumor-associated macrophages. They are a safe and effective drug delivery system of antineoplastic agents for targeted therapy of lymphatic metastatic tumors.

title: In vivo tumor cell targeting with "click" nanoparticles.
abstract: The in vivo fate of nanomaterials strongly determines their biomedical efficacy. Accordingly, much effort has been invested into the development of library screening methods to select targeting ligands for a diversity of sites in vivo. Still, broad application of chemical and biological screens to the in vivo targeting of nanomaterials requires ligand attachment chemistries that are generalizable, efficient, covalent, orthogonal to diverse biochemical libraries, applicable under aqueous conditions, and stable in in vivo environments. To date, the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition or "click" reaction has shown considerable promise as a method for developing targeted nanomaterials in vitro. Here, we investigate the utility of "click" chemistry for the in vivo targeting of inorganic nanoparticles to tumors. We find that "click" chemistry allows cyclic LyP-1 targeting peptides to be specifically linked to azido-nanoparticles and to direct their binding to p32-expressing tumor cells in vitro. Moreover, "click" nanoparticles are able to stably circulate for hours in vivo following intravenous administration (>5 h circulation time), extravasate into tumors, and penetrate the tumor interstitium to specifically bind p32-expressing cells in tumors. In the future, in vivo use of "click" nanomaterials should expedite the progression from ligand discovery to in vivo evaluation and diversify approaches toward multifunctional nanoparticle development.

title: Quantitative SPECT/CT: SPECT joins PET as a quantitative imaging modality
abstract: The introduction of combined modality single photon emission computed tomography (SPECT)/CT cameras has revived interest in quantitative SPECT. Schemes to mitigate the deleterious effects of photon attenuation and scattering in SPECT imaging have been developed over the last 30 years but have been held back by lack of ready access to data concerning the density of the body and photon transport, which we see as key to producing quantitative data. With X-ray CT data now routinely available, validations of techniques to produce quantitative SPECT reconstructions have been undertaken. While still suffering from inferior spatial resolution and sensitivity compared to positron emission tomography (PET) imaging, SPECT scans nevertheless can be produced that are as quantitative as PET scans. Routine corrections are applied for photon attenuation and scattering, resolution recovery, instrumental dead time, radioactive decay and cross-calibration to produce SPECT images in units of kBq.ml−1. Though clinical applications of quantitative SPECT imaging are lacking due to the previous non-availability of accurately calibrated SPECT reconstructions, these are beginning to emerge as the community and industry focus on producing SPECT/CT systems that are intrinsically quantitative.

title: Radiolabeling LyP-1 peptide and preliminary biodistribution evaluation in mice bearing MDA-MB-435 xenografts.
abstract: Recent studies have shown the LyP-1 peptide can home to either tumor lymphatics or the tumor cells and be internalized by targeted cells. This study aimed to investigate the possibility of using Na(131)I labeled LyP-1 peptide as an imaging agent or a therapeutic radiopharmaceutical in breast carcinoma and its metastasis.



The 10-mer cyclic peptide contained the LyP-1 sequence (YCGNKRTRGC) was synthesized by the solid phase method. Disulfide bonds between the cysteines maintain the cyclic structure. The LyP-1 peptide was labeled with Na(131)I using the chloramine-T method. The [(131)I] LyP-1 peptide and a [(131)I] control peptide were injected via tail vein into nude mice bearing MDA-MB-435 tumor xenografts. Biodistribution and imaging results in vivo were obtained.



The labeling efficiencies of LyP-1 peptide reached 80% ± 5% (n = 5). The radiochemical purity was about 96%. The radiochemical purity of the labeled compound remains 92% at 24 hours in human serum at 37°C. In the biodistribution studies, the [(131)I] LyP-1 peptide accumulated in the tumor to a higher level than in other organs. The [(131)I] LyP-1 peptide can successfully image the tumor in nude mice bearing MDA-MB-435 tumor xenografts.



The LyP-1 peptide could be effectively labeled with Na(131)I and the labeled compound is stable in human serum at 37°C for 24 hours. The high specificity of [(131)I] LyP-1 peptide suggests it may be a promising new radiotracer for identifying tumors.

title: Applications and potential toxicity of magnetic iron oxide nanoparticles.
abstract: Owing to their unique physical and chemical properties, magnetic iron oxide nanoparticles have become a powerful platform in many diverse aspects of biomedicine, including magnetic resonance imaging, drug and gene delivery, biological sensing, and hyperthermia. However, the biomedical applications of magnetic iron oxide nanoparticles arouse serious concerns about their pharmacokinetics, metabolism, and toxicity. In this review, the updated research on the biomedical applications and potential toxicity of magnetic iron oxide nanoparticles is summarized. Much more effort is required to develop magnetic iron oxide nanoparticles with improved biocompatible surface engineering to achieve minimal toxicity, for various applications in biomedicine.

title: Superparamagnetic iron oxide nanoparticles: magnetic nanoplatforms as drug carriers
abstract: A targeted drug delivery system is the need of the hour. Guiding magnetic iron oxide nanoparticles with the help of an external magnetic field to its target is the principle behind the development of superparamagnetic iron oxide nanoparticles (SPIONs) as novel drug delivery vehicles. SPIONs are small synthetic γ-Fe2O3 (maghemite) or Fe3O4 (magnetite) particles with a core ranging between 10 nm and 100 nm in diameter. These magnetic particles are coated with certain biocompatible polymers, such as dextran or polyethylene glycol, which provide chemical handles for the conjugation of therapeutic agents and also improve their blood distribution profile. The current research on SPIONs is opening up wide horizons for their use as diagnostic agents in magnetic resonance imaging as well as for drug delivery vehicles. Delivery of anticancer drugs by coupling with functionalized SPIONs to their targeted site is one of the most pursued areas of research in the development of cancer treatment strategies. SPIONs have also demonstrated their efficiency as nonviral gene vectors that facilitate the introduction of plasmids into the nucleus at rates multifold those of routinely available standard technologies. SPION-induced hyperthermia has also been utilized for localized killing of cancerous cells. Despite their potential biomedical application, alteration in gene expression profiles, disturbance in iron homeostasis, oxidative stress, and altered cellular responses are some SPION-related toxicological aspects which require due consideration. This review provides a comprehensive understanding of SPIONs with regard to their method of preparation, their utility as drug delivery vehicles, and some concerns which need to be resolved before they can be moved from bench top to bedside.

title: Superparamagnetic iron oxide nanoparticles for delivery of therapeutic agents: opportunities and challenges
abstract: Bearing in mind that many promising drug candidates have the problem of reaching their target site, the concept of advanced drug delivery can play a significant complementary role in shaping modern medicine. Among other nanoscale drug carriers, superparamagnetic iron oxide nanoparticles (SPIONs) have shown great potential in nanomedicine. The intrinsic properties of SPIONs, such as inherent magnetism, broad safety margin and the availability of methods for fabrication and surface engineering, pave the way for diverse biomedical applications. SPIONs can achieve the highest drug targeting efficiency among carriers, since an external magnetic field locally applied to the target organ enhances the accumulation of magnetic nanoparticles in the drug site of action. Moreover, theranostic multifunctional SPIONs make simultaneous delivery and imaging possible. In spite of these favorable qualities, there are some toxicological concerns, such as oxidative stress, unpredictable cellular responses and induction of signaling pathways, alteration in gene expression profiles and potential disturbance in iron homeostasis, that need to be carefully considered. Besides, the protein corona at the surface of the SPIONs may induce few shortcomings such as reduction of SPIONs targeting efficacy.  In this review, we will present recent developments of SPIONs as theranostic agents. The article will further address some barriers on drug delivery using SPIONs.  One of the major success determinants in targeted in vivo drug delivery using SPIONs is the adequacy of magnetic gradient. This can be partially achieved by using superconducting magnets, local implantation of magnets and application of magnetic stents. Other issues that must be considered include the pharmacokinetics and in vivo fate of SPIONs, their biodegradability, biocompatibility, potential side effects and the crucial impact of protein corona on either drug release profile or mistargeting. Surface modification of SPIONs can open up the possibility of drug delivery to intracellular organelles, drug delivery across the blood–brain barrier, modifying metabolic diseases and a variety of other multimodal and/or theranostic applications.

title: LyP-1 Conjugated Nanoparticles for Magnetic Resonance Imaging of Triple Negative Breast Cancer
abstract: PurposeTriple-negative breast cancer (TNBC) does not express estrogen receptor, progesterone receptor, or Her2/neu. Both diagnosis and treatment of TNBC remain a clinical challenge. LyP-1 is a cyclic 9 amino acid peptide that can bind to breast cancer cells. The goal of this study was to design and characterize LyP-1 conjugated to fluorescent iron oxide nanoparticles (LyP-1-Fe3O4-Cy5.5) as a contrast agent for improved and specific magnetic resonance imaging (MRI) in a preclinical model of TNBC.ProceduresThe binding of LyP-1-Fe3O4-Cy5.5 to MDA-MB-231 TNBC cells was evaluated and compared to scrambled peptide bio-conjugated to iron oxide nanoparticles (Ctlpep-Fe3O4-Cy5.5) as a negative control. Following the in vitro study, the MDA-MB-231 cells were injected into mammary glands of nude mice. Mice were divided into two groups: control group received Ctlpep- Fe3O4-Cy5.5 and LyP-1 group received LyP-1-Fe3O4-Cy5.5 (tail vein injection at 2 mg/kg of Fe3O4). Mice were imaged with an in vivo fluorescence imager and a 9.4 T MRI system at various time points after contrast agent injection. The T2 relaxation time was measured to observe accumulation of the contrast agent in breast tumor and muscle for both targeted and non-targeted contrast agents.ResultsImmunofluorescence revealed dense binding of the LyP-1-Fe3O4-Cy5.5 contrast agent to MDA-MB-231 cells; while little appreciable binding was observed to the scrambled negative control (Ctlpep-Fe3O4-Cy5.5). Optical imaging performed in tumor-bearing mice showed increased fluorescent signal in mammary gland of animals injected by LyP-1-Fe3O4-Cy5.5 but not Ctlpep- Fe3O4-Cy5.5. The results were confirmed ex vivo by the 2.6-fold increase of fluorescent signal from LyP-1-Fe3O4-Cy5.5 in extracted tumors when compared to the negative control. In MR imaging studies, there was a statistically significant (P < 0.01) difference in normalized T2 between healthy breast and tumor tissue at 1, 2, and 24 h post injection of the LyP-1-Fe3O4-Cy5.5. In animals injected with LyP-1-Fe3O4, distinct ring-like structures were observed with clear contrast between the tumor core and rim.ConclusionThe results demonstrate that LyP-1-Fe3O4 significantly improves MRI contrast of TNBC, hence has the potential to be exploited for the specific delivery of cancer therapeutics.

title: Cooperative nanomaterial system to sensitize, target, and treat tumors
abstract: A significant barrier to the clinical translation of systemically administered therapeutic nanoparticles is their tendency to be removed from circulation by the mononuclear phagocyte system. The addition of a targeting ligand that selectively interacts with cancer cells can improve the therapeutic efficacy of nanomaterials, although these systems have met with only limited success. Here, we present a cooperative nanosystem consisting of two discrete nanomaterials. The first component is gold nanorod (NR) “activators” that populate the porous tumor vessels and act as photothermal antennas to specify tumor heating via remote near-infrared laser irradiation. We find that local tumor heating accelerates the recruitment of the second component: a targeted nanoparticle consisting of either magnetic nanoworms (NW) or doxorubicin-loaded liposomes (LP). The targeting species employed in this work is a cyclic nine-amino acid peptide LyP-1 (Cys-Gly-Asn-Lys-Arg-Thr-Arg-Gly-Cys) that binds to the stress-related protein, p32, which we find to be upregulated on the surface of tumor-associated cells upon thermal treatment. Mice containing xenografted MDA-MB-435 tumors that are treated with the combined NR/LyP-1LP therapeutic system display significant reductions in tumor volume compared with individual nanoparticles or untargeted cooperative system.

title: X-ray-computed tomography contrast agents.
abstract: X-ray computed tomography (CT) is a well-established tissue imaging technique employed in a variety of research and clinical  Specifically, CT is a non-invasive clinical diagnostic tool that allows for 3D visual reconstruction and segmentation of tissues of interest. High resolution CT systems can be used to perform non-destructive 3D imaging of a variety of tissue types and organ systems, such as: the gastrointestinal tract, cardiovascular system, renal tract, liver, lungs, bone, cartilage, tumorous tissue, etc. CT is one of the most prevalent diagnostic tools in terms of frequency-of-use and hospital availability.2 The use of CT is on the rise and the number of clinical CT scanners in operation worldwide is estimated at over 45,000.1b Today, over 70 million clinical CT scans are performed yearly in the U.S. alone. For a recent detailed analysis of the use of clinical CT imaging and data regarding the number of regular and contrast-enhanced CT scans performed annually in the U.S. we refer the reader to the “Nationwide Evaluation of X-ray Trends” survey published by the Conference for Radiation Control Program Directors (CRCPD).3 
 
The idea of using tomography (Greek: tomos = slice, graphein = draw) as a diagnostic tool in medicine was adopted soon after the discovery of X-rays by W. C. Roentgen in 1895. However, several more decades passed before the technology advanced sufficiently to bring those ideas to fruition. The first successful CT imaging device was built in 1972 by G. N. Hounsfield, at Electric and Musical Industries Ltd. In 1979, G. N. Hounsfield and South African physicist A. M. Cormack shared a Nobel Prize in medicine for their contributions to the field of X-ray CT imaging and diagnostics.4 
 
X-rays are a form of electromagnetic radiation with wavelengths roughly between 0.01 nm and 10 nm. Traditionally, X-rays are generated by a vacuum tube using high voltage to accelerate electrons from a cathode to a (usually) tungsten-alloy anode. In the process, the accelerated electrons release electromagnetic radiation in the form of X-rays and the maximum energy of the radiation is limited by the energy of the incident electron. Operating voltages of modern clinical CT scanners differ among instrument models and manufacturers, but generally fall between 80 kVp to 150 kVp. 
 
As a rule, materials possessing higher density (ρ) or high atomic number (Z) tend to better absorb X-rays. The relationship is best expressed in the formula for X-ray absorption coefficient (μ): 
 
 
μ≈ρZ4AE3 
 
(1) 
 
 
where “A” is the atomic mass and “E” is the X-ray energy. The strong relationship between absorption and atomic number is of significant importance in clinical applications. The Z4 factor allows for contrast levels of several orders of magnitude between different tissues and types of contrast agents. 
 
When an incident X-ray has energy equal or slightly greater than the binding energy of the K-shell electron of the atom, a large sudden increase in absorption coefficient is observed. This energy value is known as absorption edge (k), and the k value increases with atomic number of the element. Consequently, X-ray attenuating contrast media containing atoms of high atomic number (most commonly iodine or barium), are frequently used in clinical settings to obtain images of soft tissues. To generate images with the highest contrast to the surrounding tissue, the energy of the X-ray source can be adjusted to closely match the absorption edge value (k) of the relevant imaging-agent atoms (i.e., iodine, barium, gold, etc.). Thus, it is also possible to do selective X-ray imaging and to differentiate between attenuating materials by fine tuning the energy source to the appropriate absorption edge value. 
 
A CT image is obtained by rotating an X-ray source around an object, with a detector positioned directly opposite the radiation source. Alternatively, in many preclinical CT scanners the object sometimes is rotated around its own axis. Such preclinical scanners are often being used for small animal in vivo imaging. Generally, X-ray scans are taken at small angular increments during rotation around the object over 360°. A series of attenuation profiles or projections is thus obtained. The projections are then processed mathematically to create a 3D rendition of the scanned object. An in depth description of the engineering principles underlying modern CT imaging instruments is beyond the scope of this manuscript, and the reader is referred to other published works.1c,5 
 
A diagnostic imaging method related to CT is X-ray fluoroscopy. Fluoroscopy allows for the acquisition of real-time, continuous images of the internal organs. Like in CT, imaging agents are often used in fluoroscopy for better contrast resolution. Small iodinated agents are commonly injected into blood vessels for use in fluoroscopic angiography, allowing for the evaluation of blood flow and visualization of the vasculature system, while barium contrast media are introduced orally or with an enema to investigate the anatomy (and pathology) of the gastrointestinal tract. 
 
The introduction of magnetic resonance imaging (MRI) resulted in a loss of interest and reduction in CT contrast agent development throughout the 1980s. However, advances in computer technology, and the introduction and widespread adoption of spiral-CT in the mid-1990s have sparked a revival of interest in CT imaging and CT contrast media. Current clinical CT scanners are capable of acquiring high resolution 3D isotropic images of the body within several minutes. CT imaging today is less time consuming, less expensive, and more readily available than other medical imaging technologies such as MRI and positron emission tomography (PET). In the last several years, the emergence of novel technologies such as dual-source CT, and multi-detector CT has advanced the field of CT imaging even further. As a comparison to X-ray imaging diagnostic methods, PET imaging employs gamma-ray emitting radioactive nuclei “tracers” as contrast agents while MRI takes advantage of nuclear magnetic resonance principles by applying high magnetic fields to align magnetization of certain atomic nuclei. In contrast to CT and PET imaging, MRI uses no ionizing radiation and it is therefore often deemed safer than the other two.

title: Ultrasmall Semimetal Nanoparticles of Bismuth for Dual-Modal Computed Tomography/Photoacoustic Imaging and Synergistic Thermoradiotherapy.
abstract: Multifunctional nanomaterials with integrated diagnostic and therapeutic functions, combination therapy to enhance treatment efficacy, as well as low toxicity have drawn tremendous attentions. Herein, we report a multifunctional theranostic agent based on peptide (LyP-1)-labeled ultrasmall semimetal nanoparticles of bismuth (Bi-LyP-1 NPs). Ultrasmall Bi NPs (3.6 nm) were facilely synthesized using oleylamine as the reducing agent and exhibited a higher tumor accumulation after being conjugated with the tumor-homing peptide LyP-1. The abilities to absorb both ionizing radiation and the second near-infrared (NIR-II) window laser radiation ensured that Bi-LyP-1 NPs are capable of dual-modal computed tomography/photoacoustic imaging and efficient synergistic NIR-II photothermal/radiotherapy of tumors. Moreover, Bi-LyP-1 NPs could be rapidly cleared from mice through both renal and fecal clearance and almost completely cleared after 30 days. Such multifunctional nanoparticles as efficient cancer theranostic agents, coupled with fast clearance and low toxicity, shed light on the future use of semimetal nanoparticles for biomedicine.

title: Ultrasound Microbubbles for Molecular Diagnosis, Therapy, and Theranostics
abstract: Ultrasound imaging is clinically established for routine screening examinations of breast, abdomen, neck, and other soft tissues, as well as for therapy monitoring. Microbubbles as vascular contrast agents improve the detection and characterization of cancerous lesions, inflammatory processes, and cardiovascular pathologies. Taking advantage of the excellent sensitivity and specificity of ultrasound for microbubble detection, molecular imaging can be realized by binding antibodies, peptides, and other targeting moieties to microbubble surfaces. Molecular microbubbles directed against various targets such as vascular endothelial growth factor receptor-2, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, selectins, and integrins were developed and were shown in preclinical studies to be able to selectively bind to tumor blood vessels and atherosclerotic plaques. Currently, the first microbubble formulations targeted to angiogenic vessels in prostate cancers are being evaluated clinically. However, microbubbles can be used for more than diagnosis: disintegrating microbubbles emit acoustic forces that are strong enough to induce thrombolysis, and they can also be used for facilitating drug and gene delivery across biologic barriers. This review on the use of microbubbles for ultrasound-based molecular imaging, therapy, and theranostics addresses innovative concepts and identifies areas in which clinical translation is foreseeable in the near future.

title: Current status and prospects for microbubbles in ultrasound theranostics.
abstract: Encapsulated microbubbles have been developed over the past two decades to provide improvements both in imaging as well as new therapeutic applications. Microbubble contrast agents are used currently for clinical imaging where increased sensitivity to blood flow is required, such as echocardiography. These compressible spheres oscillate in an acoustic field, producing nonlinear responses which can be uniquely distinguished from surrounding tissue, resulting in substantial enhancements in imaging signal-to-noise ratio. Furthermore, with sufficient acoustic energy the oscillation of microbubbles can mediate localized biological effects in tissue including the enhancement of membrane permeability or increased thermal energy deposition. Structurally, microbubbles are comprised of two principal components--an encapsulating shell and an inner gas core. This configuration enables microbubbles to be loaded with drugs or genes for additional therapeutic effect. Application of sufficient ultrasound energy can release this payload, resulting in site-specific delivery. Extensive preclinical studies illustrate that combining microbubbles and ultrasound can result in enhanced drug delivery or gene expression at spatially selective sites. Thus, microbbubles can be used for imaging, for therapy, or for both simultaneously. In this sense, microbubbles combined with acoustics may be one of the most universal theranostic tools.

title: LyP-1 ultrasonic microbubbles targeting to cancer cell as tumor bio-acoustics markers or drug carriers: Targeting efficiency evaluation in, microfluidic channels
abstract: Using ultrasonic contrast microbubbles as acoustic biomarkers and drug carrier vehicles by conjugating tumor specific antibody to microbubbles has shown great potential in ultrasonic tumor molecular imaging or drug-delivery and therapy. Microbubble probe targeting efficiency is one of the major challenges. In this study, we developed a novel method to evaluate the targeting capability and efficiency of microbubbles to cells, and more specifically, microbubbles binding LyP-1 (a cyclic nonapeptide acid peptide) target to cancer cell within a microfluidic system. The micro cell sieves within the microfludic channels could trap the tumor cells and enhance the microbubble's interaction with the cell. Assisted with the controllable fluid shear stress, the microbubble's targeting to the cell and the corresponding affinity efficiency could be quantitatively evaluated under a florescent microscope. The system provides a useful low-cost high efficient in vitro platform for studying microbubble-cell interaction for ultrasonic tumor molecular imaging or drug-delivery and therapy.

title: A novel microfluidic chip for assessing dynamic adhesion behavior of cell-targeting microbubbles.
abstract: The primary aim of this study was to develop a microfluidic chip to study the dynamic adhesion behavior of cell-targeted microbubbles. The microfluidic device is composed of polydimethylsiloxane and is fabricated using the soft lithography technique. Each chamber of the microfluidic chip comprises eight U-shaped microsieves, by which various flow velocity distributions are generated. LyP-1-conjugated microbubbles were prepared by coating the surface of the phospholipid shell of microbubbles with LyP-1 peptides via biotin-avidin linkage. Under static conditions, the resulting targeted microbubbles are able to bind onto the surface of cells on incubation with breast cancer cells. Under dynamic fluid conditions, the cell targeting efficiency of the microbubbles was assessed at various flow velocity distributions in a chamber. Accumulation of targeted microbubbles was strongly influenced by flow velocity. Better retention of targeted microbubbles on cell surfaces was achieved at low mean flow velocities (<0.03 cm/s), in agreement with our computer simulation results. In conclusion, our results indicate that the microfluidic system is a useful platform for studying the microbubble-cell adhesive interaction.

title: The lymph node pre-metastatic niche
abstract: Lymph node metastases occur frequently during the progression of many types of cancer, and their presence often reflects poor prognosis. The drainage of tumor-derived factors such as antigens, growth factors, cytokines, and exosomes through the lymphatic system to the regional lymph nodes plays an important role in the pre-metastatic conditioning of the microenvironment in lymph nodes, making them receptive and supportive metastatic niches for disseminating tumor cells. Modified immunological responses and remodeling of the vasculature are the most studied tumor-induced pre-metastatic changes in the lymph node microenvironment that promote metastasis, although other metastasis-relevant alterations are also starting to be studied. Here, I review our current understanding of the lymph node pre-metastatic niche, how tumors condition this niche, and the relevance of this conditioning for our understanding of the process of metastasis.

title: LyP-1-conjugated nanoparticles for targeting drug delivery to lymphatic metastatic tumors.
abstract: Active tumor targeting by biodegradable nanoparticles has been widely studied for cancer diagnosis and therapy. However, target-specific nanoparticles for drug delivery to lymphatic metastases have not been reported yet due to the lack of specific markers in the tumor lymphatics. Recently, peptide LyP-1 has been recognized for its specific home to tumors and their lymphatics. In this study, we tested the possibility of LyP-1 serving as a target-specific peptide of PEG-PLGA nanoparticles to tumor lymph metastases. LyP-1 was synthesized by using Boc-protected amino acids. The copolymers of maleimide-PEG-PLGA were formed by the conjugation of maleimide-PEG-NH(2) to PLGA-COOH, which were applied to prepare pegylated nanoparticles with mPEG-PLGA by means of double emulsion/solvent evaporation technique. LyP-1 with sulfhydryl group was conjugated to the maleimide function located at the distal end of PEG surrounding the nanoparticle surface. LyP-1-conjugated PEG-PLGA nanoparticle (LyP-1-NPs) had a round and regular shape with a diameter around 90 nm. In vitro, cellular uptake of LyP-1-NPs was about four times of that of PEG-PLGA nanoparticles without LyP-1 (NPs). In vivo, the uptake of LyP-1-NPs in metastasis lymph nodes was about eight times of that of NPs. This study indicates that LyP-1-NP is a promising carrier for target-specific drug delivery to lymphatic metastatic tumors.

title: Expression of gC1q-R/p33 and its major ligands in human atherosclerotic lesions.
abstract: A growing body of evidence supports the hypothesis that atherosclerosis has an inflammatory component, and that immune mechanisms, including complement activation, are likely to be involved. gC1q-R/p33 (gC1q-R) is a multifunctional and multicompartmental cellular protein, which is postulated to play a role in inflammation and thrombosis by interacting with C1q and high molecular weight kininogen (HK). To examine the expression of gC1q-R and its major ligands, C1q and HK, in human atherosclerotic lesions, sections of carotid arteries removed during endarterectomy and coronary arteries obtained at autopsy were stained with specific polyclonal or monoclonal antibodies. Control sections were stained with irrelevant rabbit IgG or isotype matched murine monoclonal antibody (MOPC), respectively. Tissue sections were counterstained with hematoxylin and examined by light microscopy. Specific staining for gC1q-R, C1q, and HK was observed in and around atherosclerotic lesions. In contrast to control antibodies, antibodies directed against gC1q-R reacted with endothelial cells, foam cells, smooth muscle cells, and inflammatory cells present in the intima and media of atherosclerotic lesions. In addition, the necrotic central core of advanced lesions with calcifications, fibrin, and lipids, stained intensely for gC1q-R, and negligibly with control antibodies. HK demonstrated a similar staining pattern, whereas C1q was most heavily expressed in the fibrous cap and necrotic core of atherosclerotic lesions. The localization of gC1q-R and its ligands C1q and HK in atherosclerotic lesions, and the previously described ability of gC1q-R to modulate complement, kinin, and coagulation cascades, suggest that gC1q-R may play an important role in promoting inflammation and thrombosis in atherosclerotic lesions.

title: Stabilization of atherosclerotic plaques: New mechanisms and clinical targets
abstract: Research points to pivotal roles for lipids in the development of atherosclerotic plaques. Lipid-lowering statins substantially reduce acute coronary events resulting from plaque development, but only modestly reduce arterial stenosis. This apparent paradox has shifted the goal of therapy towards plaque stabilization rather than enlargement of the lumen. More thorough understanding of the biology of atherosclerosis should enable us to manipulate plaque stability, and reduce further the acute complications of atherosclerosis.

title: Atherosclerosis - A matter of unresolved inflammation.
abstract: Atherosclerosis is commonly looked upon as a chronic inflammatory disease of the arterial wall arising from an unbalanced lipid metabolism and a maladaptive inflammatory response. However, atherosclerosis is not merely an inflammation of the vessel wall. In fact, the cardinal signs of unstable atherosclerotic lesions are primarily characteristics of failed resolution of a chronic inflammation. In contrast to acute inflammatory events which are typically self-limiting, atherosclerosis is an unresolved inflammatory condition, lacking the switch from the pro-inflammatory to the pro-resolving phase, the latter characterized by termination of inflammatory cell recruitment, removal of inflammatory cells from the site of inflammation by apoptosis and dead cell clearance, reprogramming of macrophages toward an anti-inflammatory, regenerative phenotype, and finally egress of effector cells and tissue regeneration. Here we present an overview on mechanisms of failed resolution contributing to atheroprogression and deliver a summary of novel therapeutic strategies to restore resolution in inflamed arteries.

title: Biomarkers of Atherosclerotic Plaque Instability and Rupture
abstract: Basic research over the last two decades has identified a large number of molecules pertinent to the atherosclerotic process, which have clearly improved our understanding of the underlying pathology. It is now well established that inflammation represents a major feature which is present in the vessel wall throughout all stages of the disease until the final pathophysiologic steps, representing plaque destabilization and eventually plaque rupture. Several cells typical for the atherosclerotic plaque, like monocyte-derived macrophages and T-lymphocytes are able to produce and secrete such mediator molecules, like cytokines, chemokines, growth-factors, enzymes, and disintegrins, which lead to activation of endothelial cells, proliferation of smooth muscle cells, lesion progression, and finally to the weakening of a vulnerable plaque by matrix degradation of its fibrous cap. Today, many of these molecules involved can be measured systemically by sensitive assays, and elevated concentrations in the circulation have been shown to be associated with future cardiovascular events. Determination of several of these molecules carries important prognostic information, independent of traditional risk factors, and may turn out to be useful in improving risk stratification. However, for most of these biomarkers the clinical utility has not yet been established.

title: Paclitaxel formulations: challenges and novel delivery options.
abstract: Paclitaxel (PTX), a taxane plant product, is one of the most effective broad-spectrum anti-cancer agents and approved for the treatment of a variety of cancers including ovarian, breast, lung, head and neck as well as Kaposi's sarcoma. Poor aqueous solubility and serious side effects associated with commercial preparation of PTX (Taxol®) triggered the development of alternative PTX formulations. Over past three decades, plethora of research work has been published towards the development of cremophor free and efficient formulations. Various nanocarrier systems including nanoparticles, liposomes, micelles, bioconjugates and dendrimers have been employed in order to improve PTX solubility and eliminate undesired side effects. These nanocarriers offer the advantage of high degree of encapsulation and cellular uptake, escape from elimination by P-glycoprotein (P-gp) mediated efflux, and can be explored for targeted drug delivery. The potential of these nanocarriers is reflected by the fact that various nanocarriers of PTX are in different stages of clinical trials and a few have already been commercialized including Abraxane®, Lipusu and Genexol PM®. This review focuses on the various challenges associated with PTX formulation development, limitations of existing formulations and novel approaches for the development of alternative formulations for PTX and also highlights the development of novel formulations in clinical settings.

title: New insights and evolving role of pegylated liposomal doxorubicin in cancer therapy.
abstract: We herein review various pharmacological and clinical aspects of pegylated liposomal doxorubicin (PLD), the first nanomedicine to be approved for cancer therapy, and discuss the gap between its potent antitumor activity in preclinical studies and its comparatively modest achievements in clinical studies and limited use in clinical practice. PLD is a complex formulation of doxorubicin based on pharmaceutical nanotechnology with unique pharmacokinetic and pharmacodynamic properties. Its long circulation time with stable retention of the payload and its accumulation in tumors with high vascular permeability both result in important advantages over conventional chemotherapy. The ability of PLD to buffer a number of undesirable side effects of doxorubicin, including a major risk reduction in cardiac toxicity, is now well-established and confers a major added value in a number of disease conditions. PLD is approved for the treatment of ovarian cancer, breast cancer, multiple myeloma, and Kaposi sarcoma. In addition, clinically significant antitumor activity of PLD has been reported in a number of other cancer types, including lymphomas and soft tissue sarcomas. In spite of this, PLD has not replaced conventional doxorubicin in common applications such as the adjuvant and neoadjuvant treatment of breast cancer, and its use in the clinic has not become as widespread as one may have predicted. Exploiting the unique pharmacology of PLD, analyzing its selective biodistribution and homing to tumors in cancer patients with proper theranostic tools, and harnessing its complex interaction with the immune system, will lead to a more selective and rational use of PLD that may have great impact on future clinical results and may help realize its largely untapped potential.

title: Multistage Continuous Targeting with Quantitatively Controlled Peptides on Chitosan-Lipid Nanoparticles with Multicore-Shell Nanoarchitecture for Enhanced Orally Administrated Anticancer In Vitro and In Vivo.
abstract: A DOX-loaded polysaccharide-lecithin reverse micelles triglyceride-based oral delivery nanocarrier (D-PL/TG NPs) conjugated with (i) RGD peptide for targeting to β1 integrin of M cells and (ii) Lyp-1 peptide for targeting to the p32 receptor of MDA-MB-231 cells is used to investigate the multistage continuous targeting capabilities of these peptide-conjugated nanocarriers (GLD-PL/TG NPs) for tumor therapy. Variations in the targeting efficacy and pharmacokinetic properties are investigated by quantitatively controlling the surface density of different peptides on the nanoparticles. In vitro permeability in a human follicle-associated epithelium model and cytotoxicity against MDA-MB-231 cells indicate that the nanocarriers conjugated with high RGD peptide concentrations display a higher permeability due to the existence of M cells with higher transcytosis activity, but a higher concentration of conjugated Lyp-1 peptide exhibits the lowest cell viability. Being benefited from specific targeting of peptide conjugation, improved bioavailability and enhanced tumor accumulation are achieved by the GLD-PL/TG NPs, leading to better antitumor efficacy. The results of in vivo biodistribution and antitumor studies reveal that the effect of LyP-1 peptide is more predominant than that of RGD peptide. This proof of multistage continuous targeting may open the door to a new generation of oral drug delivery systems in targeted cancer therapy.

title: Tailored design of multifunctional and programmable pH-responsive self-assembling polypeptides as drug delivery nanocarrier for cancer therapy.
abstract: Breast cancer has become the second leading cause of cancer-related mortality in female wherein more than 90% of breast cancer-related death results from cancer metastasis to distant organs at advanced stage. The purpose of this study is to develop biodegradable nanoparticles composed of natural polypeptides and calcium phosphate (CaP) with sequential pH-responsivity to tumor microenvironments for active targeted drug delivery. Two different amphiphilic copolymers, poly(ethylene glycol)3400-aconityl linkage-poly(l-glutamic acid)15-poly(l-histidine)10-poly(l-leucine)10 and LyP1-poly(ethylene glycol)1100-poly(l-glutamic acid)15-poly(l-histidine)10-poly(l-leucine)10, were exploited to self-assemble into micelles in aqueous phase. The bio-stable nanoparticles provide three distinct functional domains: the anionic PGlu shell for CaP mineralization, the protonation of PHis segment for facilitating anticancer drug release at target site, and the hydrophobic core of PLeu for encapsulation of anticancer drugs. Furthermore, the hydrated PEG outer corona is used for prolonging circulation time, while the active targeting ligand, LyP-1, is served to bind to breast cancer cells and lymphatic endothelial cells in tumor for inhibiting metastasis. Mineralized DOX-loaded nanoparticles (M-DOX NPs) efficiently prevent the drug leakage at physiological pH value and facilitate the encapsulated drug release at acidic condition when compared to DOX-loaded nanoparticles (DOX NPs). M-DOX NPs with LyP-1 targeting ligand effectively accumulated in MDA-MB-231 breast cancer cells. The inhibition effect on cell proliferation also enhances with time, illustrating the prominent anti-tumor efficacy. Moreover, the in vitro metastatic inhibition model shows the profound inhibition effect of inhibitory nanoparticles. In brief, this self-assembling peptide-based drug delivery nanocarrier with multifunctionality and programmable pH-sensitivity is of great promise and potential for anti-cancer therapy.



This tailored-design polypeptide-based nanoparticles with self-assembling and programmable stimulus-responsive properties enable to 1) have stable pH in physiological value with a low level of drug loss and effectively release the encapsulated drug with pH variations according to the tumor microenvironment, 2) enhance targeting ability to hard-to-treat breast cancer cells and activate endothelial cells (tumor region), 3) significantly inhibit the growth and prevent from malignant metastasis of cancer cells in consonance with promising anti-tumor efficacy, and 4) make tumors stick to localized position so that these confined solid tumors can be more accessible by different treatment modalities. This work contributes to designing a programmable pH-responsive drug delivery system based on the tailor-designed polypeptides.

title: Targeted treatment of cancer with artemisinin and artemisinin-tagged iron-carrying compounds
abstract: Artemisinin is a chemical compound that reacts with iron to form free radicals which can kill cells. Cancer cells require and uptake a large amount of iron to proliferate. They are more susceptible to the cytotoxic effect of artemisinin than normal cells. Cancer cells express a large concentration of cell surface transferrin receptors that facilitate uptake of the plasma iron-carrying protein transferrin via endocytosis. By covalently tagging artemisinin to transferrin, artemisinin could be selectively picked up and concentrated by cancer cells. Futhermore, both artemisinin and iron would be transported into the cell in one package. Once an artemisinin-tagged transferrin molecule is endocytosed, iron is released and reacts with artemisinin moieties tagged to transferrin. Formation of free radicals kills the cancer cell. The authors have found that artemisinin-tagged transferrin is highly selective and potent in killing cancer cells. Thus, artemisinin and artemisinin-tagged iron-carrying compounds could be developed into powerful anticancer drugs.

title: Development of artemisinin compounds for cancer treatment
abstract: SummaryArtemisinin contains an endoperoxide moiety that can react with iron to form cytotoxic free radicals. Cancer cells contain significantly more intracellular free iron than normal cells and it has been shown that artemisinin and its analogs selectively cause apoptosis in many cancer cell lines. In addition, artemisinin compounds have been shown to have anti-angiogenic, anti-inflammatory, anti-metastasis, and growth inhibition effects. These properties make artemisinin compounds attractive cancer chemotherapeutic drug candidates. However, simple artemisinin analogs are less potent than traditional cancer chemotherapeutic agents and have short plasma half-lives, and would require high dosage and frequent administration to be effective for cancer treatment. More potent and target-selective artemisinin-compounds are being developed. These include artemisinin dimers and trimers, artemisinin hybrid compounds, and tagging of artemisinin compounds to molecules that are involved in the intracellular iron-delivery mechanism. These compounds are promising potent anticancer compounds that produce significantly less side effect than traditional chemotherapeutic agents.

title: Therapeutic hyperthermia: The old, the new, and the upcoming.
abstract: Hyperthermia has long been used for cancer treatment, either alone or in combination with chemotherapy, radiation therapy, or both. Its efficacy and versatility continue to be well demonstrated in randomized trials across a number of primary cancers, but barriers to its widespread adoption persist including effective delivery and verification systems. This article describes hyperthermia, details its biological mechanisms of action and immunological effects, and summarizes select preclinical data and key clinical trials combining hyperthermia with standard cancer treatments. Current challenges and emerging technologies that have the potential to make this translational therapy more accessible to a greater number of patients are also described.

title: Principles, applications, risks and benefits of therapeutic hyperthermia.
abstract: Hyperthermia as a heat therapy is the procedure of raising the temperature of a part of or the whole body above normal for a certain period of time. Based largely on delivery methods, therapeutic hyperthermia falls under three major categories: local, regional, and whole-body. It may be applied alone or jointly with other modalities such as radiotherapy, chemotherapy, radiochemotherapy, and gene therapy. Because of the individual characteristics of each type of treatment, different types of heating systems have evolved. This paper provides an overview of possible mechanisms of heat-induced cell death and the way heating exerts its beneficial effect. It also discusses various heating devices as well as other modalities used with hyperthermia. The paper concludes with a summary of benefits and risks, obstacles encountered in the treatment process, and future research directions.

title: Mild photothermal therapy/photodynamic therapy/chemotherapy of breast cancer by Lyp-1 modified Docetaxel/IR820 Co-loaded micelles.
abstract: Patients suffering from cancer have benefited from combination therapy. Nanocarriers are the ideal candidates for combination therapy. In this study, we constructed docetaxel (DTX) loaded micellar nanomedicines co-loaded with near infrared (NIR) dye-IR820 for photothermal therapy (PTT)/photodynamic therapy (PDT)/chemotherapy of breast cancer. Lyp-1, a tumor homing peptide, was introduced into the nanosystems to construct the active targeting nanomedicine. In order to deliver IR820 to the tumor site and overcome its short lifetime in vivo, a PEI derivative-PCL-g-PEI was introduced. IR820 with negative charge was formed stable static interaction with the amine groups, meanwhile, the absorption of IR820 in the NIR region was weakened. It indicated that the nanosystem constructed in this study may provide an alternative candidate for mild PTT. By the evaluation of the photothermal conversion in vivo, we can confirm that IR820 has been successfully delivered and effectively accumulated in the tumor site. Furthermore, the tumor cells targeting and anticancer performances of this nanosystem have been studied in vitro and in vivo. The results demonstrated Lyp-1 modification has enhanced the tumor targeting delivery of DTX and IR820. By combining PTT and PDT, DTX nanomedicine efficiently inhibited the growth and metastasis of breast cancer in mice. This nanosystem is a promising candidate for combination therapy of breast cancer.

title: Enhanced Baculovirus-Mediated Transduction of Human Cancer Cells by Tumor-Homing Peptides
abstract: 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.

title: Tumor targeting of baculovirus displaying a lymphatic homing peptide
abstract: Tumor‐associated cells and vasculature express attractive molecular markers for site‐specific vector targeting. To attain tumor‐selective tropism, we recently developed a baculovirus vector displaying the lymphatic homing peptide LyP‐1, originally identified by ex vivo/in vivo screening of phage display libraries, on the viral envelope by fusion to the transmembrane anchor of vesicular stomatitis virus G‐protein.

title: Delivery of gene targeting siRNAs to breast cancer cells using a multifunctional peptide complex that promotes both targeted delivery and endosomal release
abstract: RNA interference has been used to dissect the importance of individual gene products in various human disease processes, including cancer. Small-interfering RNA, or siRNA, is one of the tools utilized in this regard, but specially-designed delivery agents are required to allow the siRNA to gain optimal access to the cell interior. Our laboratory has utilized two different siRNA-binding delivery peptides containing a polyarginine core, and modified by myristoylation and targeting motifs (iRGD or Lyp-1). A third peptide was designed to assist with endosomal release. Various ratios of the peptides and siRNA were combined and assayed for the ability to form stable complexes, and optimized ratios were determined. The complexes were found to form particles, with the majority having a diameter of 100–300 nm, as visualized by electron microscopy. These siRNA complexes have enhanced protection from nucleases present in serum, as compared to “naked” unprotected siRNA. The particles were internalized by the cells and could be detected in the cell cytoplasm by confocal fluorescence microscopy. In functional assays, peptide/siRNA complexes were shown to cause the knock down of corresponding targeted proteins. The peptide with the LyP-1 targeting motif was more effective at knockdown in MDA-MB-231 breast cancer cells than the peptide with the iRGD motif. Inclusion of the endosomal release peptide in the complexes greatly enhanced the peptide/siRNA effects. Peptide/siRNA complexes simultaneously targeting Stat3 and c-Myc caused a marked reduction in anchorage-independent growth, a property correlated with tumorigenicity. This study demonstrates the ability of a peptide-based siRNA-delivery system to deliver siRNA into breast cancer cells and cause both protein knockdown and suppression of the malignant phenotype. Such peptide complexes are likely to become highly useful siRNA-delivery vehicles for the characterization, and potentially for the treatment, of human cancer.

title: In vivo cleavability of a disulfide-based chimeric opioid peptide in rat brain.
abstract: Brain delivery of systemically administered neuropeptide drugs may be achieved by the synthesis of chimeric peptides, wherein the peptide is coupled to transport vectors via avidin-biotin technology. The present study focuses on factors that optimize the linkage of drugs to transport vectors. The vector is the OX26 monoclonal antibody to the transferrin receptor, and the model peptide used in these studies is [Lys7]dermorphin (K7DA). The K7DA is monobiotinylated at the epsilon-amino group of the Lys7 residue with either a cleavable linker, e.g., disulfide, using NHS-SS-biotin, or a noncleavable linker, e.g., amide, using NHS-XX-biotin. Disulfide cleavage of the biotinylated derivative yields the desbiotinylated peptide, which is thiolated. Structures of the K7DA analogues were confirmed by secondary ion mass spectrometry. The biotinylated peptides were coupled to a thiol-ether conjugate of the OX26 antibody and either neutral avidin (NLA) or streptavidin. The binding constants (Ki) of the K7DA, the biotinylated K7DA (bio-XX-K7DA), the desbiotinylated K7DA, and the bio-XX-KD7A conjugated to NLA-OX26 were 0.62 +/- 0.14, 1.59 +/- 0.27, 1.24 +/- 0.24, and > 10 nM, respectively, and were determined with a mu-opioid peptide radioreceptor assay. Comparable results were obtained with in vivo tail-flick analgesia testing following intracerebroventricular (icv) injection of opioid chimeric peptides. Reversibility of pharmacologic action of thiolated peptide was demonstrated by icv naloxone administration. The cleavability of the disulfide linker in vivo in rat plasma and brain was assessed with gel filtration HPLC and internal carotid artery perfusion of labeled opioid chimeric peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Design, characterization and in vitro evaluation of SMEDDS containing an anticancer peptide, linear LyP-1
abstract: (CGNKRTRGC) is a nine amino acid peptide that shows high specificity for tumor lymphatics. The aim of this study was to develop lipid-based formulations containing the linear form of LyP-1 for lymphatic targeting. Self-microemulsifying drug delivery systems (SMEDDS) were designed for the delivery of linear LyP-1 by itself and as a solid dispersion (SD). Formulations were characterized in terms of physical stability, pH, morphological properties, droplet size distribution and zeta potential. Thermodynamically stable microemulsions were obtained with an average droplet size around 20 nm and zeta potential near neutrality. Cytotoxicity studies of blank and peptide-containing SMEDDS were carried out on Caco-2 cell line. Bioactivity studies of peptide-containing formulations were carried out on MDA-MB-231 breast cancer cell line. It was shown that blank and peptide-containing SMEDDS formulations were not cytotoxic to Caco-2 cell line. However, formulations containing the peptide and peptide SD led to a significant decrease in cell viability on breast cancer cells. It could be concluded that the SMEDDS formulations containing the linear LyP-1 were successfully developed for the lymphatic targeting of solid tumors in vitro.

title: Tumor-targeted liposomal drug delivery mediated by a diseleno bond-stabilized cyclic peptide
abstract: Peptide ligands have played an important role in tumor-targeted drug delivery as targeting moieties. The in vivo fate of peptide-mediated drug delivery systems and the following antitumor effects may greatly depend on the stability of the peptide ligand. In the current study, a tumor-targeting cyclic peptide screened by phage display, Lyp-1 (a peptide that specifically binds to tumor and endothelial cells of tumor lymphatics in certain tumors), was structurally modified by replacement of the original intramolecular disulfide bond with a diseleno bond. The produced analog Syp-1 (seleno derivative of Lyp-1) maintained specific binding ability to the target protein p32 (Kd = 18.54 nM), which is similar to that of Lyp-1 (Kd = 10.59 nM), indicated by surface plasmon resonance assay. Compared with Lyp-1, Syp-1 showed significantly improved stability against serum. After the peptide attached onto the surface of fluorophore-encapsulating liposomes, the more efficient tumor uptake of liposomal fluorophore mediated by Syp-1 was observed. Furthermore, Syp-1 modified liposomal doxorubicin presented the most potent tumor growth inhibitory ability among all the therapeutic groups, with a low half maximal inhibitory concentration of 588 nM against MDA-MB-435 cells in vitro and a high tumor inhibition rate of 73.5% in vivo. These findings clearly indicated that Syp-1 was a stable and effective tumor targeting ligand and suggest that the sulfur-to-selenium replacement strategy may help stabilize the phage-displayed cyclic peptide containing disulfide-bond under physiological conditions and strongly support the validity of peptide-mediated drug targeting.