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

The targeted delivery of nanoparticles to solid tumors is one of the most important and challenging problems in cancer nanomedicine, but the detailed delivery mechanisms and design principles are still not well understood. Here we report quantitative tumor uptake studies for a class of elongated gold nanocrystals (called nanorods) that are covalently conjugated to tumor-targeting peptides. A major advantage in using gold as a "tracer" is that the accumulated gold in tumors and other organs can be quantitatively determined by elemental mass spectrometry (gold is not a natural element found in animals). Thus, colloidal gold nanorods are stabilized with a layer of polyethylene glycols (PEGs), and are conjugated to three different ligands: (i) a single-chain variable fragment (ScFv) peptide that recognizes the epidermal growth factor receptor (EGFR); (ii) an amino terminal fragment (ATF) peptide that recognizes the urokinase plasminogen activator receptor (uPAR); and (iii) a cyclic RGD peptide that recognizes the a v b 3 integrin receptor. Quantitative pharmacokinetic and biodistribution data show that these targeting ligands only marginally improve the total gold accumulation in xenograft tumor models in comparison with nontargeted controls, but their use could greatly alter the intracellular and extracellular nanoparticle distributions. When the gold nanorods are administered via intravenous injection, we also find that active molecular targeting of the tumor microenvironments (e.g., fibroblasts, macrophages, and vasculatures) does not significantly influence the tumor nanoparticle uptake. These results suggest that for photothermal cancer therapy, the preferred route of gold nanorod administration is intra-tumoral injection instead of intravenous injection. The targeted delivery of nanoparticles to solid tumors is a key task in the development of cancer nanomedicine for in-vivo molecular imaging and targeted therapy.1 -8 Despite extensive research and significant progress in the last 10-15 years, there are still major fundamental and technical barriers that need to be understood and overcome.1 -9 These problems include the complex interactions between naoparticles and biological systems in-vivo, rapid uptake and clearance of nanoparticles by the reticuloendothelial system (RES) organs (such as the liver and spleen), and very limited penetration of nanoparticles to poorly vascularized or necrotic tumor regions.2 , 10 Current methods for nanoparticle delivery are mainly based on an "active" mechanism and a "passive" mechanism.9 , 10 In the active mode, molecular ligands such as antibodies, peptides, or small molecules are used to recognize specific receptors on the tumor cell surface, often followed by receptor-mediated endocytosis and nanoparticle internalization. KeywordsIn the passive mode, nanoparticles without targeting ligands are accumulated and retained in the tumor interstitial space mainly through the enhanced permeability and retention (EPR) effect.11 -13 In both mechanisms, a common feature is t...

Epidermal growth factor receptor (EGFR) targeted nanoparticle are developed by conjugating a single-chain anti-EGFR antibody (ScFvEGFR) to surface functionalized quantum dots (QDs) or magnetic iron oxide (IO) nanoparticles. The results show that ScFvEGFR can be successfully conjugated to the nanoparticles, resulting in compact ScFvEGFR nanoparticles that specifically bind to and are internalized by EGFR-expressing cancer cells, thereby producing a fluorescent signal or magnetic resonance imaging (MRI) contrast. In vivo tumor targeting and uptake of the nanoparticles in human cancer cells is demonstrated after systemic delivery of ScFvEGFR-QDs or ScFvEGFR-IO nanoparticles into an orthotopic pancreatic cancer model. Therefore, ScFvEGFR nanoparticles have potential to be used as a molecular-targeted in vivo tumor imaging agent. Efficient internalization of ScFvEGFR nanoparticles into tumor cells after systemic delivery suggests that the EGFR-targeted nanoparticles can also be used for the targeted delivery of therapeutic agents.

Although increasing evidence supports a link between epidermal growth factor receptor (EGFR) signaling and resistance to apoptosis, the mechanism by which the EGFR signaling pathway inhibits apoptosis is not well understood. In this study, we found that epidermal growth factor ( The EGFR 2 signaling pathway plays a key role in the regulation of cell proliferation, survival, and differentiation (1, 2). It has been shown that the level of EGFR is up-regulated in many human tumor tissues. Activation of EGFR signaling has been associated with highly aggressive cancer types and poor responses to therapeutic agents (3-7). Prior preclinical and clinical studies have shown that blocking the EGFR signaling via monoclonal antibodies or inhibition of the EGFR tyrosine kinase with small molecules reduces the growth of breast cancers and sensitizes responses to chemotherapy (8 -10).Recently, we and others have shown that activation of the EGFR signaling pathway leads to the up-regulation of survivin, a member of the inhibitor of apoptosis (IAP) protein family (11)(12)(13)(14). Survivin is broadly expressed in fetal tissues but is undetectable in the most normal adult tissues (15). However, a high level of survivin is found in most common tumor types, including over 70% of human breast cancer tissues at all stages of cancer development (16 -18). It has been shown that in breast cancer cells, levels of survivin expression correlate with susceptibility to apoptosis (17). At present, the mechanism by which this up-regulation of survivin occurs in tumor cells having activated EGFR signaling is not fully understood. Recent studies have suggested, however, that activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway by EGFR signaling causes up-regulation of survivin expression (12, 13). It is still unknown how PI3K/AKT signaling leads to survivin gene transcription.Several studies have shown that under normoxic conditions, activation of EGFR signaling also increases the level of hypoxiainducible factor 1␣ (HIF-1␣) through the PI3K/AKT pathway (19 -21). HIF-1␣, a member of the basic helix-loop-helix-PAS protein family (22) 2 The abbreviations used are: EGFR, epidermal growth factor receptor; IAP, inhibitor of apoptosis; EGF, epidermal growth factor; HIF, hypoxia-inducible factor; Doc, docetaxel; HRE, hypoxia-responsive element; 7-AAD, 7-amino-actinomycin D; PI3K, phosphoinositide 3-kinase; siRNA, small interfering RNA; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PE, phycoerythrin; nt, nucleotide(s); MAPK, mitogen-activated protein kinase; VEGF, vascular endothelial growth factor.

Gold nanorods (AuNRs)-assisted plasmonic photothermal therapy (AuNRs-PPTT) is a promising strategy for combating cancer in which AuNRs absorb near-infrared light and convert it into heat, causing cell death mainly by apoptosis and/or necrosis. Developing a valid PPTT that induces cancer cell apoptosis and avoids necrosis in vivo and exploring its molecular mechanism of action is of great importance. Furthermore, assessment of the long-term fate of the AuNRs after treatment is critical for clinical use. We first optimized the size, surface modification [rifampicin (RF) conjugation], and concentration (2.5 nM) of AuNRs and the PPTT laser power (2 W/cm) to achieve maximal induction of apoptosis. Second, we studied the potential mechanism of action of AuNRs-PPTT using quantitative proteomic analysis in mouse tumor tissues. Several death pathways were identified, mainly involving apoptosis and cell death by releasing neutrophil extracellular traps (NETs) (NETosis), which were more obvious upon PPTT using RF-conjugated AuNRs (AuNRs@RF) than with polyethylene glycol thiol-conjugated AuNRs. Cytochrome and p53-related apoptosis mechanisms were identified as contributing to the enhanced effect of PPTT with AuNRs@RF. Furthermore, Pin1 and IL18-related signaling contributed to the observed perturbation of the NETosis pathway by PPTT with AuNRs@RF. Third, we report a 15-month toxicity study that showed no long-term toxicity of AuNRs in vivo. Together, these data demonstrate that our AuNRs-PPTT platform is effective and safe for cancer therapy in mouse models. These findings provide a strong framework for the translation of PPTT to the clinic.