Nanomedicine is an emerging technology that combines the fields of biology, chemistry, engineering, and medicine to develop new solutions for major clinical problems. Cancer is one disease where the application of nanomedicine has potential to provide clinicians the ability to overcome many existing shortcomings in screening and treatment. At the heart of nanomedicine is the development of precisely engineered nanomaterials (e.g., NPs) with desired properties. Typically, NPs in nanomedicine have dimensions of tens to hundreds of nanometers across, putting them on the same size scale as biomolecules. For example, proteins are typically in the size range of 1–20 nm, DNA has a diameter of 2 nm, and cell surface receptors are approximately 10 nm. Therefore, the size of NPs affords them the opportunity to interact with biomolecules on a scale that can modulate biological pathways elusive in medicine, such as the cell invasion pathway.
Another advantage of NPs is the unique properties of the material that arise only at the nanoscale. The most well studied phenomenon is that nanoscaled materials have a high surface area to volume ratio. This implies that the percentage of atoms on the surface of an NP is high compared to a macroscaled or even microscaled particles of the same material. This physical property renders NP surfaces highly reactive and amendable. Using nanoengineering strategies researchers can tailor the unique physical properties (e.g., size, charge, biocompatibility, solubility, hydrophilicity/hydrophobicity) of NPs to modulate their behavior in biological systems. Through these approaches, critical pharmacokinetic properties such circulation half-life, biodistribution, non-specific adsorption, premature degradation, and toxicity can be dictated. A number of other physical phenomena can occur in nanoscaled versions of materials such as the development of unique optical, electronic, and magnetic properties depending on their core material and size. These properties are highly desirable for sensing, tracking, and activation applications.
NPs can be synthesized from myriad different material formulations to create numerous nanoarchitectures. Examples from the various common classes of NP formulations developed to date can be summarized into the following categories: liposomes, albumin-based particles, nanocrystals, polymeric micelles, polymer-based NPs, dendrimers, inorganic NPs, nanotubes, and/or other solid NPs.
Another desirable property of NPs is that they are amenable to chemical modification, and through organic chemistries, can be engineered as multifunctional devices that carry multiple detection signals, tumor cell recognizing targeting ligands, and therapeutic cargos. Multifunctional devices are capable of delivering precisely targeted treatments to tumor cells, avoiding healthy tissues, and being tracked non-invasively through incorporated detection signals (contrast agents). shows a cartoon diagram depicting the general architecture of a multifunctional NP device and its assembly. A typical multifunctional NP device comprises a NP core, a biocompatible coating, surface bound or encapsulated targeting and therapeutic payloads, and/or additional detection signals.
Figure 2 General architecture and assembly of a multifunctional NP. Generally, a solid NP core is coated with a biocompatible polymer coating which can then be derivatized with targeting agents, fluorophores, radionuclides, gene therapeutics, and chemotherapy (more ...)
Many NP formulations have been examined for clinical use and some formulations have already been approved for use in humans. Less complex formulations, such as liposomes loaded with chemotherapeutic drugs, have been approved for cancer therapy for more than a decade. In these early NP formulations, the liposome enhanced the solubility of the chemotherapeutic for improved biodistributions and extended blood circulation time, which ultimately led to a higher therapeutic index for the delivered drug.
These liposomal formulations have also been used to overcome cancer cell drug resistance. This drug resistance generally occurs due to the overexpression of ATP-binding cassette (ABC) transporters which increase the efflux of a broad class of hydrophobic drugs from cancer cells. Nanotechnology provides an alternative strategy to circumvent drug resistance by encapsulating or attaching drugs to nanomaterials that are resistant to drug efflux. Indeed, several NP-based chemotherapies (e.g. Doxil, Caelyx, DaunoXome) have been approved for clinical use or are in clinical trials.
Formulations of crystalline NPs have also been examined for clinical applications. For example, a number of iron oxide NPs are in early-stage clinical trials or experimental study stages. Several formulations have already been approved for widespread clinical use in medical imaging and therapy. Some examples include: Lumiren® for bowel imaging , Feridex IV® for liver and spleen imaging , and Combidex® for lymph node metastases imaging. Iron oxide NPs are desirable because of their magnetic properties that can be exploited for non-invasive tracking through magnetic resonance imaging (MRI). Furthermore, in contrast to many other inorganic NP formulations, iron oxide NPs are biocompatible, and iron from degraded NPs are used in the body’s natural iron stores such as hemoglobin in red blood cells. In fact, a formulation of iron oxide NPs (Ferumoxytol®) was recently approved for iron replacement therapy.
Recently, more complex formulations of NPs, such as multifunctional devices that incorporate both cancer specific targeting and therapeutic delivery functionalities, have emerged in the clinical setting. One example is the multifunctional polymeric NP formulation CALAA-01 (Calando Pharmaceuticals, Inc.). This formulation consists of: (1) a linear, cyclodextrin-based polymer, (2) a human transferrin protein (TF) targeting ligand displayed on the exterior of the NP to engage TF receptors on the surface of the cancer cells, (3) a hydrophilic polymer (polyethylene glycol) used to promote NP stability in biological fluids, and (4) siRNA designed to reduce the expression of ribonucleotide reductase M2 (RRM2), a critical biomolecule in DNA synthesis. In a recently completed phase I clinical trial, this NP formulation showed favorable tolerability and therapeutic efficacy in patients with solid cancers. Most notably, the trial revealed that incorporating a targeting ligand could drastically improve the amount of NPs internalized by cancer cells and lead to higher therapeutic efficacy.
These advancements highlight the promise of nanomedicine being translated into clinical practice. Further, this emergence is opening up new avenues in nanomedicine for targeting more specialized cancer-specific pathways, such as cancer cell invasion, for more effective therapy with reduced side effects. Cancer cell invasion is highly complex and involves numerous environmentally and temporally regulated processes. This makes the multifunctional nature of nanomedicine well suited to tackle this phenomenon. By simultaneously targeting various molecular targets in the progression of cell invasion, we can produce a much more effective therapy that is less prone to development of resistance. Furthermore, the sensing and tracking capabilities that can be developed through nanomedicine provide opportunities to monitor and study the progression of cancer metastasis.
The development of NPs for the treatment and monitoring of cancer cell invasion is a new and emerging application in the field of nanomedicine. Traditionally, cancer nanomedicine strategies focus on delivering therapies and imaging contrast agents to the solid tumor mass. Tackling cell invasion will require novel NP formulations and new strategies for targeting NPs specifically to the invasive cells that have become segregated from the bulk tumor. In the following sections we will review examples of currently developed and emerging nanoparticle directing strategies, and evaluate their applicability for targeting cancer cell invasion. Furthermore, we will describe several examples of nanoparticle formulations which show promise for monitoring and treatment of cancer cell invasion.
4.2 Directing nanoparticles to cancer cells
Nanoparticles developed for cancer applications are typically administered systemically through intravenous injection. If properly engineered, the NPs travel as discrete, individual entities through the blood, bypass biological barriers (e.g., vascular tumor barriers, extracellular matrices, cell membranes), and reach their molecular target for biorecognition and activation. Directing NPs in vivo has been the focus of tremendous amounts of research and many innovative strategies have been introduced and investigated. Various reviews have specifically focused on this engineerable feature of NPs. Here we will examine several of the generalized strategies utilized and discuss their utility for targeting cancer cell invasion.
Many earlier NP-directing strategies focused on modifying the NP’s physiochemical properties to promote uptake by tumor cells. One well-studied approach is to enhance the circulation time of NPs through surface modification strategies. Long-circulating NPs can passively target tumors through a phenomenon known as the enhance permeability and retention (EPR) effect. This effect arises due to the poorly functioning blood and lymphatic vessels in tumor tissue that enable macromolecules of 1–500 nm in size to leak into tumor tissue over time. Due to inefficient lymphatic drainage, there is poor clearance of NPs leading to their prolonged accumulation. The most widely known method to impart the long-circulating property onto NPs is through surface modification with polymers such as polyethylene glycol (PEG) that possess non-fouling properties. These polymers help limit protein absorption onto the NP and the recognition of NPs by the body’s immune system. This strategy has been exploited in numerous studies to passively direct numerous NP formulations including liposomes , polymers , and crystalline NPs to tumor cells.
There are a number of disadvantages in solely relying on the EPR effect to direct NPs to tumor cells for treatment of cancer cell invasion. First, tumors are heterogeneous in vascularization, blood flow, and lymphatic drainage rate, which make delivering drugs to the entire tumor difficult. Second, not all tumors will develop an EPR effect, and in fact, certain types of solid cancers, including those of the brain, are protected by more restrictive vasculature that prohibits passively targeted NPs from reaching tumor cells. Lastly, the EPR effect is limited to the bulk tumor which means NPs cannot interact with metastasized/invasive cancer cells that have migrated away from the tumor bulk. Therefore, more specific and active targeting approaches are necessary to improve the NP uptake by invading cells dissociated from the bulk tumor.
Active targeting relies on the use of specific targeting ligands which can recognize and bind to receptors that are upregulated on cancer cells or associated stromal cells. Incorporated onto the surface of NPs, these targeting ligands can direct NPs to specific cells. Numerous targeting ligands have been evaluated to actively deliver NPs specifically to cancer cells. Of note, many crystalline systems have implemented active tumor targeting strategies with varying success, including ligands such as small organic molecules , peptides , proteins , antibodies , and aptamers. Some of these examples include ligands which recognize molecular receptors involved in cancer cell invasion, such as CTX which binds to MMP-2 upregulated on the surface of cancer cells during invasion , and the peptide RGD, which binds integrin receptors upregulated on endothelial cells associated with tumor neovasculature. Other examples include antibodies directed against ion channels upregulated on the surface of invading cells which inhibit channel function. Attaching one of these ion channel-targeting antibodies to the surface of a NP could provide the dual function of ion channel inhibition and NP mediated imaging and/or therapy.
In addition to enhancing specificity of NPs to cancer cells, targeting agents can help initiate endocytosis of the NPs to which they are attached. Therefore, targeting ligands can improve the delivery of drugs into cancer cells and the therapeutic index of the nanotherapeutic formulation. A notable study by Bartlett et al. evaluated this phenomenon by comparing the in vivo efficacy of delivering RNAi-based therapeutics using actively targeted versus passively targeted NPs. They found that active targeting can enhance the therapeutic efficacy by 50%. Interestingly, this study revealed that although similar amounts of both NP formulations were delivered to the tumor tissue, the therapeutic efficacy was enhanced for the actively targeted formulation due to improved cancer cell uptake and tumor distribution.
Active targeting strategies also improve the percentage of cancer cells that are exposed to NPs. Our group demonstrated this concept in two recent studies utilizing magnetic NPs prepared with and without the active targeting ligand CTX to compare their efficacy in delivering nucleic acids (siRNA and plasmid DNA) to brain cancer cells. These in vitro and in vivo studies revealed that the percentage of cancer cells that received therapies was two-fold higher with the actively targeted CTX modified nanovector in comparison to the passively targeted nanovector. In a recent landmark study, Sugahara et al. demonstrated that co-administration of the tumor penetrating peptide iRGD with NPs can improve their therapeutic index in tumor bearing mice. Here the peptide iRGD has the capacity to increase tumor vascular permeability. This peptide functions by first associating with integrins that are specifically expressed on the endothelium of tumor vessels, and then the peptide is proteolytically cleaved in the tumor to produce a truncated sequence that has no integrin-binding activity, but gains affinity for neuropilin-1 (NRP-1), and thus enhances tissue permeability. Notably the iRGD peptide was just as effective when co-administered with NPs as when chemically bound. This strategy opens up new opportunities for multistage therapy whereby numerous levels of targeting are included.
Other tumor directing strategies for nanomedicine include systems that can recognize tumor specific microenvironmental cues for activation of the NPs. In a series of recent of studies by, Nguyen et al. and Olson et al., protease activatable cell penetrating peptides (ACPPs) which respond to the activity of MMPs in tumors were incorporated onto the surface of dendrimeric NPs. In the presence of proteinases, a 4- to 15-fold higher cell internalization of ACPP modified NPs was observed in comparison to the passively targeted version of the same NP. Their studies revealed the ability to use the invasive tumor environment to activate nanotherapeutics.
Ultimately, it is likely that successful formulations designed to target invasive cancer cells will exploit a combination of strategies to direct NPs to cancer cells. In the next two sections we will evaluate current strategies that have been utilized for delivering nanoparticles to cancer cells for imaging cancer cell invasion and for therapy.
4.3 Nanomedicine in Imaging Cancer Cell Invasion
Non-invasive monitoring of cancer cell progression and metastasis is of great interest to clinicians. Until recently, most studies of metastasis only measured the end point of the process: macroscopic metastases. Although these studies have provided much useful information, the details of the metastatic process remain somewhat mysterious owing to difficulties in studying cell behavior with high spatial and temporal resolution in vivo. Nanomedicine provides an avenue for monitoring cancer cell invasion and metastasis in situ through various imaging platforms and can aid clinicians in visual representation, characterization, and quantification of this biological process at the cellular and molecular levels. NPs have been developed for imaging application across different platforms including MR, optical, and nuclear imaging systems. In some cases these platforms can be combined to offer clinicians the ability to obtain a variety of pathologic information using the unique imaging capabilities of each system with a common NP formulation.
Visualizing cancer cell invasion is especially critical in tumors arising at anatomical sites where surgery is complex (e.g., head and neck tumors, brain tumors, and others). Here, having the ability to visualize the extent of cancer cell infiltration into the brain could provide improved guidance to neurosurgeons in planning and executing surgical resection. In many brain tumors the extent of resection is predicative of outcome, with more complete resections correlating to improved progression free survival. This added information could drastically aid in improving the outcome of surgery as a result of a more radical resection, and thus numerous multifunctional NP formulations have been developed. Our group recently demonstrated that multifunctional magnetic/optical detectable NPs modified with CTX could safely permeate across the blood brain barrier (BBB) and highlight the extent of tumor cell infiltration into normal brain tissue under both MRI and fluorescence optical imaging. In this formulation, the combination of using CTX to actively target MMP-2 on brain tumor cells and engineering NPs to have extended blood circulation time facilitated access of the NP across the BBB to brain tumor cells. shows the imaging data obtained through this study in medulloblastoma brain tumor bearing mice with intact BBBs.
Figure 3 Summary of data obtained through this original study that demonstrated the applicability of NPCP-CTX NP’s for delineating tumor boundaries through in vivo MRI, and in vivo fluorescence imaging. a) In vivo MR images of autochthonous medulloblastoma (more ...)
As described in the preceding section, NP formulations have been developed to sense biochemical changes and molecular activity of cancer cells. This approach was recently demonstrated in imaging the extent of tumor infiltration through a series of studies performed by Nguyen et al. and Olson et al.. shows a series of images from this study depicting how this NP formulation can be applied to improve the outcome of tumor resection by highlighting tumor margins under pre-operative MRI and intra-operative optical imaging. This nanomedicine based diagnostic tool was evaluated in its ability to improve surgical outcome by aiding surgeons in identifying and resecting residual metastatic cancer cells both pre- and intra-operatively. Their formulation consisted of protease-activatable cell penetrating peptides linked to dendrimers dually labeled with a fluorophore for optical imaging and gadolinium for MR imaging. Thus, MMPs in the tumor microenvironment cleave and activate the cell penetrating peptide which promotes uptake into cancer cells. Once internalized, the optical and MR signatures associated with the nanoprobes provide navigation to aid in complex surgical resection of large and invasive tumors. This approach demonstrated a 90% reduction in residual cancer cells left after surgery.
Figure 4 Dual-Labeled ACPPD. (A–D) Example of HT1080 xenograft treated with ACPPD dually labeled with gadolinium and Cy5. Preoperative MR image of mouse showing contrast uptake in tumor (A, black arrow). Following skin incision and retraction, the tumor (more ...)
Outside of clinical screening and staging applications, nanomedicine approaches can be used to further understand cell invasion and metastasis processes in vivo in animal models. For example, cancer cells loaded with magnetic NPs have been implanted in rat brains and monitored through MRI which has provided insights into brain tumor cell invasion. Furthermore, advances in microscopic imaging techniques now provide opportunities to monitor single cells in vivo. These emerging techniques include spatiotemporally resolved imaging, fluorescent reporter reagents, and multiparametric image analysis, which can contribute to a better insight into single cell migration and invasion. Gonda et al. recently illustrated these concepts in a study where cancer cells labeled with semiconductor quantum dots (QDs) were temporally tracked in vivo through the process of invasion and metastasis. exemplifies how this approach can characterize individual cell migration over an extended period of time. In this study, QDs were labeled with an anti-PAR1 antibody and used to target and track metastatic breast cancer cells in a mouse model. Imaging was performed with a spatial precision of 7–9 nm under a confocal microscope, which provided information on membrane dynamics of invading and metastasizing cells. For example, the membrane fluidity of metastasizing cells in the blood was 1100-fold greater than that of cells in the bulk tumor, which indicates a lack of cytoskeletal actin structure near the cell membrane. This bit of information can direct therapeutic strategies towards inhibiting actin polymerization in these metastasizing cells to prevent their invasion into secondary locations.
Figure 5 Membrane dynamics in metastatic cancer cells in vessels. A, imaging of cells in the bloodstream. Cells are shown after 1 s, 17 s, and 41 s. Yellow lines show outlines of cancer cells. Red dotted lines show outlines of vessels determined by superimposed (more ...)
4.4 Nanomedicine in Treating Cancer Cell Invasion
There are several immediate benefits of using NPs as drug carriers. Most nanotechnology-based drug formulations aim to increase the therapeutic index for established chemotherapeutic drugs via improving pharmacokinetics, biodistribution, and selectivity in delivery to cancerous tissue. Combined, these formulations have utilized nanotechnology-based strategies for tumor targeting, imaging, and delivery of therapeutics. In most of these cases, well-established chemotherapeutic drug molecules (e.g., paclitaxel, doxorubicin, docetaxel, and methotrexate) have been combined with liposomal or polymeric NP platforms. More recently, biotherapeutic agents (e.g., therapeutic peptides, antibodies, genes, and siRNAs) have been combined with nanomedicine to treat cell invasion more specifically.
While there is a wealth of studies focused on developing NPs for cancer therapy, there are only a limited number of nanoformulations reported in treating cancer cell invasion. However, there is tremendous potential to combine NP formulations with known inhibitors of cancer cell invasion to curb tumor metastasis. One example of this approach is a recently published study by our group where the therapeutic effect of CTX bound to NPs was compared to free CTX in its ability to inhibit glioma tumor cell invasion. CTX is an inhibitor of MMP-2 (Section 3 above) and also plays a role in inhibiting volume regulating ion channels. In this in vitro study we demonstrated that when bound to NPs, CTX provided enhanced therapeutic potency compared to free CTX. summarizes the data obtained through this study describing comparative effect of free CTX vs. NP bound CTX. Notably, NP-CTX can simultaneously interact with numerous MMP-2 receptors expressed on glioma cell surfaces. This multivalent binding promotes cellular internalization of a larger portion of lipid rafts which contain MMP-2 receptors and volume regulating ion channels. Combined, these interactions and processes lead to inhibition of MMP-2 and ion channel activity in targeted glioma cells. Thus, an enhanced ability of NP formulation to inhibit glioma cell invasion is observed.
Figure 6 Schematic representations of CTX-enabled nanoparticles (NPCs) inhibiting tumor cell invasion and summary of MMP-2 and cell invasion inhibition data from. a) Surface chemistry of NPC conjugate. b) NPC binding to lipid rafts of glioma cells containing MMP-2 (more ...)
The inhibition of ion channels is an exciting strategy to treat invasive tumors. The use of CTX and other ion channel inhibitors in clinical trials have shown promising results. As shown above, nanotechnology can enhance the inhibition of ion channels solely through the multivalent effect wherein a larger portion of the cell membrane is internalized. Recent studies have also utilized the small scale of NPs to directly interact with ion channels to diminish cells' ability to regulate cell volume. For example, Park et al. showed that single-walled carbon nanotubes (SWCNTs) are able to inhibit K+ ion channels in Chinese hamster ovary cells if engineered properly. They found that the SWCNTs with an inner diameter of 0.9 nm had the highest K+ ion channel blocking ability, but in a reversible manner indicating the effect was highly concentration-dependent. This work was followed up by the same group in a paper by Chhowalla et al. who developed functionalized SWCNTs for irreversible inhibition of K+ ion channels. By attaching the chemical 2-trimethylammoniumethylmethane thiosulfonate (MTSET) to the SWCNTs, they showed this functionalized nanotube was able to specifically interact with the cysteine groups of amino acids within the ion channel for higher binding affinity and irreversible channel inhibition. K+ channel inhibition has also been established with multi-walled carbon nanotubes (MWCNTs). Likewise, Kraszewski et al. modeled the interaction of fullerenes (C60) with K+ ion channels and proposed that this carbon based nanomaterial has an affinity towards the transmembrane domain of K+ ion channels, and that the K+ ion current could be greatly inhibited through the attachment of hydrophobic drugs. Since invading cells rely on the intracellular concentration of K+ ions to regulate cell volume, inhibiting these channels could provide significant treatment efficacy.
Actively targeted nanoparticles have also been evaluated as carriers of conventional drug therapies designed to treat cancer metastasis. For example, Murphy et al. evaluated the use of polymeric nanoparticels loaded with the chemotherapeutic agent doxorubicin and modified with the targeting ligand RGD peptide which binds œvβ3 integrins expressed on neovascular endothelial cells. In this system, RGD was integrated to direct the doxorubicin loaded NPs to a subset of tumor blood vessels associated with angiogenesis. In the study, the NP formulation was shown to produce a therapeutic index that was 15-fold more superior to the free drug for treating cancer metastasis, and furthermore contrary to the free drug no toxicity was observed in mice treated with the NP formulation. This study demonstrates the potential of NP formulations for improving the therapeutic index of conventional drugs while minimizing their related toxicity.
NPs have also proven to be effective vehicles in delivering DNA or siRNAs for gene therapy, a powerful tool that could simultaneously affect multiple pathways leading to invasion. For example, Alshamsan et al. delivered anti-STAT3 siRNA using PLGA NPs to melanoma tumors and showed this knockdown of STAT3 diminished tumor growth. While they did not directly correlate this to inhibition of tumor cell invasion, this study shows the utility of nanotechnology in disrupting pathways involved in cancer cell invasion as an anticancer therapy.
A study by Han et al. actually showed the correlation of NP mediated gene therapy with reduced cell invasion. They used magnetic NPs coated with polyamidoamine dendrimers to carry anti-epidermal growth factor receptor (EGFR) siRNA to brain tumor cells. Knockdown of EGFR lead to the downstream reduction in expression of pro-invasion biomolecules, namely MMP-2 and MMP-9, and reduced tumor cell invasion in a transwell migration assay. Gao et al. also showed reduced cell invasion through siRNA treatment using NPs. In this study they used PEGylated liposomes to deliver anti-RhoA siRNA to breast cancer cells and showed that knockdown of RhoA lead to reduced cell invasion through a migration assay. These studies highlight the potential of nanotechnology to treat specific cellular functions that lead to invasion.
In a study that showed the knockdown of a pro-invasion gene does, in fact, lead to reduced metastases, Villares et al. employed liposomal NPs as their gene delivery vehicle. In this study they delivered anti-PAR-1 siRNA loaded into DOPC liposomes to melanoma cells and monitored lung metastases. Mice receiving intravenously injected melanoma cells treated with anti-PAR-1 siRNA showed a dramatically reduced number of lung metastases indicating this treatment prevented these cells from invading into potentially metastatic lung sites. This exciting finding demonstrates the ability of nanotechnology to inhibit cell invasion, and thus reduce cancer metastasis.