EGF-coated gold nanoparticles provide an efficient nano-scale delivery system for the molecular radiotherapy of EGFR-positive cancer.
ABSTRACT: Purpose Radiolabeled antibodies and peptides hold promise for molecular radiotherapy but are often limited by a low payload resulting in inadequate delivery of radioactivity to tumour tissue and, therefore, modest therapeutic effect. We developed a facile synthetic method of radiolabeling indium-111 (111In) to epidermal growth factor (EGF)-gold nanoparticles (111In-EGF-Au NP) with a high payload. Materials and methods EGF-Au NP were prepared via an interaction between gold and the disulphide bonds of EGF and radiolabeled using 111InCl3. Targeting efficiency was investigated by quantitating internalized radioactivity and by confocal imaging following exposure of MDA-MB-468 (1.3 × 106 EGFR/cell) and MCF-7 (104 EGFR/cell) cells to Cy3-EGF-Au NP. Cytotoxicity was evaluated in clonogenic assays. Results The proportion of total administered radioactivity that was internalized by MDA-MB-468 and MCF-7 cells was 15% and 1.3%, respectively (mixing ratio of EGF:Au of 160). This differential uptake in the two cell lines was confirmed using confocal microscopy. 111In-EGF-Au NP were significantly more radiotoxic to MDA-MB-468 than MCF-7 cells with a surviving fraction of 17.1 ± 4.4% versus 89.8 ± 1.4% (p < 0.001) after exposure for 4?h. Conclusions An 111In-labeled EGF-Au nanosystem was developed. It enabled targeted delivery of a high 111In payload specifically to EGFR-positive cancer cells leading to radiotoxicity that can be exploited for molecularly targeted radiotherapy.
Project description:The successful use of targeted radionuclide therapy in the treatment of solid tumours may be limited by radioresistance, which necessitates delivery of a high dose of radioactivity. Nanoparticle (NP)-based delivery systems possess a large surface area for attachment of radioisotopes and so offer a solution to this challenge. However, tumour uptake may be limited by rapid hepatic clearance of NP via the mononuclear phagocyte system. Liver uptake is further compounded when epidermal growth factor (EGF) is used as a targeting ligand, as EGF-tagged NP bind the EGF receptor (EGFR), which is expressed to a moderate extent by hepatocytes. This report describes an indium-111 (111In)-labelled PEGylated EGF-tagged gold (Au) NP (111In-EGF-Au-PEG) and an effective strategy of coadministration of targeting ligand to address these issues. Direct attachment of EGF to the surface of Au NP did not compromise surface coating with long-chain PEG. In vitro experiments showed that 111In-EGF-Au-PEG targets EGFR-positive cancer cells (MDA-MB-468): >11% of radioactivity was internalised after incubation for 4 h. In in vivo studies accumulation of NP was observed in MDA-MB-468 xenografts and tumour uptake was enhanced by the coadministration of 15 µg of the unlabelled targeting ligand, EGF, to block hepatic EGFR. Uptake was 3.9% versus 2.8% injected dose/g (%ID/g) of tumour tissue with and without unlabelled EGF, respectively. Coadministration of EGF reduced liver uptake by 25.95% to 7.56 %ID/g. This suggests that the coadministration of unlabelled targeting ligand with radiolabelled PEGylated NP offers a promising strategy for targeting EGFR-positive cancer and for minimising liver uptake.
Project description:Nanomedicines allow active targeting of cancer for diagnostic and therapeutic applications through incorporation of multiple functional components. Frequently, however, clinical translation is hindered by poor intratumoural delivery and distribution. The application of physical stimuli to promote tumour uptake is a viable route to overcome this limitation. In this study, ultrasound-mediated cavitation of microbubbles was investigated as a mean of enhancing the delivery of a liposome designed for chemo-radionuclide therapy targeted to EGFR overexpressing cancer. Method: Liposomes (111In-EGF-LP-Dox) were prepared by encapsulation of doxorubicin (Dox) and surface functionalisation with Indium-111 tagged epidermal growth factor. Human breast cancer cell lines with high and low EGFR expression (MDA-MB-468 and MCF7 respectively) were used to study selectivity of liposomal uptake, subcellular localisation of drug payload, cytotoxicity and DNA damage. Liposome extravasation following ultrasound-induced cavitation of microbubbles (SonoVue®) was studied using a tissue-mimicking phantom. In vivo stability, pharmacokinetic profile and biodistribution were evaluated following intravenous administration of 111In-labelled, EGF-functionalised liposomes to mice bearing subcutaneous MDA-MB-468 xenografts. Finally, the influence of ultrasound-mediated cavitation on the delivery of liposomes into tumours was studied. Results: Liposomes were loaded efficiently with Dox, surface decorated with 111In-EGF and showed selective uptake in MDA-MB-468 cells compared to MCF7. Following binding to EGFR, Dox was released into the intracellular space and 111In-EGF shuttled to the cell nucleus. DNA damage and cell kill were higher in MDA-MB-468 than MCF7 cells. Moreover, Dox and 111In were shown to have an additive cytotoxic effect in MDA-MB-468 cells. US-mediated cavitation increased the extravasation of liposomes in an in vitro gel phantom model. In vivo, the application of ultrasound with microbubbles increased tumour uptake by 66% (p<0.05) despite poor vascularisation of MDA-MB-468 xenografts (as shown by DCE-MRI). Conclusion: 111In-EGF-LP-Dox designed for concurrent chemo-radionuclide therapy showed specificity for and cytotoxicity towards EGFR-overexpressing cancer cells. Delivery to tumours was enhanced by the use of ultrasound-mediated cavitation indicating that this approach has the potential to deliver cytotoxic levels of therapeutic radionuclide to solid tumours.
Project description:The spatial distribution of radiopharmaceuticals that emit short-range high linear-energy-transfer electrons greatly affects the absorbed dose and their biological effectiveness. The purpose of this study was to investigate the effect of heterogeneous radionuclide distribution on tumor control probability (TCP) in a micrometastases model. Methods: Cancer cell lines; MDA-MB-468, SQ20B and 231-H2N were grown as spheroids to represent micrometastases. The intracellular distribution of a representative radiopeptide (111In-labelled epidermal growth factor, EGF) and radioimmunotherapeutic (111In-labelled Trastuzumab) was determined in cell internalization experiments. The intratumoral distribution was evaluated by microautoradiography of spheroids. ?H2AX staining was performed on spheroid sections to correlate DNA damage with radionuclide distribution. Experimental surviving fractions (SFexp ) were obtained using clonogenic assays. A random closed-packed algorithm, which models the random packing behavior of cells and reflects variation in the radii of cells and nuclei, was used to simulate 3-D spheroids. Calculated survival fractions (SFcal ) were generated using an iterative modelling method based on Monte Carlo determined absorbed dose with the PENELOPE code and were compared to (SFexp ). Radiobiological parameters deduced from experimental results and MC simulations were used to predict the TCP for a 3-D spheroid model. Results: Calculated SFs were in good agreement with experimental data, particularly when an increased value for relative biological effectiveness (RBE) was applied to self-dose deposited by sources located in the nucleus and when radiobiological parameters were adjusted to account for dose protraction. Only in MDA-MB-468 spheroids treated with 111In-EGF was a TCP>0.5 achieved, indicating that for this cell type the radiopeptide would be curative when targeting micrometastases. This is attributed to the relative radiosensitivity of MDA-MB-468 cells, high nuclear uptake of the radiopeptide and uniform distribution of radioactivity throughout the spheroid. Conclusion: It is imperative to include biological endpoints when evaluating the distribution of radionuclides in models emulating micrometastatic disease. The spatial distribution of radioactivity is a clear determinant of biological effect and TCP as demonstrated in this study.
Project description:Breast cancer cell invasion is influenced by growth factor concentration gradients in the tumor microenvironment. However, studying the influence of growth factor gradients on breast cancer cell invasion is challenging due to both the complexities of in vivo models and the difficulties in recapitulating the tumor microenvironment with defined gradients using in vitro models. A defined hyaluronic acid (HA)-based hydrogel crosslinked with matrix metalloproteinase (MMP) cleavable peptides and modified with multiphoton labile nitrodibenzofuran (NDBF) was synthesized to photochemically immobilize epidermal growth factor (EGF) gradients. We demonstrate that EGF gradients can differentially influence breast cancer cell invasion and drug response in cell lines with different EGF receptor (EGFR) expression levels. Photopatterned EGF gradients increase the invasion of moderate EGFR expressing MDA-MB-231?cells, reduce invasion of high EGFR expressing MDA-MB-468?cells, and have no effect on invasion of low EGFR-expressing MCF-7?cells. We evaluate MDA-MB-231 and MDA-MB-468?cell response to the clinically tested EGFR inhibitor, cetuximab. Interestingly, the cellular response to cetuximab is completely different on the EGF gradient hydrogels: cetuximab decreases MDA-MB-231?cell invasion but increases MDA-MB-468?cell invasion and cell number, thus demonstrating the importance of including cell-microenvironment interactions when evaluating drug targets.
Project description:Antibody-drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window. DS-8201a is a human epidermal growth factor receptor 2 (HER2)-targeting antibody-drug conjugate prepared using a novel linker-payload system with a potent topoisomerase I inhibitor, exatecan derivative (DX-8951 derivative, DXd). It was effective against trastuzumab emtansine (T-DM1)-insensitive patient-derived xenograft models with both high and low HER2 expression. In this study, the bystander killing effect of DS-8201a was evaluated and compared with that of T-DM1. We confirmed that the payload of DS-8201a, DXd (1), was highly membrane-permeable whereas that of T-DM1, Lys-SMCC-DM1, had a low level of permeability. Under a coculture condition of HER2-positive KPL-4 cells and negative MDA-MB-468 cells in vitro, DS-8201a killed both cells, whereas T-DM1 and an antibody-drug conjugate with a low permeable payload, anti-HER2-DXd (2), did not. In vivo evaluation was carried out using mice inoculated with a mixture of HER2-positive NCI-N87 cells and HER2-negative MDA-MB-468-Luc cells by using an in vivo imaging system. In vivo, DS-8201a reduced the luciferase signal of the mice, indicating suppression of the MDA-MB-468-Luc population; however, T-DM1 and anti-HER2-DXd (2) did not. Furthermore, it was confirmed that DS-8201a was not effective against MDA-MB-468-Luc tumors inoculated at the opposite side of the NCI-N87 tumor, suggesting that the bystander killing effect of DS-8201a is observed only in cells neighboring HER2-positive cells, indicating low concern in terms of systemic toxicity. These results indicated that DS-8201a has a potent bystander effect due to a highly membrane-permeable payload and is beneficial in treating tumors with HER2 heterogeneity that are unresponsive to T-DM1.
Project description:Multivalent presentation of ligands on nanoparticles (NPs) is considered a general strategy for enhancing receptor binding and activation through amplification of ligand-receptor interactions within the footprint of the individual NPs. The spatial clustering of ligand-functionalized NPs represents an additional, less well understood mechanism for increasing local ligand-receptor interactions, especially for receptors that form higher-order assemblies, such as the epidermal growth factor (EGF) receptor (EGFR). To shed light on the interplay between ligand density ( i.e., multivalency) and NP clustering in signal amplification, we apply EGF-functionalized 72 ± 1 nm gold nanoparticles (NP-EGF) with known ligand loading (10-200 EGF/NP) as quantifiable and experimentally tractable units of EGFR activation and characterize the NP-mediated amplification of EGFR phosphorylation as a function of both EGF surface density and NP-EGF clustering for two cancer cell lines (HeLa and MDA-MB-468). The measurements confirm a strong multivalent amplification of EGFR phosphorylation through NP-EGF on the cellular level that results in EGF-loading-dependent maximum EGFR phosphorylation levels. A microscopic analysis of NP-EGF-induced EGFR phosphorylation reveals a heterogeneous spatial distribution of EGFR activation across the cell surface. Clustering of multivalent NP-EGF on sub-diffraction-limited length scales is found to result in a local enhancement of EGFR phosphorylation in signaling "hot spots" from where the signal can spread laterally in an EGF-independent fashion. Increasing EGF loadings of the NP enhances NP-EGF clustering and intensifies EGFR phosphorylation. These observations suggest that NP-EGF clustering and the associated local enhancement of ligand-receptor interactions are intrinsic components of the multivalent amplification of phosphorylation for the heterogeneously distributed EGFR through NP-EGF.
Project description:BackgroundLipid nanocapsules (LNCs) are promising vehicles for drug delivery. However, since not much was known about cellular toxicity of these nanoparticles in themselves, we have here investigated the mechanisms involved in LNC-induced intoxication of the three breast cancer cell lines MCF-7, MDA-MD-231 and MDA-MB-468. The LNCs used were made of Labrafac™ Lipophile WL1349, Lipoid® S75 and Solutol® HS15.ResultsHigh resolution SIM microscopy showed that the DiD-labeled LNCs ended up in lysosomes close to the membrane. Empty LNCs, i.e. without encapsulated drug, induced not only increased lysosomal pH, but also acidification of the cytosol and a rapid inhibition of protein synthesis. The cytotoxicity of the LNCs were measured for up to 72 h of incubation using the MTT assay and ATP measurements in all three cell lines, and revealed that MDA-MB-468 was the most sensitive cell line and MCF-7 the least sensitive cell line to these LNCs. The LNCs induced generation of reactive free oxygen species and lipid peroxidation. Experiments with knock-down of kinases in the near-haploid cell line HAP1 indicated that the kinase HRI is essential for the observed phosphorylation of eIF2?. Nrf2 and ATF4 seem to play a protective role against the LNCs in MDA-MB-231 cells, as knock-down of these factors sensitizes the cells to the LNCs. This is in contrast to MCF-7 cells where the knock-down of these factors had a minor effect on the toxicity of the LNCs. Inhibitors of ferroptosis provided a large protection against LNC toxicity in MDA-MB-231 cells, but not in MCF-7 cells.ConclusionsHigh doses of LNCs showed a different degree of toxicity on the three cell lines studied, i.e. MCF-7, MDA-MD-231 and MDA-MB-468 and affected signaling factors and the cell fate differently in these cell lines.
Project description:Purpose:To conduct an exploratory study to identify mechanisms that differentiate Luminal A (BT474 and MCF-7) and triple-negative (MDA-MB-231 and MDA-MB-468) breast cancer (BCa) cell lines to potentially provide novel therapeutic targets based on differences in energy utilization. Methods:Cells were cultured in media containing either [U-13C]-glucose or [U-13C]-glutamine for 48 hours. Conditioned media and cellular extracts were analyzed by 1H and 13C NMR spectroscopy. Results:MCF-7 cells consumed the most glucose, producing the most lactate, demonstrating the greatest Warburg effect-associated energy utilization. BT474 cells had the highest tricarboxylic acid cycle (TCA) activity. The majority of energy utilization patterns in MCF-7 cells were more similar to MDA-MB-468 cells, while the patterns for BT474 cells were more similar to MDA-MB-231 cells. Compared to the Luminal A cell lines, TNBC cell lines consumed more glutamine and less glucose. BT474 and MDA-MB-468 cells produced high amounts of 13C-glycine from media [U-13C]-glucose which was integrated into glutathione, indicating de novo synthesis. Conclusions:Stable isotopic resolved metabolomics using 13C substrates provided mechanistic information about energy utilization that was difficult to interpret using 1H data alone. Overall, cell lines that have different hormone receptor status have different energy utilization requirements, even if they are classified by the same clinical BCa subtype; and these differences offer clues about optimizing treatment strategies.
Project description:The mevalonate pathway has emerged as a promising target for several solid tumors. Statins are inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme of this pathway, and are commonly used to treat patients with hypercholesterolemia. Pleiotropic antitumor mechanisms of statins have been demonstrated for several human cancer types. However, cancer cells differ in their individual statin sensitivity and some cell lines have shown relative resistance. In this study we demonstrate, that the human breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF-7, and T47D are differentially affected by statins. Whereas the vitality of MDA-MB-231 and MDA-MB-468 cells was reduced by up to 60% using atorvastatin, simvastatin, or rosuvastatin (p?<?0.001), only marginal effects were seen in T47D and MCF-7 cells following exposure to statins. Statin treatment led to an upregulation of HMGCR mRNA and protein expression by up to sixfolds in the statin-resistant cells lines (p?<?0.001), but no alterations of HMGCR were observed in the statin-sensitive MDA-MB-231 and MDA-MB-468 cells. The knockdown of HMGCR prior to statin treatment sensitized the resistant cell lines, reflected by a 70% reduction in vitality, increased apoptotic DNA fragmentation (sixfold) and by accumulation of the apoptosis marker cleaved poly-ADP ribose polymerase. Statins induced a cleavage of the sterol-regulatory element-binding protein (SREBP)-2, a transcriptional activator of the HMGCR, in T47D and MCF-7 cells. The inhibition of SREBP-2 activation by co-administration of dipyridamole sensitized MCF-7 and T47D cells for statins (loss of vitality by 80%; p?<?0.001). Furthermore, assessment of a statin-resistant MDA-MB-231 clone, generated by long-term sublethal statin exposure, revealed a significant induction of HMGCR expression by up to 12-folds (p?<?0.001). Knockdown of HMGCR restored statin sensitivity back to levels of the parental cells. In conclusion, these results indicate a resistance of cancer cells against statins, which is in part due to the induction of HMGCR.
Project description:In addition to the intrinsic toxicity associated with the chemical composition of nanoparticles (NP) and their ligands, biofunctionalized NP can perturb specific cellular processes through NP-cell interactions and induce programmed cell death (apoptosis). In the case of the epidermal growth factor (EGF), nanoconjugation has been shown to enhance the apoptotic efficacy of the ligand, but the critical aspects of the underlying mechanism and its dependence on the NP morphology remain unclear. In this manuscript we characterize the apoptotic efficacy of nanoconjugated EGF as a function of NP size (with sphere diameters in the range 20-80 nm), aspect ratio (A.R., in the range of 4.5 to 8.6), and EGF surface loading in EGFR overexpressing MDA-MB-468 cells. We demonstrate a significant size and morphology dependence in this relatively narrow parameter space with spherical NP with a diameter of approx. 80 nm being much more efficient in inducing apoptosis than smaller spherical NP or rod-shaped NP with comparable EGF loading. The nanoconjugated EGF is found to trigger an EGFR-dependent increase in cytoplasmic reactive oxygen species (ROS) levels but no indications of increased mitochondrial ROS levels or mitochondrial membrane damage are detected at early time points of the apoptosis induction. The increase in cytoplasmic ROS is accompanied by a perturbation of the intracellular glutathione homeostasis, which represents an important check-point for NP-EGF mediated apoptosis. Abrogation of the oxidative stress through the inhibition of EGFR signaling by the EGFR inhibitor AG1478 or addition of antioxidants N-acetyl cysteine (NAC) or tempol, but not trolox, successfully suppressed the apoptotic effect of nanoconjugated EGF. A model to account for the observed morphology dependence of EGF nanoconjugation enhanced apoptosis and the underlying NP-cell interactions is discussed.