Folate-conjugated iron oxide nanoparticles for solid tumor targeting as potential specific magnetic hyperthermia mediators: synthesis, physicochemical characterization, and in vitro experiments

New folate-conjugated superparamagnetic maghemite nanoparticles have been synthesized for the intracellular hyperthermia treatment of solid tumors. These ultradispersed nanosystems have been characterized for their physicochemical properties and tumor cell targeting ability, facilitated by surface modification with folic acid. Preliminary experiments of nanoparticles heating under the influence of an alternating magnetic field at 108 kHz have been also performed. The nanoparticle size, surface charge, and colloidal stability have been assessed in various conditions of ionic strength and pH. The ability of these folate "decorated" maghemite nanoparticles to recognize the folate receptor has been investigated both by surface plasmon resonance and in folate receptor expressing cell lines, using radiolabeled folic acid in competitive binding experiments. The specificity of nanoparticle cellular uptake has been further investigated by transmission electron microscopy after incubation of these nanoparticles in the presence of three cell lines with differing folate receptor expression levels. Qualitative and quantitative determinations of both folate nanoparticles and nontargeted control nanoparticles demonstrated a specific cell internalization of the folate superparamagnetic nanoparticles.     

Synthesis and Evaluation of a Peptide Targeted Small Molecular Gd-DOTA Monoamide Conjugate for MR Molecular Imaging of Prostate Cancer

Tumor extracellular matrix has an abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. Innovative design and development of safe and effective targeted contrast agents to these biomarkers would allow effective MR cancer molecular imaging with high spatial resolution. In this study, we synthesized a low molecular weight CLT1 peptide targeted Gd(III) chelate CLT1-dL-(Gd-DOTA)(4) specific to clotted plasma proteins in tumor stroma for cancer MR molecular imaging. CLT1-dL-(Gd-DOTA)(4) was synthesized by conjugating four Gd-DOTA monoamide chelates to a CLT1 peptide via generation 1 lysine dendrimer. The T(1) relaxivity of CLT1-dL-(Gd-DOTA)(4) was 40.4 mM(-1) s(-1) per molecule (10.1 mM(-1) s(-1) per Gd) at 37 °C and 1.5 T. Fluorescence imaging showed high binding specificity of CLT1 to orthotopic PC3 prostate tumor in mice. The contrast agent resulted in improved tumor contrast enhancement in male athymic nude mice bearing orthotopic PC3 prostate tumor xenograft at a dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for high-resolution MR molecular imaging of prostate tumor.     

Engineering tumor-targeted gadolinium hexanedione nanoparticles for potential application in neutron capture therapy

Microemulsions (oil-in-water) have been employed as templates to engineer nanoparticles containing high concentrations of gadolinium for potential application in neutron capture therapy of tumors. Gadolinium hexanedione (GdH), synthesized by complexation of Gd(3+) with 2,4-hexanedione, was used as the nanoparticle matrix alone or in combination with either emulsifying wax or PEG-400 monostearate. Solid nanoparticles (<125 nm size) were obtained by simple cooling of the microemulsions prepared at 60 degrees C to room temperature in one vessel. The feasibility of tumor targeting via folate receptors was studied. A folate ligand was synthesized by chemically linking folic acid to distearoylphosphatidylethanolamine (DSPE) via a poly(ethylene glycol) (PEG; MW 3350) spacer. To obtain folate-coated nanoparticles, the folate ligand (0.75% w/w to 15% w/w) was added to either the microemulsion templates at 60 degrees C or nanoparticle suspensions at 25 degrees C. Efficiencies of folate ligand attachment/adsorption to nanoparticle formulations were monitored by gel permeation chromatography. Cell uptake studies were carried out in KB cells (human nasopharyngeal epidermal carcinoma cell line), known to overexpress folate receptors. The uptake of folate-coated nanoparticles was about 10-fold higher than uncoated nanoparticles after 30 min at 37 degrees C. The uptake of folate-coated nanoparticles at 4 degrees C was 20-fold lower than the uptake at 37 degrees C and comparable to the uptake of uncoated nanoparticles at 37 degrees C. Folate-mediated endocytosis was further verified by the inhibition of folate-coated nanoparticles uptake by free folic acid. It was observed that folate-coated nanoparticles uptake decreased to approximately 2% of its initial value with the coincubation of 0.001 mM of free folic acid. The results suggested that these tumor-targeted nanoparticles containing high concentrations of Gd may have potential for neutron capture therapy.     

Preparation and preliminary evaluation of a biotin-targeted, lectin-targeted dendrimer-based probe for dual-modality magnetic resonance and fluorescence imaging

A novel approach for the preparation of a biotinylated dendrimer-based MRI agent 5 is described, in which a unique disulfide bond in the core of the Gd(III)-1B4M-DTPA chelated G2 PAMAM dendrimer was reduced and then attached to a maleimide-functionalized biotin. The new MRI agent 5 features a well-defined dendron structure and a unique biotin functionality. Immobilization of up to four copies of biotinylated dendrimer 5 to fluorescently labeled avidin yields a supramolecular avidin-biotin-dendrimer-Gd(III) complex. Validation of the complex in mice bearing ovarian cancer tumors demonstrates that the avidin-biotin-dendrimer targeting system efficiently targets and delivers sufficient amounts of chelated Gd(III) and fluorophores (e.g., Rhodamine green) to ovarian tumors to produce visible changes in the tumors by both MRI and optical imaging, respectively. Thus, the avidin-biotin-dendrimer complex may be used as a tumor-targeted probe for dual-modality magnetic resonance and fluorescence imaging.     

A novel cholic acid-based contrast enhancement agent for targeted MRI

The novel Gd(III) complexes of heptadentate ligands NE3TA and NE3TA-Bn were prepared, and their relaxivities were measured and favorably compared to the commercially available MRI contrast enhancement agent Gd(DOTA). NE3TA was conjugated with cholic acid (CA) to produce CA-NE3TA. TEM images of Gd(CA-NE3TA) indicate that the complex self-assembles forming nano-sized micelles and displays an over threefold increased relaxivity compared to Gd(DOTA). The new cholic acid-conjugated nanoparticle MR contrast enhancement agent, Gd(CA-NE3TA) possesses great promise for use in targeted MRI.     

The facile synthesis of multifunctional PAMAM dendrimer conjugates through copper-free click chemistry

The facile conjugation of three azido modified functionalities, namely a therapeutic drug (methotrexate), a targeting moiety (folic acid), and an imaging agent (fluorescein) with a G5 PAMAM dendrimer scaffold with cyclooctyne molecules at the surface through copper-free click chemistry is reported. Mono-, di-, and tri-functional PAMAM dendrimer conjugates can be obtained via combinatorial mixing of different azido modified functionalities simultaneously or sequentially with the dendrimer platform. Preliminary flow cytometry results indicate that the folic acid targeted nanoparticles are efficiently binding with KB cells.     

转铁蛋白导向脂质体核磁造影剂纳米粒子（TfNIR-LipNBD-Magnevist）——一种肿瘤靶向磁共振造影剂

造影剂辅助的核磁共振成像是目前肿瘤诊断的最吁方法之一。但是由于核磁共振成像内在的低灵敏性以及造影剂的非特异性,导致肿瘤早期诊断较为困难。文章将一种新的肿瘤靶向核磁造影剂纳米粒子应用于早期肿瘤的影像诊断。这种新的肿瘤靶向核磁造影剂纳米粒子由配体转铁蛋白（Tf）、纳米水平的正电脂质体（Lip）载体和临床常用的造影剂Magnevist（Tf^NIR-Lip^NBD-Magnevist）三部分构成。另外转铁蛋白和脂质体粒子上,亦标记了荧光物质用于确定转铁蛋白一脂质体一造影剂纳米粒子的靶向性,以及肿瘤的光学影像诊断。在体外实验中,利用激光共聚焦显微镜和光学影像证明了靶向纳米粒子介导的细胞内吞和特异性结合。在裸鼠肿瘤模型中,造影剂纳米粒子Tf^NIR-Lip^NBD-Magnevist经尾静脉注入后,显著增强了肿瘤内信号与周围组织的对比度。由造影剂纳米粒子介导的肿瘤内信号显著强于单独Magnevist辅助的肿瘤内信号。同时,利用光学影像方法,在肿瘤内检测到特异的荧光信号。其结果进一步支持了转铁蛋白一脂质体一造影利（Tf^NIR-Lip^NBD-Magnevist）纳米粒子的靶向性和肿瘤影像诊断的有效性。     

Functionalized amphiphilic hyperbranched polymers for targeted drug delivery

Amphiphilic hyperbranched core-shell polymers with folate moieties as the targeting groups were synthesized and characterized. The core of the amphiphilic polymers was hyperbranched aliphatic polyester Boltorn H40. The inner part and the outer shell of the amphiphilic polymers were composed of hydrophobic poly(epsilon-caprolactone) segments and hydrophilic poly(ethylene glycol) (PEG) segments, respectively. To achieve tumor cell targeting property, folic acid was further incorporated to the surface of the amphiphilic polymers via a coupling reaction between the hydroxyl group of the PEG segment and the carboxyl group of folic acid. The polymers were characterized by (1)H NMR, (13)C NMR, and combined size-exclusion chromatography and multiangle laser light scattering analysis. The nanoparticles of the amphiphilic polymers prepared by dialysis method were characterized by transmission electron microscopy and particle size analysis. Two antineoplastic drugs, 5-fluorouracil and paclitaxel, were encapsulated into the nanoparticles. The drug release property and the targeting of the drug-loaded nanoparticles to different cells were evaluated in vitro. The results showed the drug-loaded nanoparticles exhibited enhanced cell inhibition because folate targeting increased the cytotoxicity of drug-loaded nanoparticles against folate receptor expressing tumor cells.     

Selective N-alkylation of β-alanine facilitates the synthesis of a poly(amino acid)-based theranostic nanoagent

The development of functional amino acid-based polymeric materials is emerging as a platform to create biodegradable and nontoxic nanomaterials for medical and biotechnology applications. In particular, facile synthetic routes for these polymers and their corresponding polymeric nanomaterials would have a positive impact in the development of novel biomaterials and nanoparticles. However, progress has been hampered by the need to use complex protection-deprotection methods and toxic phase transfer catalysts. In this study, we report a facile, single-step approach for the synthesis of an N-alkylated amino acid as an AB-type functional monomer to generate a novel pseudo-poly(amino acid), without using the laborious multistep, protection-deprotection methods. This synthetic strategy is reproducible, easy to scale up, and does not produce toxic byproducts. In addition, the synthesized amino acid-based polymer is different from conventional linear polymers as the butyl pendants enhance its solubility in common organic solvents and facilitate the creation of hydrophobic nanocavities for the effective encapsulation of hydrophobic cargos upon nanoparticle formation. Within the nanoparticles, we have encapsulated a hydrophobic DiI dye and a therapeutic drug, Taxol. In addition, we have conjugated folic acid as a folate receptor-targeting ligand for the targeted delivery of the nanoparticles to cancer cells expressing the folate receptor. Cell cytotoxicity studies confirm the low toxicity of the polymeric nanoparticles, and drug-release experiments with the Taxol-encapsulated nanoparticles only exhibit cytotoxicity upon internalization into cancer cells expressing the folate receptor. Taken together, these results suggested that our synthetic strategy can be useful for the one-step synthesis of amino acid-based small molecules, biopolymers, and theranostic polymeric nanoagents for the targeted detection and treatment of cancer.     

Synthesis and biological evaluation of EC140: a novel folate-targeted vinca alkaloid conjugate

A novel folate conjugate of desacetylvinblastine monohydrazide (DAVLBH), herein referred to as EC140, was designed and evaluated for biological activity against folate receptor (FR)-positive cells and tumors. EC140 was produced by coupling a peptidic analogue of the vitamin folic acid to DAVLBH via an acylhydrazone bond. This water-soluble conjugate was found to retain high affinity for FR-positive cells, and it produced specific, dose-responsive activity in vitro. Initial in vivo tests confirmed EC140's activity in both syngeneic and xenograft models. Hence, enduring complete responses were observed in animals bearing established, subcutaneous tumors prior to therapy using regimens that produced minor toxicity. In contrast, treatment with the unconjugated DAVLBH drug produced nominal efficacy when dosed at its MTD. Overall, EC140's performance in vitro and in vivo warrants further preclinical study before this novel targeted chemotherapeutic is considered for clinical investigation.     

Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting of folate receptor-positive tumor cells

This paper reports the creation of Au nanoparticles (AuNP) that are soluble in aqueous solution over a broad range of pH and ionic strength values and that are capable of selective uptake by folate receptor positive (FR+) cancer cells. A novel poly(ethylene glycol) (PEG) construct with thioctic acid and folic acid coupled on opposite ends of the polymer chain was synthesized for targeting the AuNP to FR+ tumor cells via receptor-mediated endocytosis. These folic acid-PEG-thioctic acid conjugates were grafted onto 10-nm-diameter Au particles in aqueous solution. The resulting folate-PEG-coated nanoparticles do not aggregate over a pH range of from 2 to 12 and at electrolyte concentrations of up to 0.5 M NaCl with particle concentrations as high as 1.5 x 10(13) particles/mL. Transmission electron microscopy was used to document the performance of these coated nanoparticles in cell culture. Selective uptake of folate-PEG grafted AuNPs by KB cells, a FR+ cell line that overexpress the folate receptor, was observed. AuNP uptake was minimal in cells that (1) do not overexpress the folate receptor, (2) were exposed to AuNP lacking the folate-PEG conjugate, or (3) were co-incubated with free folic acid in large excess relative to the folate-PEG grafted AuNP. Understanding this process is an important step in the development of methods that use targeted metal nanoparticles for tumor imaging and ablation.     

Human aortic endothelial cell labeling with positive contrast gadolinium oxide nanoparticles for cellular magnetic resonance imaging at 7 Tesla

Positive T? contrast using gadolinium (Gd) contrast agents can potentially improve detection of labeled cells on magnetic resonance imaging (MRI). Recently, gadolinium oxide (Gd?O?) nanoparticles have shown promise as a sensitive T? agent for cell labeling at clinical field strengths compared to conventional Gd chelates. The objective of this study was to investigate Gado CELLTrack, a commercially available Gd?O? nanoparticle, for cell labeling and MRI at 7 T. Relaxivity measurements yielded r1 = 4.7 s?1 mM?1 and r?/r? = 6.2. Human aortic endothelial cells were labeled with Gd?O? at various concentrations and underwent MRI from 1 to 7 days postlabeling. The magnetic resonance relaxation times T? and T? of labeled cell pellets were measured. Cellular contrast agent uptake was quantified by inductively coupled plasma-atomic emission spectroscopy, which showed very high uptake compared to conventional Gd compounds. MRI demonstrated significant positive T? contrast and stable labeling on cells. Enhancement was optimal at low Gd concentrations, attained in the 0.02 to 0.1 mM incubation concentration range (corresponding cell uptake was 7.26 to 34.1 pg Gd/cell). Cell viability and proliferation were unaffected at the concentrations tested and up to at least 3 days postlabeling. Gd?O? is a promising sensitive and stable positive contrast agent for cellular MRI at 7 T.     

Avidin-dendrimer-(1B4M-Gd)(254): a tumor-targeting therapeutic agent for gadolinium neutron capture therapy of intraperitoneal disseminated tumor which can be monitored by MRI.

Peritoneal carcinomatosis is a late stage in cancer progress, for which no effective therapeutic modality exists. Targeting therapeutic agents to disseminated lesions may be a promising modality for treating peritoneal carcinomatosis. Gadolinium ((157,155)Gd) is known to generate Auger and internal conversion electrons efficiently by irradiation with a neutron beam. Auger electrons from neutron-activated Gd(III) are strongly cytotoxic, but only when Gd(III) atoms have been internalized into the cells. In the present investigation, we have developed a quickly internalizing tumor-targeting system to deliver large quantities of Gd(III) atoms into tumor cells to generate the Auger emission with an external neutron beam. Simultaneously, one would be able to image its biodistribution by MRI with a shortened T1 relaxation time. Avidin-G6-(1B4M-Gd)(254) (Av-G6Gd) was synthesized from generation-6 polyamidoamine dendrimer, biotin, avidin, and 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (1B4M). The Av-G6Gd was radiolabeled with Gd(III) doped with (153)Gd. All of the 1B4M's on the conjugate were fully saturated with Gd(III) atoms. An in vitro internalization study showed that Av-G6Gd accumulated and was internalized into SHIN3 cells (a human ovarian cancer) 50- and 3.5-fold greater than Gd-DTPA (Magnevist) and G6-(1B4M-Gd)(256) (G6Gd). In addition, accumulation of Gd(III) in the cells was detected by the increased signal on T1-weighted MRI. A biodistribution study was performed in nude mice bearing intraperitoneally disseminated SHIN3 tumors. Av-G6Gd showed specific accumulation in the SHIN3 tumor (103% ID/g) 366- and 3.4-fold greater than Gd-DTPA (0.28% ID/g, p < 0.001) and G6Gd (30% ID/g, p < 0.001) 1 day after i.p. injection. Seventy-eight percent of the tumor-related radioactivity of Av-G6Gd in the SHIN3 tumor was located inside the cells. The SHIN3 tumor-to-normal tissue ratio was greater than 17:1 in all organs and increased up to 638:1 at 1 day after i.p. injection. In conclusion, a sufficient amount (162 ppm) of Av-G6Gd was accumulated and internalized into the SHIN3 cells both in vitro and in vivo to kill the cell using (157/155)Gd with external irradiation with an appropriate neutron beam while monitoring with MRI. Thus, Av-G6Gd may be a promising agent for Gd neutron capture therapy of peritoneal carcinomatosis. This reagent also has the potential to permit monitoring of its pharmacokinetic progress with MRI.     

Development of SM5-1-conjugated ultrasmall superparamagnetic iron oxide nanoparticles for hepatoma detection

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are maghemite or magnetite nanoparticles currently used as contrast agent in magnetic resonance imaging (MRI). In this study, a targeted contrast agent (SM-USPIO) was prepared by conjugating coprecipitated USPIO to a humanized SM5-1 antibody which can specifically react with human hepatocellular carcinoma (HCC) cells. The binding and internalization of SM-USPIO to the HCC cell line ch-hep-3 was confirmed by flow cytometry and confocal microscopy. Furthermore, SM-USPIO was demonstrated to be able to selectively accumulate in the tumor cells, resulting in a marked decrease of MRI T2-weighted signal intensity. Biodistribution studies demonstrated the efficient accumulation of SM-USPIO in the ch-hep-3 tumor in nude mice. The in vivo study in the ch-hep-3 tumor-bearing nude mice indicated that MRI using the SM-USPIO as contrast agent possessed good diagnostic ability, suggesting that SM-USPIO had the potential to be a promising targeted contrast agent for diagnosis of HCC.     

Integrin αvβ3-Targeted Dynamic Contrast-Enhanced Magnetic Resonance Imaging Using a Gadolinium-Loaded Polyethylene Gycol-Dendrimer-Cyclic RGD Conjugate to Evaluate Tumor Angiogenesis and to Assess Early Antiangiogenic Treatment Response in a Mouse Xenograft Tumor Model

AbstractThe purpose of this study was to validate an integrin αvβ3-targeted magnetic resonance contrast agent, PEG-G3-(Gd-DTPA)6-(cRGD-DTPA)2, for its ability to detect tumor angiogenesis and assess early response to antiangiogenic therapy using dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI). Integrin αvβ3-positive U87 cells and control groups were incubated with fluorescein-labeled cRGD-conjugated dendrimer, and the cellular attachment of the dendrimer was observed. DCE MRI was performed on mice bearing KB xenograft tumors using either PEG-G3-(Gd-DTPA)6-(cRGD-DTPA)2 or PEG-G3-(Gd-DTPA)6-(cRAD-DTPA)2. DCE MRI was also performed 2 hours after anti-integrin αvβ3 monoclonal antibody treatment and after bevacizumab treatment on days 3 and 6t. Using DCE MRI, the 30-minute contrast washout percentage was significantly lower in the cRGD-conjugate injection groups. The enhancement patterns were different between the two contrast injection groups. In the antiangiogenic therapy groups, a rapid increase in 30-minute contrast washout percentage was observed in both the LM609 and bevacizumab treatment groups, and this occurred before there was an observable decrease in tumor size. The integrin αvβ3 targeting ability of PEG-G3-(Gd-DTPA)6-(cRGD-DTPA)2 in vitro and in vivo was demonstrated. The 30-minute contrast washout percentage is a useful parameter for examining tumor angiogenesis and for the early assessment of antiangiogenic treatment response.     

Folate receptor-targeted aggregation-enhanced near-IR emitting silica nanoprobe for one-photon in vivo and two-photon ex vivo fluorescence bioimaging

A two-photon absorbing (2PA) and aggregation-enhanced near-infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes the fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong a signal as the unaggregated dye, a 3-fold increase in two-photon absorption relative to the DFP in solution, and approximately 4-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and (1)H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing a folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FR-targeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 μm deep in the HeLa tumor.     

Tumor-targeted pH-low insertion peptide delivery of theranostic gadolinium nanoparticles for image-guided nanoparticle-enhanced radiation therapy

Tumor targeting studies using metallic nanoparticles (NPs) have shown that the enhanced permeability and retention effect may not be sufficient to deliver the amount of intratumoral and intracellular NPs needed for effective in vivo radiosensitization. This work describes a pH-Low Insertion Peptide (pHLIP) targeted theranostic agent to enable image-guided NP-enhanced radiotherapy using a clinically feasible amount of injected NPs. Conventional gadolinium (Gd) NPs were conjugated to pHLIPs and evaluated in vitro for radiosensitivity and in vivo for mouse MRI. Cultured A549 human lung cancer cells were incubated with 0.5 mM of pHLIP-GdNP or conventional GdNP. Mass spectrometry showed 78-fold more cellular Gd uptake with pHLIP-GdNPs, and clonogenic survival assays showed 44% more enhanced radiosensitivity by 5 Gy irradiation with pHLIP-GdNPs at pH 6.2. In contrast to conventional GdNPs, MR imaging of tumor-bearing mice showed pHLIP-GdNPs had a long retention time in the tumor (>9 h), suitable for radiotherapy, and penetrated into the poorly-vascularized tumor core. The Gd-enhanced tumor corresponded with low-pH areas also independently measured by an in vivo molecular MRI technique. pHLIPs actively target cell surface acidity from tumor cell metabolism and deliver GdNPs into cells in solid tumors. Intracellular delivery enhances the effect of short-range radiosensitizing photoelectrons and Auger electrons. Because acidity is a general hallmark of tumor cells, the delivery is more general than antibody targeting. Imaging the in vivo NP biodistribution and more acidic (often more aggressive) tumors has the potential for quantitative radiotherapy treatment planning and pre-selecting patients who will likely benefit more from NP radiation enhancement.     

Polyvalent saccharide-functionalized generation 3 poly(amidoamine) dendrimer-methotrexate conjugate as a potential anticancer agent

A saccharide-terminated generation 3 (G3) polyamidoamine (PAMAM) dendrimer was synthesized as a drug carrier. Utilizing this dendritic platform, we have successfully synthesized polyvalent conjugates (G3-MTX) containing the drug methotrexate (MTX). Surface Plasmon Resonance (SPR) results showed that G3-MTX presented three orders of magnitude enhancement in binding avidity to folate-binding protein (FBP) as compared to the free folic acid (FA). Flow cytometric and confocal microscopic analysis showed that conjugate (G3-MTX-FI) containing imaging agent fluorescein-5(6)-carboxamidohexanoic acid (FI) was internalized into folate receptor (FR)-expressing KB cells in dose-dependent and receptor-mediated fashion. The G3-MTX induced a dose-dependent cytotoxicity in the KB cells. Therefore, the polyvalent G3-MTX may have potential as an anticancer nanodevice for the specific targeting and killing of FR-expressing tumor cells.     

Design of (Gd-DO3A)n-polydiamidopropanoyl-peptide nucleic acid-D(Cys-Ser-Lys-Cys) magnetic resonance contrast agents

We hypothesized that chelating Gd(III) to 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A) on peptide nucleic acid (PNA) hybridization probes would provide a magnetic resonance genetic imaging agent capable of hybridization to a specific mRNA. Because of the low sensitivity of Gd(III) as an magnetic resonance imaging (MRI) contrast agent, a single Gd-DO3A complex per PNA hybridization agent could not provide enough contrast for detection of cancer gene mRNAs, even at thousands of mRNA copies per cell. To increase the Gd(III) shift intensity of MRI genetic imaging agents, we extended a novel DO3An-polydiamidopropanoyl (PDAPm) dendrimer, up to n = 16, from the N-terminus of KRAS PNA hybridization agents by solid phase synthesis. A C-terminal D(Cys-Ser-Lys-Cys) cyclized peptide analog of insulin-like growth factor 1 (IGF1) was included to enable receptor-mediated cellular uptake. Molecular dynamic simulation of the (Gd-DO3A-AEEA)16-PDAP4-AEEA2-KRAS PNA-AEEA-D(Cys-Ser-Lys-Cys) genetic imaging nanoparticles in explicit water yielded a pair correlation function similar to that of PAMAM dendrimers, and a predicted structure in which the PDAP dendron did not sequester the PNA. Thermal melting measurements indicated that the size of the PDAP dendron included in the (DO3A-AEEA)n-PDAPm-AEEA2-KRAS PNA-AEEA-D(Cys-Ser-Lys-Cys) probes (up to 16 Gd(III) cations per PNA) did not depress the melting temperatures (Tm) of the complementary PNA/RNA hybrid duplexes. The Gd(III) dendrimer PNA genetic imaging agents in phantom solutions displayed significantly greater T1 relaxivity per probe (r1 = 30.64 +/- 2.68 mM(-1) s(-1) for n = 2, r1 = 153.84 +/- 11.28 mM(-1) s(-1) for n = 8) than Gd-DTPA (r1 = 10.35 +/- 0.37 mM(-1) s(-1)), but less than that of (Gd-DO3A)32-PAMAM dendrimer (r1 = 771.84 +/- 20.48 mM(-1) s(-1)) (P < 0.05). Higher generations of PDAP dendrimers with 32 or more Gd-DO3A residues attached to PNA-D(Cys-Ser-Lys-Cys) genetic imaging agents might provide greater contrast for more sensitive detection.     

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.