Effect of Dose and Selection of Two Different Ligands on the Deposition and Antitumor Efficacy of Targeted Nanoparticles in Brain Tumors.

Deposition of nanoparticles to tumors often can be enhanced by targeting receptors overexpressed in a tumor. However, a tumor may exhibit a finite number of a biomarker that is accessible and targetable by nanoparticles, limiting the available landing spots. To explore this, we selected two different biomarkers that effectively home nanoparticles in brain tumors. Specifically, we used either an αvß3 integrin-targeting peptide or a fibronectin-targeting peptide as a ligand on nanoparticles termed RGD-NP and CREKA-NP, respectively. In mouse models of glioblastoma multiforme, we systemically injected the nanoparticles loaded with a cytotoxic drug at different doses ranging from 2 to 8 mg/kg drug. The upper dose threshold of RGD-NP is ∼2 mg/kg. CREKA-NP reached its upper dose threshold at 5 mg/kg. For both targeted nanoparticle variants, higher dose did not ensure higher intratumoral drug levels, but it contributed to elevated off-target deposition and potentially greater toxicity. A cocktail combining RGD-NP and CREKA-NP was then administered at a dose corresponding to the upper dose threshold for each formulation resulting in a 3-fold higher intratumoral deposition than the individual formulations. The combination of the two different targeting schemes at the appropriate dose for each nanoparticle variant facilitated remarkable increase in intratumoral drug levels that was not achievable by a sole targeting nanoparticle alone.

Effective treatment of cancer metastasis using a dual-ligand nanoparticle.

Metastasis is responsible for the majority of deaths of breast cancer patients. While cytotoxic drugs are available with high potency to kill breast cancer cells, they are not designed to specifically seek and navigate in the dynamic and continuously changing microenvironment of metastatic disease. To effectively delivery chemotherapeutic agents to metastasis, we designed a dual-ligand nanoparticle loaded with doxorubicin by using two different types of ligands targeting EGFR and αvß3 integrin. Metastatic cancer cells continuously change resulting in heterogeneity even across adjacent micrometastatic regions with variable expression of these targetable receptors. Using a mouse model of breast cancer metastasis, in vivo and ex vivo imaging showed that both EGFR and αvß3 integrin-targeting were required to reliably direct the nanoparticle to metastasis and capture the spread and exact topology of the disease. Survival studies compared the anticancer efficacy of the standard drug, EGFR-targeting nanoparticle, αvß3 integrin-targeting nanoparticle and the dual-ligand nanoparticle. While all the other treatments produced moderate therapeutic outcomes, treatment with the dual-ligand nanoparticle yielded significant improvement and event-free survival in a mouse model of breast cancer metastasis.

Imaging breast cancer using a dual-ligand nanochain particle.

Nanoparticles often only exploit the upregulation of a receptor on cancer cells to enhance intratumoral deposition of therapeutic and imaging agents. However, a single targeting moiety assumes that a tumor is homogenous and static. Tumoral microenvironments are both heterogenous and dynamic, often displaying variable spatial and temporal expression of targetable receptors throughout disease progression. Here, we evaluated the in vivo performance of an iron oxide nanoparticle in terms of targeting and imaging of orthotropic mouse models of aggressive breast tumors. The nanoparticle, a multi-component nanochain, was comprised of 3-5 iron oxide nanoparticles chemically linked in a linear chain. The nanoparticle's surface was decorated with two types of ligands each targeting two different upregulated biomarkers on the tumor endothelium, P-selectin and fibronectin. The nanochain exhibited improved tumor deposition not only through vascular targeting but also through its elongated structure. A single-ligand nanochain exhibited a ~2.5-fold higher intratumoral deposition than a spherical nanoparticle variant. Furthermore, the dual-ligand nanochain exhibited higher consistency in generating detectable MR signals compared to a single-ligand nanochain. Using a 7T MRI, the dual-ligand nanochains exhibited highly detectable MR signal within 3h after injection in two different animal models of breast cancer.

Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis.

Various targeting strategies and ligands have been employed to direct nanoparticles to tumors that upregulate specific cell-surface molecules. However, tumors display a dynamic, heterogeneous microenvironment, which undergoes spatiotemporal changes including the expression of targetable cell-surface biomarkers. Here, we investigated a dual-ligand nanoparticle to effectively target two receptors overexpressed in aggressive tumors. By using two different chemical specificities, the dual-ligand strategy considered the spatiotemporal alterations in the expression patterns of the receptors in cancer sites. As a case study, we used two mouse models of metastasis of triple-negative breast cancer using the MDA-MB-231 and 4T1 cells. The dual-ligand system utilized two peptides targeting P-selectin and αvß3 integrin, which are functionally linked to different stages of the development of metastatic disease at a distal site. Using in vivo multimodal imaging and post mortem histological analyses, this study shows that the dual-ligand nanoparticle effectively targeted metastatic disease that was otherwise missed by single-ligand strategies. The dual-ligand nanoparticle was capable of capturing different metastatic sites within the same animal that overexpressed either receptor or both of them. Furthermore, the highly efficient targeting resulted in 22% of the injected dual-ligand nanoparticles being deposited in early-stage metastases within 2 h after injection.

Vascular Targeting of a Gold Nanoparticle to Breast Cancer Metastasis.

The vast majority of breast cancer deaths are due to metastatic disease. Although deep tissue targeting of nanoparticles is suitable for some primary tumors, vascular targeting may be a more attractive strategy for micrometastasis. This study combined a vascular targeting strategy with the enhanced targeting capabilities of a nanoparticle to evaluate the ability of a gold nanoparticle (AuNP) to specifically target the early spread of metastatic disease. As a ligand for the vascular targeting strategy, we utilized a peptide targeting alpha(v) beta(3) integrin, which is functionally linked to the development of micrometastases at a distal site. By employing a straightforward radiolabeling method to incorporate Technetium-99m into the AuNPs, we used the high sensitivity of radionuclide imaging to monitor the longitudinal accumulation of the nanoparticles in metastatic sites. Animal and histological studies showed that vascular targeting of the nanoparticle facilitated highly accurate targeting of micrometastasis in the 4T1 mouse model of breast cancer metastasis using radionuclide imaging and a low dose of the nanoparticle. Because of the efficient targeting scheme, 14% of the injected AuNP deposited at metastatic sites in the lungs within 60 min after injection, indicating that the vascular bed of metastasis is a viable target site for nanoparticles.

Treatment of Invasive Brain Tumors Using a Chain-like Nanoparticle.

Glioblastoma multiforme is generally recalcitrant to current surgical and local radiotherapeutic approaches. Moreover, systemic chemotherapeutic approaches are impeded by the blood-tumor barrier. To circumvent limitations in the latter area, we developed a multicomponent, chain-like nanoparticle that can penetrate brain tumors, composed of three iron oxide nanospheres and one drug-loaded liposome linked chemically into a linear chain-like assembly. Unlike traditional small-molecule drugs or spherical nanotherapeutics, this oblong-shaped, flexible nanochain particle possessed a unique ability to gain access to and accumulate at glioma sites. Vascular targeting of nanochains to the αvß3 integrin receptor resulted in a 18.6-fold greater drug dose administered to brain tumors than standard chemotherapy. By 2 hours after injection, when nanochains had exited the blood stream and docked at vascular beds in the brain, the application of an external low-power radiofrequency field was sufficient to remotely trigger rapid drug release. This effect was produced by mechanically induced defects in the liposomal membrane caused by the oscillation of the iron oxide portion of the nanochain. In vivo efficacy studies conducted in two different mouse orthotopic models of glioblastoma illustrated how enhanced targeting by the nanochain facilitates widespread site-specific drug delivery. Our findings offer preclinical proof-of-concept for a broadly improved method for glioblastoma treatment.

Treatment of cancer micrometastasis using a multicomponent chain-like nanoparticle.

While potent cytotoxic agents are available to oncologists, the clinical utility of these agents is limited due to their non-specific distribution in the body and toxicity to normal tissues leading to use of suboptimal doses for eradication of metastatic disease. Furthermore, treatment of micrometastases is impeded by several biobarriers, including their small size and high dispersion to organs, making them nearly inaccessible to drugs. To circumvent these limitations in treating metastatic disease, we developed a multicomponent, flexible chain-like nanoparticle (termed nanochain) that possesses a unique ability to gain access to and be deposited at micrometastatic sites. Moreover, coupling nanochain particles to radiofrequency (RF)-triggered cargo delivery facilitated widespread delivery of drug into hard-to-reach cancer cells. Collectively, these features synergistically facilitate effective treatment and ultimately eradication of micrometastatic disease using a low dose of a cytotoxic drug.

Nanobubble ultrasound contrast agents for enhanced delivery of thermal sensitizer to tumors undergoing radiofrequency ablation.

PURPOSE: Pluronic has been shown to sensitize various tumor cell lines to chemotherapy and hyperthermia by altering the membrane fluidity, depleting ATP, and modulating the heat shock protein 70 expression. In our prior work, Pluronic was also used to formulate nanosized ultrasound contrast agents. In the current study we evaluate the use of these contrast agents as vehicles for image-guided delivery of Pluronic to improve outcomes of tumor radiofrequency (RF) ablation. METHODS: Lipid-shelled Pluronic nanobubbles were prepared and examined for size distribution, zeta potential, stability, biodistribution, accumulation of nanobubbles in the tumor, and treatment efficacy. LS174-T xenograft tumor-bearing mice were used to evaluate tumor growth suppression and measure treatment efficacy after RF ablation. RESULTS: The average diameter of Pluronic bubbles was 230 nm, and initial bubble echogenicity was 16 dB. In vitro, cells exposed to Pluronic nanobubbles exhibited low cytotoxicity in the absence of ultrasound, even if heat (43 ºC) was applied. When the cells were exposed to Pluronic nanobubbles, heat, and ultrasound; viability was significantly reduced. In vivo, tumors treated with ultrasound-modulated nanobubbles prior to RF ablation showed a significant reduction in growth compared to the RF alone (P<0.05). CONCLUSION: Lipid and Pluronic-shelled, echogenic nanobubbles combined with ultrasound modulation can serve as an effective theranostic method for sensitization of tumors to RF ablation.

On-command drug release from nanochains inhibits growth of breast tumors.

PURPOSE: To evaluate the ability of radiofrequency (RF)-triggered drug release from a multicomponent chain-shaped nanoparticle to inhibit the growth of an aggressive breast tumor. METHODS: A two-step solid phase chemistry was employed to synthesize doxorubicin-loaded nanochains, which were composed of three iron oxide nanospheres and one doxorubicin-loaded liposome assembled in a 100-nm-long linear nanochain. The nanochains were tested in the 4T1-LUC-GFP orthotopic mouse model, which is a highly aggressive breast cancer model. The 4T1-LUC-GFP cell line stably expresses firefly luciferase, which allowed the non-invasive in vivo imaging of tumor response to the treatment using bioluminescence imaging (BLI). RESULTS: Longitudinal BLI imaging showed that a single nanochain treatment followed by application of RF resulted in an at least 100-fold lower BLI signal compared to the groups treated with nanochains (without RF) or free doxorubicin followed by RF. A statistically significant increase in survival time of the nanochain-treated animals followed by RF (64.3 days) was observed when compared to the nanochain-treated group without RF (35.7 days), free doxorubicin-treated group followed by RF (38.5 days), and the untreated group (30.5 days; n=5 animals per group). CONCLUSIONS: These studies showed that the combination of RF and nanochains has the potential to effectively treat highly aggressive cancers and prolong survival.

Shaping cancer nanomedicine: the effect of particle shape on the in vivo journey of nanoparticles.

Recent advances in nanoparticle technology have enabled the fabrication of nanoparticle classes with unique sizes, shapes and materials, which in turn has facilitated major advancements in the field of nanomedicine. More specifically, in the last decade, nanoscientists have recognized that nanomedicine exhibits a highly engineerable nature that makes it a mainstream scientific discipline that is governed by its own distinctive principles in terms of interactions with cells and intravascular, transvascular and interstitial transport. This review focuses on the recent developments and understanding of the relationship between the shape of a nanoparticle and its navigation through different biological processes. It also seeks to illustrate that the shape of a nanoparticle can govern its in vivo journey and destination, dictating its biodistribution, intravascular and transvascular transport, and, ultimately, targeting of difficult to reach cancer sites.

Multimodal in vivo imaging exposes the voyage of nanoparticles in tumor microcirculation.

Tumors present numerous biobarriers to the successful delivery of nanoparticles. Decreased blood flow and high interstitial pressure in tumors dictate the degree of resistance to extravasation of nanoparticles. To understand how a nanoparticle can overcome these biobarriers, we developed a multimodal in vivo imaging methodology, which enabled the noninvasive measurement of microvascular parameters and deposition of nanoparticles at the microscopic scale. To monitor the spatiotemporal progression of tumor vasculature and its vascular permeability to nanoparticles at the microcapillary level, we developed a quantitative in vivo imaging method using an iodinated liposomal contrast agent and a micro-CT. Following perfusion CT for quantitative assessment of blood flow, small animal fluorescence molecular tomography was used to image the in vivo fate of cocktails containing liposomes of different sizes labeled with different NIR fluorophores. The animal studies showed that the deposition of liposomes depended on local blood flow. Considering tumor regions of different blood flow, the deposition of liposomes followed a size-dependent pattern. In general, the larger liposomes effectively extravasated in fast flow regions, while smaller liposomes performed better in slow flow regions. We also evaluated whether the tumor retention of nanoparticles is dictated by targeting them to a receptor overexpressed by the cancer cells. Targeting of 100 nm liposomes showed no benefits at any flow rate. However, active targeting of 30 nm liposomes substantially increased their deposition in slow flow tumor regions (∼12-fold increase), which suggested that targeting prevented the washout of the smaller nanoparticles from the tumor interstitium back to blood circulation.

Imaging metastasis using an integrin-targeting chain-shaped nanoparticle.

While the enhanced permeability and retention effect may promote the preferential accumulation of nanoparticles into well-vascularized primary tumors, it is ineffective in the case of metastases hidden within a large population of normal cells. Due to their small size, high dispersion to organs, and low vascularization, metastatic tumors are less accessible to targeted nanoparticles. To tackle these challenges, we designed a nanoparticle for vascular targeting based on an α(v)ß(3) integrin-targeted nanochain particle composed of four iron oxide nanospheres chemically linked in a linear assembly. The chain-shaped nanoparticles enabled enhanced "sensing" of the tumor-associated remodeling of the vascular bed, offering increased likelihood of specific recognition of metastatic tumors. Compared to spherical nanoparticles, the chain-shaped nanoparticles resulted in superior targeting of α(v)ß(3) integrin due to geometrically enhanced multivalent docking. We performed multimodal in vivo imaging (fluorescence molecular tomography and magnetic resonance imaging) in a non-invasive and quantitative manner, which showed that the nanoparticles targeted metastases in the liver and lungs with high specificity in a highly aggressive breast tumor model in mice.

Enhanced delivery of chemotherapy to tumors using a multicomponent nanochain with radio-frequency-tunable drug release.

While nanoparticles maximize the amount of chemotherapeutic drug in tumors relative to normal tissues, nanoparticle-based drugs are not accessible to the majority of cancer cells because nanoparticles display patchy, near-perivascular accumulation in tumors. To overcome the limitations of current drugs in their molecular or nanoparticle form, we developed a nanoparticle based on multicomponent nanochains to deliver drug to the majority of cancer cells throughout a tumor while reducing off-target delivery. The nanoparticle is composed of three magnetic nanospheres and one doxorubicin-loaded liposome assembled in a 100 nm long chain. These nanoparticles display prolonged blood circulation and significant intratumoral deposition in tumor models in rodents. Furthermore, the magnetic particles of the chains serve as a mechanical transducer to transfer radio frequency energy to the drug-loaded liposome. The defects on the liposomal walls trigger the release of free drug capable of spreading throughout the entire tumor, which results in a widespread anticancer effect.

Assembly of linear nano-chains from iron oxide nanospheres with asymmetric surface chemistry.

Besides the multifunctionality, another equally important aspect of nanoparticles is their engineerability to control the geometrical and chemical properties during fabrication. In this work, we exploited this aspect to define asymmetric surface chemistry of an iron oxide nanosphere by controlling the topology of ligand expression on its surface resulting in a particle with two faces, one displaying only amines and the other only thiols. Specifically, amine-functionalized iron oxide nanospheres were attached on a solid support via a crosslinker containing a disulfide bridge. Liberation of the nanosphere using thiolytic cleavage created thiols on the portion of the particle's surface that interacted with the solid support. Employing a solid-phase strategy and a step-by-step addition of particles, the two unique faces on the same nanosphere served as fittings to assemble them into linear nano-chains. Assembly of chains with various lengths and aspect ratios was controlled by the size and number of the added nanospheres. The characteristics of those chains showed a high degree of uniformity indicating the exceptional control of the synthetic process. Notably, one of the unique properties of the iron oxide nano-chains was an increased magnetic relaxivity, indicating their potential use as contrast agents for magnetic resonance imaging.