Convergence of Nanotechnology and Cancer Prevention: Are We There Yet?

Nanotechnology by definition involves the study and use of materials between 1 and 100 nanometers (nm) in size (Fig. 1). The idea of nanotechnology began as theoretical concepts posed by physicists and other scientists involving nanoscale assembly of materials (1, 2). The term nanotechnology was first used to describe semiconductor generation (3). The early discipline began with the invention of the scanning probe microscope and the discovery of molecular structures such as fullerenes (4, 5). The field has more recently expanded into various specialized areas of nanomaterials and nanomedicine (6–10).

Existing nanomaterials include liposomes, natural polymers (chitosan), synthetic polymers [polyethylene glycol (PEG), etc.], carbon, carbon fullerenes such as buckyballs and carbon nanotubes (CNT), graphene, ceramic, crystals, metal, silica, and quantum dots (Fig. 2 and Table 1). They generally range in size between 5 and 200 nm, but some may exceed this range. When these technologies are coupled with molecular-based targeting methods, they can potentially achieve selective gene targeting. For example, coupling of chitosan with hydrogel technology and/or selective epitope targeting with siRNA-based gene silencing are exciting developments in selective gene targeting (11, 12). The use of thioaptamer-based molecular epitope recognition also holds significant promise for selective targeting (13). The use of phage display–based peptides as well can facilitate selectively “nanotargeting” cancers (14, 15). Selective targeting can also employ the use of specific promoter-based gene expression only in premalignant or malignant cells (16–20). Any single or combination approach for molecularly enhancing target recognition in conjunction with nanoscale payload delivery is likely to facilitate selectively targeting the various stages of cancer.

Knowledge about uptake and distribution of nanoparticles comes from studies involving various types of environmental exposures (21). Nanoparticle internalization depends on the exposure time, carrier vehicle, mode of access, and tissue interface involved in a given exposure (21). Tissue interfaces include dermal surfaces, any exposed mucous membrane, and the respiratory airways (21). The biodistribution of nanoparticles in the body typically depends on the material and its size, shape, and charge (21–24). In the case of dermal exposures, nanoparticle penetration typically occurs at hair follicles (25) and in flexed (26) and broken skin (27). As one example, 10 to 50 nm sized TiO₂ nanoparticles found in sunscreen, when suspended in oil-in-water emulsions, can penetrate hairy skin at the hair follicle sites or pores when compared with water-based suspensions (28, 29). In the case of gastrointestinal (GI) mucous membranes, absorption depends on size, which diminishes for larger particles ranging from 50 to 1,000 nm (30), exposure time, and enterocyte involvement (31). As a potential consequence, GI uptake of dietary nanoparticles (100–1,000 nm) may also influence chronic inflammation in the colon (32). In the case of aerosol delivery to the lung in contrast, noncationic nanoparticles larger than 34 nm are retained within the lungs, whereas smaller nanoparticles enter the regional lymph nodes (33). In the same report, neutrally charged nanoparticles 6 nm or less entered the bloodstream from the alveolar air-spaces followed by renal clearance (33). Similarly, blood-borne particles 10 nm or less in size usually undergo elimination by renal clearance, whereas particles 100 nm or greater in size are taken up by the reticuloendothelial system (34). The shape and surface properties of nanoparticles can also influence uptake and distribution, which can be experimentally optimized at the molecular level to enhance targeting properties (35, 36).

The relative success of nanoparticle preclinical and clinical use has evolved with the introduction of technology (Table 1). The use of nanoencapsulated agents helps reduce the toxicity of chemotherapeutic drugs. Many clinically approved approaches involved the early introduction of lipid liposomes, including ONC-TCS, DepoCyt, Myocet, and DaunXome (37–40). Similarly, a number of PEGylated/lipid liposome–based approaches are either clinically approved or in trial, including Lipoplatin, Doxil, and Thermodox (41–43). One approach to overcome multidrug resistance integrates therapy and preventive approaches by combining doxorubicin–curcumin into nanoparticles known as NanoDoxCurc (NDC; ref. 44). The delivery of RNAi therapy using nanoparticles is another important use of nanotechnology. Alnylam Pharmaceuticals, for example, has two liposomal siRNA in non–cancer-related clinical trials (45, 46). Alnylam also produced polymeric nanoparticles for in vivo delivery of siRNA to target endothelial cells, primary tumor growth, and metastasis (47). Another nanoformulation consisting of albumin-based (Nab)–paclitaxel or Abraxane is the first drug delivery system approved for the treatment of metastatic breast cancer, metastatic non–small cell lung cancer (NSCLC) and first-line treatment of patients with metastatic pancreatic cancer (48). A variety of synthetic polymeric nanoparticles are also in clinical settings, such as Genexol-PM, Oncaspar, and Prolindac (49–51). In the targeted realm, BIND-14 is a nanoencapsulated composite that includes a controlled-release synthetic polymer containing docetaxel. This nanoparticle composite binds to the prostate-specific membrane antigen (PSMA) targets for treating solid tumors (52). In contrast, chitosan is a natural polymer used orally to decrease high-serum LDL cholesterol (53). Since chitosan was introduced, it has enjoyed numerous uses as a nanoparticle platform in preclinical and clinical studies (12, 54–56).

Inert carbon-based modalities are also receiving significant interest as drug, nucleotide, and protein-protective delivery approaches. Fullerenes and nanotubes are nanoparticles that hold much promise in preclinical settings (57–63). Mesoporous silica is another biologically inert platform for building complex nanoparticle identification and delivery of drug, nucleotide, and protein agents (64–66). Within the silica realm, Cornell (C)-dots received phase I approval for melanoma targeting (64). In contrast, uses of inert quantum dots are focused on targeted delivery and imaging due to their high quantum fluorescence yield (67–69).

In the heavy metal space, gold nanoparticles may be particularly useful for targeting tumors (70–73). In one study, unmodified gold nanoparticles inhibit tumor growth and metastasis through abrogation of MAPK signaling and reversal of the epithelial–mesenchymal transition in two preclinical mouse models of ovarian cancer (70). The authors suggested that these findings laid the foundation for further research in the use of inorganic nanoparticles as a class of antitumor and antimetastatic agents (70). In addition to serving as drug carriers, gold nanoparticles are finding use as photothermal agents, contrast agents, and radiosensitisers (74).

Other reports highlight some advantages of nanoparticles (75). Chauhan and Jain have applied nanodelivery methods to decrease the toxicity of therapeutic drugs (76). Inhalation of retinoids, steroids, or certain therapeutics used to treat lung cancer may also be more effective if applied in a nanoized form (77–80). Because a primary goal of nanoprevention is sustained release along with significant reduction in toxicity, the use of low-dose nanoized chemotherapeutic drugs might be useful in high-risk preventive settings.

Identifying biocompatible nontoxic nanomaterials is vital for the application of nanotechnology in prevention. The mode of delivery greatly influences uptake and toxicity (81). Nanotherapies are often injected directly via the blood stream or peritoneum (Table 1 and Fig. 3). Other common delivery methods include the skin, respiratory, or GI systems, as they are more acceptable for nanoprevention modalities (81). Mucous membrane surfaces of the eye, mouth, nose, upper GI tract, and vaginal surfaces can also take up nanoparticles. The particulate nature of nanomaterials leads to local accumulation followed by systemic dissemination via the cardiovascular or lymphatic systems. Among a variety of toxic effects, acute responses can begin with the generation of reactive oxygen species coupled with inflammatory reactions (82).

The blood stream is often used for delivering nanotherapy (Fig. 3). It is a closed system that is not directly exposed to the environment. As a closed system, the blood stream has specific processes identifying and mitigating pathogens and foreign bodies, which are processed further by the liver or excreted by the kidneys (33, 83). Once in the blood stream, platelets are among the first responders to many foreign bodies that initiate emboli formation (84). Nanoparticles in flowing blood also interact with antibodies, circulating immune cells, coagulation factors, and the surfaces of endothelial cells (85, 86). Nanomaterials can bind proteins through noncovalent interactions to form a protein corona (87, 88). This corona influences biologic activity and interactions with platelets and probably liver clearance (87, 88). Chemically modifying CNT surfaces, for example, to preferentially bind albumin versus fibrinogen influences protein corona formation and platelet interactions (87). Similarly, nanoparticle protein coronas can activate the circulating macrophages and trigger inflammasome formation by immune cells (89). Preformed albumin nanoparticle coronas are another method of limiting toxicity in the blood by reducing complement activation (90). Other mechanisms of toxicity occur with metal and metal oxides that generate reactive oxygen species and proinflammatory oxidative stress (91, 92). Depending on the routes of exposure, metal and metal oxide nanoparticles can affect cells and organ pathophysiology (91, 92). Similarly, graphene-based (93) and other inorganic nanomaterials (94) exhibit various toxicities to consider in the exposure and safer design of these materials. Nanoparticles are also being used to facilitate vaccinations and immunotherapy (95). Functionalizing polymeric nanoparticles using membrane coating derived from cancer cells is a different approach to selectively target tumors, but any effects on cardiovascular toxicity remain to be determined (96). The structure, size, and surface properties that affect efficacy and toxicity should be carefully considered before using the blood stream as a portal for delivering nanomaterials.

Intraperitoneal (i.p.) injection is another common mode of nanoparticle delivery to a closed-body compartment (97, 98). Far less is known about intraperitoneal uptake and toxicity associated with this delivery method. Among the various abdominal-cell targets are peritoneal macrophages (99). After intraperitoneal injections in one study, silver nanoparticles accumulated most heavily in liver Kupffer cells and hepatocytes along with kidney podocytes and other cells in the peritoneum (100). These changes were accompanied by cell death and the infiltration of white blood cells, lymphocytes, granulocytes, and hemoglobin (100). Chemical modification of surface charges can minimize toxicity while enhancing biodistribution and tumor targeting of some intraperitoneal injected nanoparticles (101).

In contrast with the blood and peritoneum, the lungs, skin, and intestinal tract are in direct contact with the environment (102). The upper airway and lungs continuously interface with the atmosphere (ref. 103; Fig. 3). A well-developed immune system helps process inhaled atmospheric particulates (104). In the lung, inhaled nanoparticles initially encounter pulmonary surfactants and then pulmonary cells (105). The very thin air–blood barrier (<1 μm) combined with the collective lung-alveolar cell-surface area provides high systemic nanoparticle bioavailability (105). Once engaged by cell surfaces, caveolae-mediated endocytosis translocates nanoparticles into alveolar epithelial or immune cells (106). Depending on the type and size of inhaled nanoparticles (107), pyroptosis-based toxicity can occur (108). Pyroptosis is driven by alveolar macrophages that cause a specialized inflammasome-dependent form of cell death (108). Pulmonary effects are further influenced by the volumes of material inhaled and mass transfer (109). Clinicopathologic responses involve follicular hyperplasia, protein effusion, alveolar lipoproteinosis, and pulmonary capillary vessel hyperemia that lead to fibrosis and emphysema with sustained exposure (110). Modification of nanoparticle size and charge can reduce toxicity while providing rapid sustained release in the lung tissue, thereby resulting in reduced dosing frequency and improved patient compliance (104, 111, 112). As long as toxicity and side effects are minimized, inhalation is a highly efficient mode of nanomaterial delivery for preventive treatments.

Skin, in contrast, is the largest organ and protective barrier of the body that can serve as a topical, regional, and transdermal mode of drug delivery (113). The use of skin-based nanomedical delivery methods may help reduce toxicity, improve sustained release, and penetration (113, 114). Skin exposure to nanoparticles as pollution, antibacterials, and sun screen each have some toxicity concerns (115–117). Nanoparticle uptake measurements using quantum dots showed that penetration into the dermal layer is limited to the uppermost stratum corneum layers and the hair follicles (118). Those nanoparticles that enter the blood stream accumulate in the liver and kidney with poor clearance rates (118). Toxicity can directly affect skin cells by forming reactive oxygen species along with autophagic vacuole accumulation and mitochondria damage (119). Localized inflammatory responses lead to macrophage-, monocyte-, and dendritic-cell responses that cause cytotoxicity (120). Additional oxidative stress, Ca²⁺ flux and decreased mitochondrial membrane potential are accompanied by production of IL1beta and chemokine CXCL9 (120). A variety of approaches hold promise for decreasing dermal toxicity and improving delivery as potential preventive modalities (121–123). Similar approaches hold promise for directly improving melanoma treatment (124).

Oral delivery of nanomaterials is the most common and well-tolerated method for delivering preventive or other agents (125). Other than distribution studies, the toxicology of nanoparticles is not well understood (126). Although oral delivery of nanomaterials is well integrated into the food and drug industry, their long-term fate in the GI tract remains unclear (127, 128). Nanoparticle delivery methods are showing improvements in stabilizing oral delivery and survival of in low gastric pH (129, 130). The intestinal tract maintains elaborate mechanisms for simultaneously taking in nutrients while preserving healthy gut flora and controlling micro- and nanopathogens (131–134). The uptake and distribution of nanoparticles in the gut is also influenced by mucin produced as a protective barrier (135). The mucosal component of the GI tract includes the gut-associated lymphoid tissue that is responsible for antigen sampling (131, 136, 137). One of the primary endocytic pathways for pathogen sampling includes the follicle-associated epithelium of the Peyer's patches found in the upper GI tract (Fig. 4; refs. 131–134). Peyer's Patches contain specialized microfold (M) cells that actively internalize particulate material (131–134). This process involves endocytosis in clathrin-coated vesicles, actin-dependent phagocytosis, or macropinocytosis (131–134). Once internalized, M cells form “intraepithelial pockets” through an expanded basolateral domain. This transcytosis process culminates in shipment and presentation of pathogen-laden exosomes to immune cells (131–134). M-cell exosomal antigens are presented to subepithelial dendritic cells (DC) or are directly sampled by LysoM⁺ dome DCs. These antigens are subsequently presented to T- and B lymphocytes within Peyers' Patches (138, 139). Some efforts are under way to specifically target the M cells (131).

In other areas of the gut, enterocytes absorb nutrients via microvilli of the brush border that can be disrupted by exposure to nanomaterials (140). Villous bearing areas of the gut also exhibit a different set of mechanisms for transepithelial antigen sampling or microbiota/antigen presentation to cells and vessels in the microenvironment of the lamina propria (Fig. 3). This process can occur directly as CX3CR1⁺ lamina propria cells (LPC) cross the epithelium in a basolateral-to-luminal direction (141). Another mechanism involves direct sampling from the gut lumen by mucus-producing goblet cells (142). After the goblet cells have sampled the gut lumen, goblet cell–associated antigen passages' (GAP) transfer various antigens to CD103(+) DCs in the lamina propria (142). These CD103(+) DCs within the lamina propria then induce T-cell responses (142). In other cases, CD11c⁺ DCs or CXCR1⁺ LPC cells in the lamina propria can extend transepithelial dendrites (TED) between enterocytes directly into the gut to sample luminal contents and ensnare microbiota (143–145). As a completely different mechanism of transcytosis, vesicle passage into the lamina propria also occurs directly through intestinal epithelial cells (138, 146). Once exosomes enter the lamina propria, they can gain access to the various cells, including myofibroblasts, pericytes, and capillaries or lacteal vessels of the lymphatic system (147–150). Functionalizing the outer nanoparticle surface can make them an ideal vector suited to traverse brush borders, mucosal lymphoid tissue, and other surfaces (140). Nanoparticles can also be used to deliver vaccines via the gut (151). Nanotargeting of chronic inflammation in the GI tract also holds promise for prevention (152). As with the other delivery methods, minimizing toxicity while maximizing tolerability are essential in generating acceptance of GI delivery approaches for prevention.

Vital differences exist between various precancerous and cancer lesions that can profoundly influence how they are targeted using nanotechnology. Morphologic differences based on gross pathology and histology are the most obvious distinctions (153). The site of origin influences the morphologic characteristics and biologic differences along with the unique characteristics of the individual tumor. Each type of cancer presents its own unique challenges for effective targeting (154). These challenges are influenced further by variations in the anatomic, physiologic, microenvironmental, cellular, and molecular features of the involved tissue site (155). The stage of progression is also critical (156). Advanced-stage cancers that have metastasized can originate in a totally different organ from the one in which they are discovered. In contrast, premalignancies are confined to the site of origin.

Complex multistep biology combined with long progression time frames provide multiple potential points for early detection or intervention using nanotechnology-based approaches. Nanotheranostic tools that simultaneously identify and selectively target premalignant lesions would be extremely useful. This is likely to require modification of probes that selectively identify specific premalignant lesions. Ideally, we will be able personalize theranostic approaches and nanotarget premalignancies by implementing “-omics” approaches for profiling as a first step and selective targeting as a second step (157–159). Some recent advances in theranostics incorporate magnetic resonance (MRI) imaging for early detection along with targeting (160, 161). A molecular imaging strategy using a new triple-modality MRI photoacoustic-Raman nanoparticle is a unique theranostic approach directed at brain tumors (162).

Premalignancies are generally considered the primary targets for cancer prevention (163). Several characteristics are used to distinguish between premalignant and malignant lesions. These characteristics can potentially affect the effectiveness of nanotechnology-based targeting (see Table 2). In addition to being the most common form of cancer, carcinomas arising from epithelial origins are the easiest to identify on the basis of the morphologic distinctions between precancers and cancers. They are often referred to as carcinoma in situ or intraepithelial neoplasias (IEN). Epithelial precancers are by nature organ-confined and generally remain restricted to ducts or the epithelial strata of tissues (164). Cancers, in contrast, are often identified as having breached or invaded through a fine fibrous complex that forms a sheet-like barrier known as the basement membrane (165). The basement membrane can undergo extensive remodeling or thickening during inflammatory responses (166, 167) or become disorganized in tumor vasculature (168) and in various cancers (169). How early this occurs is not clear, particularly with respect to precancerous lesions. Because the basement membrane serves as a structural base for normal epithelia/endothelia, but can potentially accrue abnormalities during carcinogenesis, its status demands adequate consideration when devising nanotargeting approaches.

Premalignant IEN lesions are often accompanied by hyperplasia, or the physiologic proliferation of cells to a greater extent than normal (153). These precancerous lesions may also exhibit dysplasia or the abnormal maturation of cells. Along the continuum of progression from precancerous to cancerous lesions, the cells may begin to exhibit anaplasia. Anaplastic lesions contain cells that have lost their functional tissue identity and reverted to a more primitive or undifferentiated form. IEN lesions also can display pleomorphism that includes large, darkly stained nuclei, and the ratio of nuclear material to cytoplasmic material increases. These precancers are sometimes widely distributed and/or very small in extent and size and, thus, hard to identify until they grow larger (164). If IENs arise in a ductal structure, they can begin to fill in the vacant space or lumen cavity of the duct. Unless this proliferation is accompanied by the stimulation of angiogenesis, these lesions may begin to show signs of apoptosis (170). These IEN lesion characteristics are likely to influence the delivery of nanotechnology to the target site and should be considered during the design and analytic phases of any proposed prevention study (171).

Although angiogenesis may contribute to premalignancy (172), exactly how early it occurs and to what extent it plays a role in the genesis and progression of precancerous lesions is not fully understood (Fig. 5). The biomarkers used to immunochemically identify increased microvascular density include factor VIII, CD31, and CD34 (172). Another biomarker, CD105, is also used to identify newly formed vessels (173–177). More advanced tumor-mediated angiogenesis is highly deregulated and leads to disorganized, poorly formed, and leaky or highly permeable blood vessels (178).

The “angiogenic switch” that initiates angiogenesis was shown by Folkman and colleagues to occur before cells achieve the invasive state (179). This switch was recently shown to involve microRNA, among other factors (180), and may serve as a useful target for cancer prevention (181). Recent evidence indicates that angiogenesis coincides with the onset of dysplasia during adenoma formation in colorectal cancer progression (182). In the breast, angiogenesis seems to begin with the onset of hyperplasia in the mammary duct and progresses through ductal carcinoma in situ and invasive disease (183). Whether angiogenesis starts as a normal inflammatory or tissue repair process during premalignancy and then progresses toward an abnormal state during tumorigenesis or begins as an abnormal process is not known.

Abnormal or leaky blood vessels that arise during tumor-initiated angiogenesis are likely to be attractive targets for nanotechnology-based interventions using vascular delivery approaches (184). However, further work is needed to determine whether vascular-based targeting will be a useful approach for cancer prevention.

Various nanotools could potentially help identify any connections that may exist between the angiogenic switch and premalignancy. Perhaps, nanotools might help identify early changes in endothelial cells and/or tumor cells that signify the beginning of neoangiogenesis. Optimally, early-stage biomarker recognition epitopes or ligands involved in neoangiogenesis will be incorporated onto nanotools as surface molecules that recognize premalignant changes. The involvement of interendothelial transport of modified nanotools may facilitate identifying not only the early changes in these lesions but also the interconnections between endothelial cells and tumor cells (185). These processes may also be identified by biologic changes in the pericytes (186, 187) or vascular endothelial tip cells (188, 189) that are heavily linked to angiogenesis and vascular sprouting. The ability to image and target early-stage changes in the microvasculature of premalignant lesions will help to significantly advance this emerging field.

Determining the usefulness of preventive interventions relies heavily on the therapeutic index of a given treatment (190). This index can be broken down into a risk–benefit ratio that assesses its effectiveness (191, 192). In the case of prevention, the risk to a given population is typically minimized to the greatest extent possible. Any risk assessment needs to be carefully examined, not only for the legal ramifications but also for cost effectiveness (193). It is also important to identify a reasonable balance between safety, efficacy, and affordability, particularly because nanoscale devices are often very complex and can add substantial production costs. Ultimately, a risk assessment also encompasses identifying a given agent's intended and unintended effects based on an individual's/cohort's susceptibilities to both disease development and the agent's effects. If a subset of patients is more susceptible to the adverse effects of a nanopreventive agent than the general population, these patients must be identified and excluded from any study. Moreover, any nanoscale preventive treatment with severe side effects would generally not be suitable for use. This type of intervention must also entail tolerable risk that is suitable for long-term use. However, if a nanopreventive approach heightens sustained local release at the target site, substantial benefit may be realized. In the final analysis, any intervention can exhibit risk, but the risk must be worth the reward.

Chemoprevention is a term describing the use of agents to reverse, suppress, or prevent carcinogenesis and malignancy (194). Subjects for chemoprevention trials are stratified into multiple levels of risk (195). The lowest level of risk encompasses subjects in the general population who are seemingly healthy but may contract sporadic cancers. The allowable risk increases for subjects who present with precancerous lesions, such as IENs (195). The more advanced the premalignancy, the higher the accompanying risk (195). Subjects at high risk, such as those with a genetic predisposition or familial inheritance, are more likely to tolerate more severe side effects. This class of subjects can also include cancer survivors or those at high risk for second cancers, in which case a higher risk of adverse effects becomes more tolerable (196). The same principles that apply to identifying and stratifying subjects for chemoprevention studies should apply to nanopreventive studies. The unique nature of any given nanopreventive intervention is likely to dictate how it is used. This is probably most applicable to nanodevices that might require special monitoring procedures to ensure clearance from the subject's system.

There is a growing number of “at-risk” individuals due to demographics, the aging of the general population, and the adoption of unhealthy lifestyles (196, 197). In addition, enhanced screening can lead to identification of more individuals with precancers; and the number of cancer survivors continues to grow with enhanced early detection and treatment (196). The public tends to have a limited understanding of cancer development, risk, and prevention, let alone nanotechnology (198). Educating the public should help improve the acceptance and adoption of both prevention and nanotechnology in fighting cancer. Once implemented, nanoprevention strategies may increase the need for insurance coverage/reimbursement for prevention, necessitating the passage of legislation.

New research venues and funding opportunities are needed to improve the insights and options for the nano-based prevention of cancer. Advances in genomics, proteomics, lipidomics, metabolomics, and biospecimen-based risk assessment and prevention have had limited impact to date, perhaps because of concerns about the risk:benefit balance (199). A clear demonstration of enhanced benefit with minimized risk is needed to move this area forward at both the basic and translational levels.

Cancer prevention involves overcoming more complex biology in comparison with successes in the cardiovascular prevention area. In cancer prevention, multiple disease sites, each with their own unique challenges, must be taken into account, along with the difficulty of identifying premalignant lesions. Nanotechnology-based preventive interventions may help solve some of these issues, if they improve the early detection and targetability of premalignancies and cancers as well as the delivery and efficacy of preventive agents.

The U.S. Food and Drug Administration approved various agents for treatment of precancerous lesions or cancer risk reduction (Table 3). These vary in the type of agent and mechanism of action, depending upon the target site. There may be opportunities to make these treatments more effective by applying nanotechnology to achieve more targeted release or uptake of some of the smaller molecules or to enhance the immune response to some of the more complex molecules.

Multiple areas of cancer prevention can benefit from the application of nanotechnology. The easiest application to implement and the most useful for helping to collect scientific data is probably early detection. Nanoscale devices that effectively identify premalignancies or cancer signatures hold great promise.

Early forays into device development have targeted blood analysis. A prime example is the barcode chip (200). Devices such as these that integrate microfluidics and a barcoded protein-biomarker capture mechanism on a single chip are ideal for point-of-care detection using a finger-prick sample. Other systems use single-walled CNTs as multicolor Raman labels to achieve highly sensitive, multiplexed protein detection (201). Still other systems have coupled nanoporous silica chips that selectively enrich and stabilize low-molecular-weight peptides with mass spectrometry for highly sensitive biomarker detection (202). Coupling these nanotechnologies with stabile isotope mass spectrometry–based quantitation methods and/or specific gene subsets to plasma analyses exhibiting predictive or preventive value may significantly advance this field (203, 204). One example of this approach was used to monitor the progression of metastatic breast cancer in mouse xenografts (205). Analysis of urine also is under intensive development. Evaluation of urine biomarkers has been accomplished using near-infrared fluorescent core-shell silica-based nanoparticles (C dots; ref. 206). Similar approaches have been applied to feces (207) and saliva (208). Obtaining these samples is minimally invasive and their use will afford effective monitoring, but are, for the most part, indirect. More direct measures include isolating circulating cells or DNA from blood coupled with next-generation sequencing to identify inherent risk.

Nanotechnology may also be directly applied to the analysis of tissue biopsies or exfoliated cells for early detection. The key advantage to having cellular material is the presence of a primary source of DNA, RNA, and proteins. However, obtaining tissue biopsies is generally invasive and involves substantial discomfort and risk to the patient. Thus, obtaining biopsies is usually reserved for high-risk subjects. Another source of cellular material is exfoliated cells. The early detection of oral cancer using exfoliated cells is one example. A nanobiochip sensor technique was applied to exfoliative cytology specimens; these studies illustrate the advantage of targeting both biochemical and morphologic changes associated with early oral tumorigenesis (209). Other sources of exfoliated cells include the colon (210–212), lung (213, 214), bladder (215), cervix (216, 217), and breast (218). Ideally, the source of cells themselves or factors they produce would be suited to point-of-care monitoring (219).

Nanotechnology-based procedures are expected to significantly improve the imaging of cancer (220), including early precancerous lesions. In addition to the imaging uses already described (Table 1), other examples include magnetic nanoparticles (221), Q-dots (222), gold nanoparticles (223), nanoshells (224), and nanotubes (225). When these technologies are coupled with the modification of cell-surface chemistry, adding epitope recognition or antibody/ligand binding sites can enable targeted homing. Coupling homing with unique magnetic signatures, tunable absorption and emission spectral properties, and advanced synthesis and physical characteristics can enhance the usefulness of nanoparticles as probes. A particularly exciting breakthrough involves hyperpolarized MRI of select molecules and/or nanoparticles at up to 20,000-fold greater signal level for early detection of metabolic changes that occur in both premalignancies and cancer. (226, 227).

Achieving targeted imaging using nanoparticles in many cases affords the advantage of also delivering a site-specific chemopreventive payload. The selectiveness of these targeting systems can be driven on two levels. The first level includes the surface ligands involved in homing to the lesion site, and the second level relies on the specificity of released molecules that act directly on the target. In some cases, optimized delivery of novel nanoparticles to the tumor microenvironment may take advantage of unique or abnormal vasculature (178). In other cases, the presence of fibrotic stroma may enhance uptake and delivery (228). Furthermore, other cells such as mesenchymal stem cells may be harnessed to act as a targeted delivery system (229).

Another potential approach involves multistage nanovector delivery (230). The rationale for the use of these more complex delivery systems involves many factors. They are expected to overcome endothelial and epithelial barriers or to be taken up by the reticuloendothelial system (231), are biodegradable, and exhibit favorable hemorheology characteristics in circulation (201). The largest component of these nanovectors acts as a porous shuttle that bears the payload to a particular site. This porous shuttle is loaded with smaller nanoliposomes that contain the targeted payload, such as siRNA (232). This type of delivery enables the sustained release of an agent at a specific target site.

The immune system is capable of recognizing, homing to, and killing premalignant and malignant cells. Harnessing the immune system and enhancing the immune response is another important application of nanotechnology. Nanotechnology can enhance the antibody response (233) and the response of specific immune cells, such as T-helper-17 cells, cytokines (234), and DCs (235). Enhancing the effectiveness of vaccines is another objective (236). These efforts include nanoencapsulation to achieve transcutaneous delivery (237) as well as mucosal immunization (238). Targeted immune system recruitment may also enhance the antitumor immune response (239). These and other strategies may involve personalized regimens to eliminate tumors.

Vaccines can be very effective preventive measures. The prevention of cervical cancer through vaccine development against human papilloma virus (HPV) and early immunization is a good example. However, those individuals already infected with HPV may need other options for controlling or eliminating disease. The delivery of siRNA against NFκB through “photothermal transfection” was used to successfully treat nude mice bearing HeLa cervical cancer xenografts (240). This involved using hollow gold nanospheres, to deliver siRNA in conjunction with near-infrared light irradiation to elicit a photothermal effect along with micro-positron emission tomography/computed tomography imaging (240). Engineering viral nanoparticles may also be effective in treating cervical cancer. The use of RNAi/RGD-based mimoretrovirus to target the Zbtb7 gene is one example (241). This mimoretrovirus exhibited excellent antitumor capacity in vivo in a nude mouse model of cervical cancer (241). The utility of using nanotools to target human HPV–infected tissues for preventive management or virus eradication remains to be fully tested.

Nanoparticle-mediated delivery is expected to limit the toxicity of chemopreventive agents while simultaneously enhancing bioavailability and sustained release. Various success stories continue to emerge involving the use of nanodelivery approaches. The application of nano-epigallocatechin-3-gallate (EGCG) is one example (242). EGCG encapsulated in polylactic acid–PEG nanoparticles enhanced the biologic effectiveness of EGCG at inducing apoptosis and reducing angiogenesis by 10 fold. The use of EGCG in a sustained food release setting has proven useful in a cancer nanochemoprevention setting (243).

Chemoprevention using nonsteroidal anti-inflammatory drugs (NSAID) has shown proven efficacy in treating high-risk colorectal cancer patients (244, 245). Cyclooxygenase-2 (COX-2) selective inhibitors, such as celecoxib, are associated with increased cardiovascular risk (246). Various attempts have been made to incorporate celecoxib into nanoparticles to control release and minimize GI and cardiovascular toxicity (150, 247–251). When applied to arthritis, the compartmentalized injection of a nanolipid–celecoxib formulation directly into joints enabled a localized and gradual sustained release of celecoxib, without any significant increase in cardiovascular drug levels (251). These nanoized formulations of celecoxib also show promise for treating cancer (250). Other investigators used an ibuprofen-conjugated phosphatidylcholine formulation to enhance GI safety and analgesic efficacy in osteoarthritic patients (252). Combinatorial nanoencapsulation of NSAIDs with other drugs may also be effective. For example, when aspirin, folic acid, and calcium (AFAC) were incorporated into nanoparticles, the combination greatly reduced the formation of premalignant aberrant crypt foci in the colon tissues of azoxymethane-treated rats (156). Encapsulation of various NSAIDs or combination treatments continues to progress as promising approaches for the future of nanochemoprevention.

Another example of a combinatorial nanoparticle approach has also had a major impact on pancreatic cancer prevention. Grandhi and colleagues generated an aspirin, curcumin, and sulforaphane (ACS) combination in solid liquid nanoparticles (SLN; ref. 253). These ACS/SLNs were used to perform multimodal targeting of pancreatic cancer. A hamster pancreatic cancer model was generated using N-nitrosobis (2-oxopropyl) amine (BOP) treatment that developed pancreatic intraepithelial neoplasms (PanIN) and tumors. Nanoencapsulated ACS regimens reduced tumor incidence by as high as 75% at doses 10-times lower than free-drug combinations (253).

Other success stories involve nanoencapsulation of proven chemopreventive agents that help solve poor bioavailability problems. Curcumin, the active component of turmeric, is a prime example. Polymeric nanoparticle–encapsulated curcumin (NanoCurc; ref. 254) inhibited tumor growth and systemic metastases in orthotopic pancreatic cancer xenograft models. This approach was also effective in delivering cocktails containing ACS to treat pancreatic cancer (253). Similarly, other forms of curcumin nanoparticles were effective in treating human lung tumor xenografts (255, 256). Enhancing bioavailability and the sustained release of poorly bioavailable compounds may serve as a successful formula for nanoprevention.

We are just beginning our journey into the realm of nanoprevention. A good first step is to clearly identify reasonable goals for this fledgling area of research. High-priority areas where an impact could be made will depend on the type of premalignancy targeted. Finding ways to effectively couple “-omics” into rapid profiling and selective targeting of nanotools for individualized treatment is an ambitious long-term goal. In the near term, improving theranostics based on more generalized molecular recognition signatures such as neoangiogenesis or viral infection may be a good start. For example, imaging and treatment of HPV as a high-risk factor for cervical or oral dysplasia/cancer by nanoparticle-mediated delivery of RNAi may be achievable. As a measurable outcome, this approach might effectively lead to complete clearance of HPV, thereby preventing subsequent disease. As another potential near-term goal, improving methods for nanoencapsulation might enhance the sustained localized release of RNAi or anti-inflammatory agents. For example, one might envision a macro–micro–nano–encapsulation approach that achieves targeted release at the appropriate site in the GI tract. For the field to be successful, it is also critical to not only identify realizable goals but also potential barriers. Ultimately, we must merge the best ideas in prevention with the most effective and least risky nanotechnology.

No potential conflicts of interest were disclosed.

The authors thank Karen L. Colbert for editorial improvements in this article.

This research was supported in part by the Boone Pickens Distinguished Chair for Early Prevention of Cancer (to E.T. Hawk) and from CPRIT RP100969 (to D.G. Menter). Additional support included 5U54CA151668-03 (to D.G. Menter, C.D. Logsdon, and A.K. Sood) and Cancer Center Support grant 5P30CA016672-37 from the NIH.