Peptide-conjugated Nanoparticles Research Articles

Peptide-conjugated Nanoparticle Platforms for Targeted Delivery, Imaging, and Biosensing Applications.

Peptides have become an indispensable tool in engineering of multifunctional nanostructure platforms for biomedical applications such as targeted drug and gene delivery, imaging and biosensing. They can be covalently incorporated into a variety of nanoparticles (NPs) including polymers, metallic nanoparticles, and others. Using different bioconjugation techniques, multifunctional peptide-modified NPs can be formulated to produce therapeutical and diagnostic platforms offering high specificity, lower toxicity, biocompatibility, and stimuli responsive behavior. Targeting peptides can direct the nanoparticles into specific tissues for targeted drug and gene delivery and imaging applications due to their specificity towards certain receptors. Furthermore, due to their stimuli-responsive features, they can offer controlled release of therapeutics into desired sites of disease. In addition, peptide-based biosensors and imaging agents can provide non-invasive detection and monitoring of diseases including cancer, infectious diseases, and neurological disorders. In this review, we covered the design and formulation of recent peptide-based NP platforms, as well as their utilization in in vitro and in vivo applications such as targeted drug and gene delivery, targeting, sensing, and imaging applications. In the end, we provided the future outlook to design new peptide conjugated nanomaterials for biomedical applications.

Improving Pharmacokinetics of Peptides Using Phage Display.

Phage display is a versatile method often used in the discovery of peptides that targets disease-related biomarkers. A major advantage of this technology is the ease and cost efficiency of affinity selection, also known as biopanning, to identify novel peptides. While it is relatively straightforward to identify peptides with optimal binding affinity, the pharmacokinetics of the selected peptides often prove to be suboptimal. Therefore, careful consideration of the experimental conditions, including the choice of using in vitro, in situ, or in vivo affinity selections, is essential in generating peptides with high affinity and specificity that also demonstrate desirable pharmacokinetics. Specifically, in vivo biopanning, or the combination of in vitro, in situ, and in vivo affinity selections, has been proven to influence the biodistribution and clearance of peptides and peptide-conjugated nanoparticles. Additionally, the marked difference in properties between peptides and nanoparticles must be considered. While peptide biodistribution depends primarily on physiochemical properties and can be modified by amino acid modifications, the size and shape of nanoparticles also affect both absorption and distribution. Thus, optimization of the desired pharmacokinetic properties should be an important consideration in biopanning strategies to enable the selection of peptides and peptide-conjugated nanoparticles that effectively target biomarkers in vivo.

VEGF mimetic peptide-conjugated nanoparticles for magnetic resonance imaging and therapy of myocardial infarction.

To reduce the mortality of myocardial infarction (MI), accurate detection of the infarct and appropriate prevention against ischemia/reperfusion (I/R) induced cardiac dysfunction are highly desired. Considering that vascular endothelial growth factor (VEGF) receptors are overexpressed in the infarcted heart and VEGF mimetic peptide QK binds specifically to VEGF receptors and activates vascularization, the PEG-QK-modified, gadolinium-doped carbon dots (GCD-PEG-QK) were formulated. This research aims to investigate the magnetic resonance imaging (MRI) capability of GCD-PEG-QK on myocardial infarct and their therapeutic effect on I/R-induced myocardial injury. These multifunctional nanoparticles exhibited good colloidal stability, excellent fluorescent and magnetic property, and satisfactory biocompatibility. Intravenous injection of GCD-PEG-QK nanoparticles post myocardial I/R displayed accurate MRI of the infarct, enhanced efficacy of QK peptide on pro-angiogenesis, and amelioration of cardiac fibrosis, remodeling and dysfunction, probably via the improvement on QK's in vivo stability and MI-targeting. Collectively, the data suggested that this theranostic nanomedicine can realize precise MRI and effective therapy for acute MI in a non-invasive manner.

Peptide-conjugated nanoparticles for targeted photodynamic therapy

Abstract Cancer is the second leading cause of death worldwide after cardiovascular disease. Depending on the type and the location of the tumor, several cancer treatments are implemented. Among these, the three most conventional therapies are surgery, radiotherapy and chemotherapy. However, there are other therapeutic approaches such as photodynamic therapy (PDT). PDT relies on the combined action of light, a photoactivable molecule called photosensitizer (PS) and molecular oxygen. Most of the PSs used for clinical applications are not cancer-cell specific. One of the solutions to overcome this problem is the use of nanoparticles (NPs) to induce a passive targeting. It is also possible to graft a vector onto the NPs to specifically target membrane receptors overexpressed in the tumor cells or neovessels surrounding the tumor. In this review, we focus on the NPs loaded with PSs and coupled to peptides for targeted PDT. We described nanosystems that targeted Neuropilin-1 (NRP-1), αvβ3 integrins, nucleolin membrane receptor, epidermal growth factor (EGF) receptor, protein-glutamine-gamma-glutamyltransferase (TGM2), p32, transferrin, PD-1, and mitochondrial membrane. The use of a cell absorbing-peptide is also described.

Nanoparticle-mediated targeting of autoantigen peptide to cross-presenting liver sinusoidal endothelial cells protects from CD8 T-cell-driven autoimmune cholangitis.

SummaryAutoimmune diseases are caused by adaptive immune responses to self‐antigens. The development of antigen‐specific therapies that suppress disease‐related, but not unrelated immune responses in general, is an important goal of biomedical research. We have previously shown that delivery of myelin peptides to liver sinusoidal endothelial cells (LSECs) using LSEC‐targeting nanoparticles provides effective protection from CD4 T‐cell‐driven autoimmune encephalomyelitis. Here, we investigated whether this methodology might also serve antigen‐specific treatment of a CD8 T‐cell‐driven autoimmune disease. As a model for CD8 T‐cell‐mediated autoimmunity, we used OT‐1 T‐cell‐driven cholangitis in K14‐OVAp mice expressing the cognate MHC I‐restricted SIINFEKL peptide in cholangiocytes. To study whether peptide delivery to LSECs could modulate cholangitis, SIINFEKL peptide‐conjugated nanoparticles were administered intravenously one day before transfer of OT‐1 T cells; five days after cell transfer, liver pathology and hepatic infiltrates were analysed. SIINFEKL peptide‐conjugated nanoparticles were rapidly taken up by LSECs in vivo, which effectively cross‐presented the delivered peptide on MHC I molecules. Intriguingly, K14‐OVAp mice receiving SIINFEKL‐loaded nanoparticles manifested significantly reduced liver damage compared with vehicle‐treated K14‐OVAp mice. Mechanistically, treatment with LSEC‐targeting SIINFEKL‐loaded nanoparticles significantly reduced the number of liver‐infiltrating OT‐1 T cells, which up‐regulated expression of the co‐inhibitory receptor PD‐1 and down‐regulated cytotoxic effector function and inflammatory cytokine production. These findings show that tolerogenic LSECs can effectively internalize circulating nanoparticles and cross‐present nanoparticle‐bound peptides on MHC I molecules. Therefore, nanoparticle‐mediated autoantigen peptide delivery to LSECs might serve the antigen‐specific treatment of CD8 T‐cell‐driven autoimmune disease.

Commentary on "Peptide-Conjugated Nanoparticles as Targeted Anti-angiogenesis Therapeutic and Diagnostic in Cancer" by Shaker A. Mousa, Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY 12144, United States - Peptide-Conjugated Nanoparticles for Multimodal Nanomedicine.

Commentary on "Peptide-Conjugated Nanoparticles as Targeted Anti-angiogenesis Therapeutic and Diagnostic in Cancer" by Shaker A. Mousa, Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY 12144, United States - Peptide-Conjugated Nanoparticles for Multimodal Nanomedicine.

Peptide-Conjugated Nanoparticles as Targeted Anti-angiogenesis Therapeutic and Diagnostic in Cancer.

Targeting angiogenesis in the microenvironment of a tumor can enable suppression of tumor angiogenesis and delivery of anticancer drugs into the tumor. Anti-angiogenesis targeted delivery systems utilizing passive targeting such as Enhanced Permeability and Retention (EPR) and specific receptor-mediated targeting (active targeting) should result in tumor-specific targeting. One targeted anti-angiogenesis approach uses peptides conjugated to nanoparticles, which can be loaded with anticancer agents. Anti-angiogenesis agents can suppress tumor angiogenesis and thereby affect tumor growth progression (tumor growth arrest), which may be further reduced with the targetdelivered anticancer agent. This review provides an update of tumor vascular targeting for therapeutic and diagnostic applications, with conventional or long-circulating nanoparticles decorated with peptides that target neovascularization (anti-angiogenesis) in the tumor microenvironment.

Detection of influenza virus by agglutination using nanoparticles conjugated with a sialic acid-mimic peptide

Influenza virus (IFV) detection in the early phase of disease is critical for effective anti-influenza therapy using neuraminidase inhibitors. Sialyloligosaccharide receptors on the surface of respiratory cells are recognized by IFV hemagglutinin (HA) in the infection. Here, we show that agglutination of IFV is detected using poly(glycidyl methacrylate) (PGMA)-coated polystyrene nanoparticles conjugated with a sialic acid-mimic peptide. The azido peptide was immobilized onto the surface of the PGMA-coated nanoparticles by click chemistry. The distribution of particle size, determined by dynamic light scattering, indicated that the peptide-conjugated nanoparticles were agglutinated in the presence of HA and IFV. Nanoparticles conjugated with the receptor-mimic peptide may be a useful alternative to red blood cells in the global surveillance and clinical diagnosis of influenza. This study examines the use of poly(glycidyl methacrylate) (PGMA)-coated polystyrene functionalized with peptides that mimic receptors found on red blood cells for detection of the influenza virus. Although the current method, the hemagglutination inhibition test, utilized by the World Health Organization, is simple, it has some limitations. We found that our peptide dimers attached to beads were efficiently agglutinated, leading us to detect the presence of the influenza virus antigens. We believe that our study makes a significant contribution to the fight against influenza because we provide an alternative method for the important task of influenza surveillance and diagnosis.

Glutathione (GSH) Peptide Conjugated Magnetic Nanoparticles As Blood–Brain Barrier Shuttle for MRI-Monitored Brain Delivery of Paclitaxel

In drug delivery science, brain delivery is one of the most important challenges because of the low efficiency of the available treatments. Nowadays, shuttle peptides have attracted more attention because of lower price, reduced immunity, and increased chemical capability. Glutathione (GSH) is one of the blood–brain barrier (BBB) shuttle peptides that has reached the most progressive steps in the path toward clinical application. This project discovered the possibility of GSH-conjugated IONPs as an MRI-monitored paclitaxel (PTX) delivery vehicle across the BBB using BALB/c mouse model. Synthesized shuttle peptide-conjugated nanoparticles were tracked over a certain time by MRI. A one-pot method was used for preparation of IONPs@Asp to form functionalized nanoparticles with two functional groups for linkage of PTX, PEG, and then GSH on the surface of nanoparticles. Afterward, they were analyzed by XRD, TGA, FTIR, TEM, VSM, and DLS techniques. In addition, histological study were performed on the key organs...

Effect of peptide-conjugated nanoparticles on cell lines

Metal nanoparticles are widely used for the delivery and targeting of pharmaceutical, therapeutic and diagnostic agents in cancer therapy in recent years. The multifunctional nanoparticles constructed currently are supposed to show superior effects on cancer cells. This study was conducted to observe the difference between the effect of a biologically important peptide, silver (AgNPs) and gold (AuNPs) nanoparticles and their conjugates on two different cancer cells. Peptide (Boc-L-DP-L-OMe) was acquired from different sources and subjected to conjugation with biosynthesized gold and silver nanoparticles under standard conditions. These conjugates were tested against the colon cancer (HT-29) and breast cancer (MDA MB-231) cell lines. The results clearly depicted the improved activity of nanoparticles in the form of conjugates. Fluorescent dye microscopy and DNA fragmentation assay substantiate the fact that the conjugated nanoparticles cause higher level of disintegration of DNA in cells that consecutively damages and causes apoptosis due to lethality.

Nano-Antagonist Alleviates Inflammation and Allows for MRI of Atherosclerosis.

Specific targeting of inflammation remains a challenge in many pathologies, because of the necessary balance between host tolerance and efficacious inflammation resolution. Here, we discovered a short, 4-mer peptide which possesses antagonist properties towards CC chemokine receptor 2 (CCR2), but only when displayed on the surface of lipid nanoparticles. According to BLAST analysis, this peptide motif is a common repeating fragment in a number of proteins of the CC chemokine family, which are key players in the inflammatory response. In this study, self-assembled, peptide-conjugated nanoparticles (CCTV) exhibited typical properties of CCR2 antagonism, including affinity to the CCR2 receptor, inhibition of chemotactic migration of primary monocytes, and prevention from CC chemokine ligand 2 (CCL2)-induced actin polymerization. Furthermore, CCTV ameliorated NFkB activation and downregulated the secondary, but not the primary, inflammatory response in cultured macrophages. When conjugated with gadolinium or europium cryptates, CCTV enabled targeted imaging (via magnetic resonance imaging and time-resolved fluorescence) of atherosclerosis, a chronic inflammatory condition in which the CCL2/CCR2 axis is highly dysfunctional. CCTV targeted CCR2hiLy6Chi inflammatory monocytes in blood and the atherosclerotic plaque, resulting in cell-specific transcriptional downregulation of key inflammatory genes. Finally, CCTV generated pronounced inflammasome inactivation, likely mediated through reactive oxygen species scavenging and downregulation of NLRP3. In summary, our work demonstrates for the first time that a short peptide fragment presented on a nanoparticle surface exhibit potent receptor-targeted antagonist effects, which are not seen with the peptide alone. Unlike commonly used cargo-carrying, vector-directed drug delivery vehicles, CCTV nanoparticles may act as therapeutics/theranostics themselves, particularly in inflammatory conditions with CCL2/CCR2 pathogenesis, including cardiovascular disease and cancer.

Follicle-stimulating hormone peptide-conjugated nanoparticles for targeted shRNA delivery lead to effective gro-α silencing and antitumor activity against ovarian cancer

The distinct hormone molecules and receptors, such as follicle-stimulating hormone receptor (FSHR) in ovarian cancer, provide opportunities for more precisely targeted therapy. We previously developed FSHR-mediated nanoparticles and found that FSH peptides on the surface of nanoparticles improved the delivery of short interfering RNA (siRNA) into ovarian cancer cells. However, the high toxicity of the nanoparticles and the transient silencing of the siRNA in vivo limited further study. Here, we developed FSH peptide-conjugated nanoparticles with an increased amount of polyethylene glycol (PEG) grafting and encapsulated short hairpin RNA (shRNA) to silence the target gene, growth-regulated oncogene α (gro-α). The nanoparticle complexes exhibited good stability over three weeks. Expression of the target gene, gro-α, was significantly down-regulated by gro-α shRNA-loaded nanoparticles conjugated with FSH peptides (FSH33-G-NP) in FSHR-positive HEY cells. Cell proliferation, migration, and invasion were also inhibited by FSH33-G-NP. Tumor growth was delayed significantly in the mice treated with FSH33-G-NP. No significant loss of body weight or severe toxic effects were observed in any groups. In conclusion, gro-α shRNA-loaded nanoparticles conjugated with FSH peptides overcame the drawbacks of the in vivo application of RNAi therapeutics and polymer-based nanocarriers and showed safe antitumor efficacy. Our study might contribute to the application of FSHR-based targeted therapy and imaging in cancer.

Transcriptional control of the MUC16 promoter facilitates follicle-stimulating hormone peptide-conjugated shRNA nanoparticle-mediated inhibition of ovarian carcinoma in vivo

Ovarian cancer is the leading cause of cancer death among gynecological malignancies. The high mortality rate has not been significantly reduced despite advances in surgery and chemotherapy. Gene therapy shows therapeutic potential, but several key issues must be resolved before clinical application. To minimize toxicity in noncancerous tissues, tumor-specific ligands are conjugated to vectors to increase the selectivity of drug delivery. The expression pattern of follicle-stimulating hormone (FSH) receptor in normal and cancer tissues provides an opportunity for highly selective drug delivery in ovarian cancer. Furthermore, tumor-specific promoters can conditionally regulate therapeutic gene expression in tumor or normal tissues. The mucin 16 (MUC16) promoter might be a potential tool to drive ovarian cancer-localized gene expression since MUC16/CA125 is overexpressed in most ovarian carcinomas. Here, we screened the possible MUC16 promoter sequences and constructed MUC16 promoter-driven gro-α shRNA plasmid vectors. The vectors were specifically delivered into ovarian cancer cells via FSH peptide-conjugated nanoparticles. The predicted promoter sequence with TAAA repeats showed high transcriptional activity. The nanoparticle complex containing MUC16 promoter-driven gro-α shRNA and FSH peptides had the ability to decrease gro-α protein secretion in ovarian cancer cells and block tumor growth without obvious toxic effects in a nude mouse model bearing ovarian cancer. Our study provides a novel gene delivery system using a MUC16 promoter trigger and FSH peptide-mediated active targeting in ovarian cancer, and this system may be a promising strategy for specific genetic therapeutic delivery.

Abstract 349: Tracking Human Pluripotent Stem Cell-Derived Cardiomyocytes in Post-Myocardial Infarction Heart with Photoacoustic Imaging

Background: Molecular imaging has helped unravel potential benefits and barriers of stem cell therapy, yet in vivo tracking of cell injection and engraftment with high 3D spatial resolution remains a challenge. This study aims to testify the feasibility of photoacoustic imaging (PAI) as an efficient method to monitor transplanted therapeutic cells in post-myocardial infarction (MI) hearts. Methods: Cell penetrating peptide-conjugated photoacoustic nanoparticles (PANPs) were synthesized and applied directly to cultured human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) at the concentration of 4nM for 6h. Post exogenous labeling, cells were injected into mouse MI hearts (n=5) guided by PAI and then monitored by PAI in vivo . These engraftments were further confirmed by immunostaining. Results: Flow cytometry and confocal imaging indicated that PANP labeling significantly increased the labeling efficiency of hPSC-CMs from 38% to 90% compared to endocytosis labeling. Moreover, calcium imaging and contractile analysis indicated that PANP labeling had no cardiotoxicity to both electrical and mechanical functions of hPSC-CMs. In addition, quantitative polymerase chain reaction (qPCR) showed no adverse effects of PANP labeling on the genes related to cardiomyocyte structures and functions. Finally, animal experiments demonstrated the feasibility of PAI for both injection guidance and followed tracking of hPSC-CMs in vivo . The real-time acquired PAI images showed a high spatial resolution (~100 μm) for mice hearts and a high intensity for the labeled cells. Conclusion: We utilized PAI for the first time to monitor cardiac regenerative therapy in a preclinical study. PAI demonstrated its superior potential to track cell therapy in vivo due to its high spatial resolution, low cost, and non-radioactivity. It will be a useful tool for the investigation of cardiac regenerative therapy.

Peptide-Conjugated Nanoparticles Reduce Positive Co-stimulatory Expression and T Cell Activity to Induce Tolerance

Targeted approaches to treat autoimmune diseases would improve upon current therapies that broadly suppress the immune system and lead to detrimental side effects. Antigen-specific tolerance was induced using poly(lactide-co-glycolide) nanoparticles conjugated with disease-relevant antigen to treat a model of multiple sclerosis. Increasing the nanoparticle dose and amount of conjugated antigen both resulted in more durable immune tolerance. To identify active tolerance mechanisms, we investigated downstream cellular and molecular events following nanoparticle internalization by antigen-presenting cells. The initial cell response to nanoparticles indicated suppression of inflammatory signaling pathways. Direct and functional measurement of surface MHC-restricted antigen showed positive correlation with both increasing particle dose from 1 to 100μg/mL and increasing peptide conjugation by 2-fold. Co-stimulatory analysis of cells expressing MHC-restricted antigen revealed most significant decreases in positive co-stimulatory molecules (CD86, CD80, and CD40) following high doses of nanoparticles with higher peptide conjugation, whereas expression of a negative co-stimulatory molecule (PD-L1) remained high. Tcells isolated from mice immunized against myelin proteolipid protein (PLP139-151) were co-cultured with antigen-presenting cells administered PLP139-151-conjugated nanoparticles, which resulted in reduced Tcell proliferation, increased Tcell apoptosis, and a stronger anti-inflammatory response. These findings indicate several potential mechanisms used by peptide-conjugated nanoparticles to induce antigen-specific tolerance.

Nanoparticle Assembly and Gelatin Binding Mediated by Triple Helical Collagen Mimetic Peptide.

Peptide-conjugated nanoparticles (NPs) have promising potential for applications in biosensing, diagnosis, and therapeutics because of their appropriate size, unique self-assembly, and specific substrate-binding properties. However, controlled assembly and selective target binding are difficult to achieve with simple peptides on NP surfaces because high surface energy makes NPs prone to self-aggregate and adhere nonspecifically. Here, we report the self-assembly and gelatin binding properties of collagen mimetic peptide (CMP) conjugated gold NPs (CMP-NPs). We show that the orientation of CMPs displayed on the NP surface can control NP assembly either by promoting or hindering triple helical folding between CMPs of neighboring NPs. We also show that CMP-NPs can specifically bind to denatured collagen by forming triple-helical hybrids between denatured collagen strands and CMPs, demonstrating their potential use for detection and selective removal of gelatin from protein mixtures. CMP conjugated NPs offer a simple and effective method for NP assembly and for targeting denatured collagens with high specificity. Therefore, they may lead to new types of functional nanomaterials for detection and study of denatured collagen associated with diseases characterized by high levels of collagen degradation.

Abstract LB-191: Photoacoustic detection and imaging of single cancer cells with peptide-conjugated gold nanoparticles

Abstract Photoacoustic imaging (PAI), which is based on the conversion of light into sound, is an emerging, hybrid and noninvasive imaging modality with highly scalable resolution. PAI has shown tremendous potential in early tumor detection and imaging by providing structural, functional and metabolic information. But to date, most work has been focused on tumor detection at the macroscopic level (i.e. tissue, organ). For early tumor diagnosis, microscopic detection of individual cancer cells is highly desirable and of special significance. However, detection and imaging of single cancer cells using PAI is very challenging because most cancer cells are colorless, thus exhibiting little endogenous contrast. Gold nanoparticles (AuNPs), which have good biocompatibility, low toxicity and high optical absorption, are an exogenous contrast agent that can facilitate PAI of single cancer cells. In this study, we synthesized peptide-conjugated AuNPs to selectively target DU145 human prostate cancer cells, and employed a laser-scanning photoacoustic microscopy (PAM) to detect and image individual cells. The used AuNPs had an average diameter of 30 nm and were physically fabricated by ultrafast pulsed laser ablation of gold target in ultrapure water. Their surface chemistry was modified through a sequential conjugation process, in which a predetermined amount of thiolated PEG 5000 molecules and RGD peptides were successively bound to the AuNPs with quantitative control and high efficiency. After incubation with DU145 cells, the functionalized PEG-RGD-AuNPs were endocytosed with the help of membrane receptors and produced sharp absorption contrast. The internalized AuNPs generated strong photoacoustic signals after absorbing excitation laser and the signals were captured by a highly sensitive hydrophone to form cell images. Combing functional AuNPs and high-resolution PAM, we successfully detected and imaged single, live DU145 cancer cells. Though transparent, the cancer cells were visibly delineated by intracellular AuNPs and were clearly distinguishable. Moreover, to achieve optimum detection efficiency, we also studied the relationship between the density of RGD peptide and the amplitude of photoacoustic signal. Experimental results reveal that, although the amount of cellular uptake of AuNPs was proportional to RGD density (from radioactivity analysis using a Gamma counter), the amplitude of photoacoustic signal did not. Photoacoustic detection efficiency reached a maximum when the molar ratio of RGD peptides to AuNPs was approximately 1000. To our best knowledge, this is the first report on photoacoustic detection and imaging of specific cancer cells using peptide-conjugated AuNPs at single-cell resolution. By translating PAI from macro to micro, the work is expected to open a new avenue for early cancer detection, imaging, diagnosis and even treatment. Citation Format: Chao Tian, Wei Qian, Xia Shao, Zhixing Xie, Xu Cheng, Shengchun Liu, Qian Cheng, Bing Liu, Xueding Wang. Photoacoustic detection and imaging of single cancer cells with peptide-conjugated gold nanoparticles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-191.

Detection of Cancer-Specific Proteases Using Magnetic Relaxation of Peptide-Conjugated Nanoparticles in Biological Environment.

Protease expression is closely linked to malignant phenotypes of different solid tumors; as such, their detection is promising for diagnosis and treatment of cancers, Alzheimer's, and vascular diseases. Here, we describe a new method for detecting proteases by sensitively monitoring the magnetic relaxation of monodisperse iron oxide nanoparticles (IONPs) using magnetic particle spectrometer (MPS). In this assay, tailored peptides functioning as activatable nanosensors link magnetic nanoparticles and possess selective sites that are recognizeable and cleaveable by specific proteases. When these linker peptides, labeled with biotin at N- and C-terminals, are added to the neutravidin functionalized IONPs, nanoparticles aggregate, resulting in well-defined changes in the MPS signal. However, as designed, in the presence of proteases these peptides are cleaved at predetermined sites, redispersing IONPs, and returning the MPS signal(s) close to its preaggregation state. These changes observed in all aspects of the MPS signal (peak intensity, its position as a function of field amplitude, and full width at half-maximum-when combined, these three also eliminate false positives), help to detect specific proteases, relying only on the magnetic relaxation characteristics of the functionalized nanoparticles. We demonstrate the general utility of this assay by detecting one each from the two general classes of proteases: trypsin (digestive serine protease, involved in various cancers, promoting proliferation, invasion, and metastasis) and matrix metalloproteinase (MMP-2, observed through metastasis and tumor angiogenesis). This MPS based protease-assay is rapid, reproducible, and highly sensitive and can form the basis of a feasible, high-throughput method for detection of various other proteases.

Peptide-conjugated nanoparticles for targeted imaging and therapy of prostate cancer

While there has been extensive development of anti-cancer drugs for treatment of prostate cancer, the therapeutic efficacy of such drugs remains inadequate in many cases. Here, we performed in vitro biopanning of the PC3 human prostate carcinoma cell line to select prostate cancer-specific peptides by phage display. We successfully identified specific peptides targeting prostate cancer cells, and their specificity was confirmed by cellular ELISA and flow cytometry. Moreover, we found that the phage clones also recognize other prostate cancer cell lines and surgical specimens from prostate cancer patients. The tumor targeting ability of these phages was validated in a xenograft model, in which high accumulation of targeting phage was observed. To investigate whether selected peptides are able to target tumors and enhance drug delivery into cancer cells, we synthesized peptide-PEGylated lipids and post-inserted them into preformed liposomal doxorubicin and vinorelbine. The results of our cellular uptake and MTT assays indicate that peptide-conjugated liposomes exhibit enhanced drug intracellular delivery and cytotoxicity. The conjugation of targeting peptide to imaging agents, such as quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs), results in more precise delivery of these agents to tumor sites. Furthermore, administration of liposomal doxorubicin and vinorelbine conjugated with targeting peptides was found to markedly increase the inhibition of human prostate tumor growth in mouse xenograft and orthotopic models. These results indicate that targeting peptide, SP204, has significant potential for targeted therapy and molecular imaging in prostate cancer.

Interrogating the Role of Receptor-Mediated Mechanisms: Biological Fate of Peptide-Functionalized Radiolabeled Gold Nanoparticles in Tumor Mice.

To get a better insight on the transport mechanism of peptide-conjugated nanoparticles to tumors, we performed in vivo biological studies of bombesin (BBN) peptide functionalized gold nanoparticles (AuNPs) in human prostate tumor bearing mice. Initially, we sought to compare AuNPs with thiol derivatives of acyclic and macrocyclic chelators of DTPA and DOTA types. The DTPA derivatives were unable to provide a stable coordination of (67)Ga, and therefore, the functionalization with the BBN analogues was pursued for the DOTA-containing AuNPs. The DOTA-coated AuNPs were functionalized with BBN[7-14] using a unidentate cysteine group or a bidentate thioctic group to attach the peptide. AuNPs functionalized with thioctic-BBN displayed the highest in vitro cellular internalization (≈ 25%, 15 min) in gastrin releasing peptide (GRP) receptor expressing cancer cells. However, these results fail to translate to in vivo tumor uptake. Biodistribution studies following intravenous (IV) and intraperitoneal (IP) administration of nanoconjugates in tumor bearing mice indicated that the presence of BBN influences to some degree the biological profile of the nanoconstructs. For IV administration, the receptor-mediated pathway appears to be outweighed by the EPR effect. By contrast, in IP administration, it is reasoned that the GRPr-mediated mechanism plays a role in pancreas uptake.