Review pubs.acs.org/bc
Nanodrug Delivery: Is the Enhanced Permeability and Retention Effect Sufficient for Curing Cancer? Yuko Nakamura,† Ai Mochida,† Peter L. Choyke,† and Hisataka Kobayashi*,† †
Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-1088, United States ABSTRACT: Nanotechnology offers several attractive design features that have prompted its exploration for cancer diagnosis and treatment. Nanosized drugs have a large loading capacity, the ability to protect the payload from degradation, a large surface on which to conjugate targeting ligands, and controlled or sustained release. Nanosized drugs also leak preferentially into tumor tissue through permeable tumor vessels and are then retained in the tumor bed due to reduced lymphatic drainage. This process is known as the enhanced permeability and retention (EPR) effect. However, while the EPR effect is widely held to improve delivery of nanodrugs to tumors, it in fact offers less than a 2-fold increase in nanodrug delivery compared with critical normal organs, resulting in drug concentrations that are not sufficient for curing most cancers. In this Review, we first overview various barriers for nanosized drug delivery with an emphasis on the capillary wall’s resistance, the main obstacle to delivering drugs. Then, we discuss current regulatory issues facing nanomedicine. Finally, we discuss how to make the delivery of nanosized drugs to tumors more effective by building on the EPR effect.
1. INTRODUCTION Solid tumors often develop drug resistance due to a number of well-known mechanisms, including alternative drug export pumps, alterations in gene expression that render the tumor insensitive, changes in the metabolic pathways that affect the metabolism of cytotoxic drugs, deregulation of DNA repair, and subsequent apoptosis induction. Along with these welldescribed mechanisms, the tumor microenvironment plays an important role in drug resistance by imposing barriers that limit drug delivery to the tumor. Thus, a comprehensive understanding of the mechanisms, microenvironment, and barriers are needed to address the issues related to the limited efficacy of cancer chemotherapy.1 Nanosized agents have a number of theoretical advantages over conventional low molecular weight agents including a large loading capacity, the ability to protect the payload from degradation, specific targeting, and controlled or sustained release.2−5 Their features can be enhanced by changing characteristics such as size, shape, payload, and surface features.6,7 Thus, the field of nanomedicine has been rapidly evolving, particularly for the diagnosis and treatment of cancer.5,8−11 However, nanosized drugs are, by definition, larger than most drugs and, therefore, leak more slowly from capillary beds. Fortunately, the vasculature of solid tumors is characterized by leaky vessels with poor lymphatic drainage. When administered intravenously, nanosized agents tend to circulate for a long period of time if they are not small enough to be excreted by the kidney or large enough to be rapidly recognized and trapped by the reticuloendothelial system (RES).12 Nanosized agents with long circulation times leak preferentially into tumor This article not subject to U.S. Copyright. Published XXXX by the American Chemical Society
tissue through the permeable tumor vasculature and are then retained in the tumor bed due to reduced lymphatic drainage. This phenomenon is known as the enhanced permeability and retention (EPR) effect.13,14 The basis for nanosized drug delivery is accumulation of the agent within tumors due to the EPR effect followed by release of their therapeutic payloads. However, EPR effects are relatively modest, offering less than a 2-fold increase in delivery compared with critical normal organs.13 The longer the drug stays in circulation, the more likely it is to extravasate into the tumor through the EPR effect, but at the same time, the drug can also extravasate into normal tissues albeit at a slower rate. Thus, methods that even temporarily increase the local EPR effect within the tumor are needed to improve the specific uptake of the drug within the tumor, thereby improving its therapeutic effect. In this Review, we first overview the barriers to delivery, focusing on the capillary wall’s resistance of tumor. Then, we discuss the current regulatory environment facing nanomedicine in general. Finally, we discuss methods that enhance the EPR effect, thereby increasing the delivery of nanosized drug to the tumor. Received: August 4, 2016 Revised: August 19, 2016
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Figure 1. Barriers for the delivery of nanosized drugs into tumors.
Matsumura and Maeda were the first to show that nanoparticles are able to extravasate through the inherent leaky and loosely compacted vasculature to reach the tumor space and stay there due to the poor lymphatic drainage of tumors.13 This phenomenon was later termed the EPR effect and paved the way for the passive targeting of tumors using nanosized drugs. However, drug delivery due to EPR is still limited, as the rate of leakage from the vessels is slow and the drug can either be excreted or metabolized during the time it takes for the accumulation to reach therapeutic levels.27 EPR effects are also modest, providing less than 2-fold increases in delivery compared with critical normal organs. This is generally insufficient for achieving therapeutic levels within the tumor, although side effects are usually greatly reduced as a result of very low accumulation within normal tissues lacking EPR.28
2. ENHANCED PERMEABILITY AND RETENTION EFFECT Intravenously injected nanosized drugs are delivered into pathological lesions through arterioles and released from capillaries. Therefore, the key mediators of intratumoral delivery are small vessels, especially capillaries.15 Normal capillaries are lined by a tightly sealed endothelium, firmly attached and supported on the abluminal side by stellateshaped pericytes, which are further enveloped in a thin layer of basement membrane (BM).16 In normal tissues, pericyte coverage of the endothelial abluminal surface varies among different organs and blood vasculatures, with a general range between 10% and 70%.16,17 The vasculature BM, with major components of type IV collagen, laminin, entactin (nidogen), and fibronectin, usually envelops blood vessels with a thickness ranging from 100 to 150 nm.18,19 In order to grow, tumor cells recruit a neovasculature to ensure an adequate supply of nutrients and oxygen. As tumors grow, they recruit new vessels or engulf existing blood vessels. Unlike normal blood vessels, the tumor vasculature usually has incomplete endothelial lining causing relatively large pores (0.1−3 μm in diameter), leading to significantly higher vascular permeability and hydraulic conductivity.20,21 In addition, the extent of pericyte coverage on tumor vessels is typically diminished compared to normal tissues.16 Both pericytes and BM are loosely associated with endothelial cells and occasionally penetrate deep in the tumor parenchyma, increasing the transendothelial permeability.16,22−25 Moreover, perivascular smooth muscle is often lacking in tumor vessels, making them poorly reactive to normal vasoregulation.26
3. BARRIERS TO THE DELIVERY OF NANOSIZED DRUGS The EPR effect improves nanosized drug accumulation in the extravascular space of tumors. In normal organs, circulating nanosized drugs are cleared from the circulation by the mononuclear phagocyte system (MPS) or by glomerular filtration in the kidney. The capillary wall’s resistance to the transport of macromolecules and resistance through the interstitium limit nanosized drug extravasation from blood vessels into the interstitial space.25,29 In spite of the EPR effect, overall permeability of the tumor endothelium is a limiting factor for the uptake of macromolecules like antibodies, and the tumor antibody concentration is always 100−1000-fold lower than the plasma concentration. Analogous to resistors in series B
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stromal cell-related extracellular matrix production and organization.46,48 Altogether, excessive growth-induced solid stress compresses tumor vessels and reduces perfusion. Tumor overgrowth may also create an interstitial barrier for efficient drug penetration into tumor tissues. Thus, alleviation of solid stress by targeting the tumor or stromal cells may provide an effective strategy for improving drug delivery.49 Solid Stress from Abnormal Stromal Matrix. In normal tissues, the composition and structure of the extracellular matrix (ECM) is unique and dynamic and functions to regulate cell growth. The major ECM components are collagen, glycoprotein, proteoglycan, elastin, and hyaluronan. The ECMassociated structural components, enzymes, and growth factors are crucial for the regulation of cell proliferation and differentiation, ultimately prolonging cell survival and homeostasis. The ECM is localized at two different sites: the basement membrane and the interstitial space. For instance, the matrix of the basement membrane contains highly compact and less porous structures compared to that of tumors formed by collagen (type IV), fibronectin, laminins, and other related proteins that help to link collagen with other matrix proteins. Type I collagen, glycoproteins, and proteoglycans are abundant in the interstitial matrix and are collectively responsible for tensile strength of normal tissues.50,51 In contrast to these normal stromal features, tumor stroma comprise modified ECM attached to multifaceted stromal cells including fibroblasts, pericytes, endothelial cells, and immune cells.52 Moreover, altered biophysical and biological characteristics of the tumor ECM in a hypoxic microenvironment contribute to tumor progression and metastases.53 Fibrosis is a hallmark of many types of cancers; it develops due to excessive ECM production or limited ECM turnover in tumor tissues.54 Severe desmoplastic or fibrotic reactions are characterized by deregulated accumulation of various types of collagen networks,50 leading to extracellular matrix abnormalities that promote tumor progression through architectural and signaling interactions.51 Cancer-associated fibroblasts (CAFs) play a major role in extracellular matrix-mediated malignant changes, which include upregulated extracellular matrix synthesis, post-translational modifications, and matrix metalloproteinase (MMP)-induced extracellular matrix remodeling, leading to the reduction of drug uptake in tumors.55,56 Resting fibroblasts are transformed into CAFs in response to tumor-associated growth factors such as TGF-β, stroma-derived factor (SDF-1), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF).49 Thus, the abnormal matrix found in tumors further interferes with nanosized drug delivery to tumor.
in an electric circuit, the capillary wall’s resistance to the transport of macromolecules is significantly greater than subsequent resistance through the tumor interstitium.30,31 Here, we will focus on the capillary wall’s resistance, in other words, barriers for extravasation of nanosized drug into tumors (Figure 1). Abnormal Tumor Vasculature. The tumor vascular network is characterized by dilated, tortuous, and saccular channels with haphazard patterns of interconnection and branching.32−34 Unlike the microvasculature of normal tissue, which has an organized and regular branching order, tumor microvasculature is disorganized and lacks the conventional hierarchy of blood vessels.35 Arterioles, capillaries, and venules are not identifiable as such, and instead, vessels are enlarged and often interconnected by bidirectional shunts.36 One physiological consequence of these vascular abnormalities is heterogeneity of tumor blood flow,37 resulting in poor and heterogeneous perfusion in the tumor and elevated interstitial fluid pressure from constant extravasation of fluid, which, in turn, creates hypoxic and acidic intratumoral conditions. This environment prevents the penetration of nanosized drugs deep within the tumor and, therefore, contributes to tumor progression, metastasis, and drug resistance.21,38−40 High Interstitial Fluid Pressure. As tumor tissues have high osmotic pressure, high interstitial fluid pressure (IFP) may hinder adequate delivery of anticancer drugs.41 IFP is elevated in solid tumors not only due to increased vessel permeability and hyperperfusion, but also due to poor lymphatic drainage which normally maintains fluid balance, as well as hyperplasia around blood vessels and increased production of extracellular matrix components.42 In normal tissues, IFP is approximately 0 mmHg; whereas in tumors, IFP can reach microvascular pressure levels (with a range of 10−40 mmHg).43 High IFP limits the convection of nanosized drugs, while paradoxically promoting passive diffusion out of the tumor.43 Diffusion is a much slower transvascular process than convection, especially for the transport of large nanosized drugs.44 Moreover, stroma cells compress intratumoral blood and lymphatic vessels, further impairing blood flow, leading to blood stasis, loss of function, and further inhibition of nanosized drug penetration.45 Finally, because of the steep drop in IFP on the edge of tumors, intratumoral fluid can escape from the tumor periphery into the surrounding tissue, thus spilling nanosized drugs from their intended target, the tumor, into surrounding tissue.44 Thus, high IFP poses a formidable barrier to both the delivery and efficacy of nanosized drugs. Growth-Induced Solid Stress. Tumor growth is associated with the production of intratumoral mechanical forces, both fluid and solid, due to the unrestrained and rapid tumor cell proliferation in a limited area. Solid stress and mechanical forces are generated when cellular and noncellular components of the tumor microenvironment interact with adjacent noncancerous cells and parts of their matrix. In certain tumor types with a less supportive stroma, solid stress-mediated vessel compression can occur due to proliferating tumor cells, hindering the distribution of bloodborne anticancer drugs into tumor tissue.45,46 Therefore, high tumor cell density is the foremost obstacle for penetration of nanosized drugs deep into tumor tissues.47 Solid stress caused either by cancer and stromal cell compression or deformation of vascular and lymphatic structures thereby contributes to tumor progression. Cell compression simultaneously results in changes in gene expression, cellular proliferation, apoptosis, invasion, and
4. CHALLENGES LIMITING CLINICAL TRANSLATION OF CANCER NANOTHERAPEUTICS Despite the huge investments by government and industry and an astonishing number of scientific publications regarding “nanotechnology for cancer treatment” in the last 10 years, the translation of nanosized drugs into clinical practice has been slow compared to that for small-molecule drugs.57,58 In fact, the majority of nanomaterials designed for clinical use are barely at the stage of in vivo evaluation, and even fewer have reached clinical trials. Existing knowledge gaps in science and technology are delaying the development of widely accepted modeling and predictive screening strategies, which would speed up the process between conception of innovative nanomedicine platforms and clinical approval. Challenges also C
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effects are paramount for their use (e.g., in electronic or optical devices). This size limitation is not usually relevant for drug delivery. In fact, the EPR effect typically operates in the range of 100−400 nm.62 Thus, the EC acknowledged that an upper limit of 100 nm is not scientifically justified across the whole range of nanomaterial applications, and noted that “special circumstances prevail in the pharmaceutical sector” by stating that the recommendation should “not prejudice the use of the term “nano” when defining certain pharmaceuticals and medical devices”.65,66 Ultimately, even though the US NNI and EC definitions of nanotechnology and nanomaterial are substantially different, the fundamental difficulty in providing a clear and unequivocal definition of a nanomedicine seems to be shared by both US and EU regulators.59 Regulating Clinical Translation. According to the FDA, regulations can only be based on the current best information, the product has to be fundamentally safe and effective, independent of whether it employs a nanomaterial, and evaluation is based on case-by-case assessment. In 2007, the FDA stated that existing regulations were sufficiently comprehensive to ensure the safety of nanoproducts because these products would undergo premarket testing and approval either as new drugs under the New Drug Application (NDA) process or, in the case of medical devices, under the Class-3 Premarket Approval (PMA) process.62,67 This conclusion was based on the assumption that the regulatory requirements in place would detect toxicity during the required safety studies, even if nanoproducts presented unique properties related to their size. Many experts criticized this view because most FDAapproved nanoproducts obtain approval based in whole or in part on studies of non-nanoversions of the drug, so they do not undergo the full PMA or NDA process.62 In 2011, the FDA reopened the dialogue on nanomedicine regulation by publishing proposed guidelines on how the agency will identify whether nanomaterials have been used in FDA-regulated products.64 The FDA’s purpose here was to help medical product developers identify when there is a need to consider the regulatory status, safety, effectiveness, or health issues that could arise from the use of nanomaterials in FDA-regulated products. In 2012, the FDA commissioner summarized in general terms a “broadly inclusive initial approach” with respect to “nano-governance” in a two-page policy paper published in Science. This paper stated that the “FDA does not categorically judge all products containing nanomaterials or otherwise involving the application of nanotechnology as intrinsically benign or harmful. As with other emerging technologies, advances in both basic and applied nanotechnology science may be unpredictable, rapid, and unevenly distributed across product applications and risk management tools. Therefore, the optimal regulatory approach is iterative, adaptive, and flexible”.68 Most experts in the nanomedicine field continue to criticize the FDA’s effort to regulate nanotechnology, pointing to the fact that delay in addressing nanospecific regulation could have a very harmful effect on investors, public confidence, and commercialization efforts.62
lie in integrating the expert input required from a wide variety of disciplines. Nanomedicine requires a focused effort to integrate all of the relevant disciplines and go beyond the limits of discipline-specific knowledge and jargon. In addition, clear regulatory guidelines for the translation of nanotechnologybased therapies into clinically marketable products are lacking.59 Practically, if nanodrugs rely on the EPR effect for delivery, their circulating half-life has to be sufficiently long for a sufficient amount of the nanodrug to enter the tumor. In general, acute and subacute toxicity must be evaluated for at least ten half-lives which, in the case of nanodrugs, mandates a lot of time and cost. Therefore, nanodrugs carry with them greater regulatory costs than typical small molecular drugs. Regulatory Challenges. Currently in the United States, nanomaterials in pharmaceuticals are regulated by the Food and Drug Administration (FDA).60 The FDA is authorized to regulate new pharmaceuticals under the Federal Food, Drug, and Cosmetic Act. New drugs undergo an extensive premarket approval process, where authorities evaluate the drug’s benefits and risks on a case-by-case basis.61 From a regulatory point of view, the lack of a clear and widely accepted definition of “nanomaterial”,62,63 and thus “nanomedicine”, is another source of uncertainty. The FDA initially adopted a definition of nanotechnology in the 2007 National Nanotechnology Initiative (NNI) as involving all of the following elements: (1) the research and technology development at the atomic, molecular or macromolecular scale leading to the controlled creation and use of structures, devices and systems with a length scale of approximately 1−100 nm; (2) creating and using structures, devices, and systems, which have novel properties and functions as a result of their small and/or intermediate size; and (3) the ability to control or manipulate these properties on an atomic scale. In 2011, the FDA issued a draft guidance for industry entitled “Considering Whether an FDA Regulated Product Involves the Application of Nanotechnology”,64 where it noted that the FDA chose not to adopt a regulatory definition of nanotechnology or related terms. In determining if an FDA-regulated product involves the use of nanotechnology, the FDA parameters are as follows: (1) whether the engineered material or end product has at least one dimension in the nanoscale range (approximately 1−100 nm); and (2) whether the engineered material or end product exhibits properties or phenomena, including physical or chemical properties or biological effects, that are attributable to its dimensions, even if these dimensions fall outside the nanoscale range, up to 1 μm. From the European perspective, the European Commission (EC) provided an official and more detailed definition of “nanomaterial” in the Recommendation 2011/696/EU. According to the EC recommendation: (1) Nanomaterial means a natural, incidental, or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1−100 nm. (2) In specific cases and where warranted by concerns for the environment, health, safety, or competitiveness, the number size distribution threshold of 50% may be replaced by a threshold between 1% and 50%. The EC recommends the above definition for all fields of application of nanotechnology, but a distinct regulatory definition is not provided as in the case of nanomedicine. It is clear that nanomedicines are often not within the 1−100 nm size range, unlike nonmedical nanomaterials where quantum
5. HOW TO IMPROVE THE EPR EFFECT The extent of the EPR effect is dependent on several factors.44,69 By manipulating either local tumor or systemic conditions, EPR effects can increase, leading to increased nanosized drug delivery. The three most important modifiable parameters that improve the tumor capillary wall’s resistance D
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Figure 2. Methods for improving cancer nanosized drug delivery based on EPR effects by manipulating intrinsic physiological barriers.
include (1) modulating the tumor blood flow; (2) modulating the tumor vasculature and stroma; and (3) killing the cancer cells to reduce their barrier function (Figure 2).31,70 Modulating Tumor Blood Flow. One of the hallmarks of solid tumors is inefficient blood flow, which limits drug
transport in the tumor and contributes to a reduced or absent transcapillary pressure gradient. Both effects reduce the uptake and distribution of antitumor drugs. In order to restore an efficient tumor blood flow, both vasoconstrictors and vasodilators have been used as promoter drugs that modulate tumor E
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blood flow. This appears paradoxical, but both approaches aim to increase the transcapillary pressure gradient through different mechanisms such as increasing blood pressure with vasoconstrictors or decreasing flow resistance with vasodilators. Vasoconstrictor. To improve perfusion in the tumor, the overall physiological condition of the patient can be altered. For example, Nagamitsu et al. induced systemic hypertension in patients with solid tumors using angiotensin II during infusion of nanoparticles.71 They found that this strategy increased the accumulation of nanoparticles and resulted in improved therapeutic response, less toxicity, and a shorter time to achieve tumor regression.72 While inducing hypertension has been shown to be a promising approach on a clinical basis, augmenting the EPR effect through a strategy that alters the physiological condition of the patient has challenges since it may affect the whole body. In the case of inducing hypertension, treatment is limited to patients not already on antihypertensive medication. In countries such as the US, where approximately one-third of the population have hypertension,73 some cancer patients may not be eligible for this treatment. Therefore, strategies augmenting the EPR effect that affect the whole body will have to take the demographics of the population into consideration.39 Normal vessels retain their ability to respond to extrinsic vasoconstrictors whereas tumor vessels lose their responsiveness to such agents. When vasoconstrictive drugs are administered, normal vessels become constricted due to contraction of muscular fibers in the vessel wall, limiting blood flow and increasing blood pressure. In contrast, tumor vessels do not respond to vasoconstrictors because of insufficient muscular structure. This leads to a relative increase in the blood flow in vessels supplying tumors.74 This phenomenon was recognized in the 1970s during diagnostic angiography for tumor localization and was termed “pharmacoangiography”.75 During diagnostic angiography, vasoconstricting agents, including α-receptor agonists, were injected via a catheter to constrict normal vessels while accentuating tumor vessels.76,77 Later, pharmaco-angiography was used to constrict vessels after the delivery of a nanodrug therapy to reduce washout and increase exposure of the tumor to the therapy.78 Diagnostic pharmaco-angiography is no longer needed for conventional diagnostic scanning because CT and MRI scans have become proficient at detecting cancers, but the effect can still be put to use to selectively increase drug delivery.70 Vasodilator. To enhance the EPR effect, a number of vascular mediators are utilized.14 Nitric oxide (NO) is an endogenous mediator that causes vessels to dilate and thereby lowers blood pressure. Many solid tumors manifest vascular embolism or vascular clogging. If nitroglycerin is administered to restore the vascular blood flow of such tumors, drug delivery is increased and hence a greater EPR effect occurs. Seki and others published investigations of the application of nitroglycerin and angiotensin-converting enzyme (ACE) inhibitors, such as enalapril, in which they reported increased delivery of Evans blue-albumin to tumors.79−84 Fang et al. also showed that carbon monoxide-releasing micelles enhanced drug delivery.85,86 Yasuda et al. reported significant benefits of using an NO-releasing agent (nitroglycerin) using conventional low-molecular-weight anticancer agents in clinical settings.87,88 Another vascular mediator involved in the EPR effect, Kinin, is a major mediator of inflammation that induces extravasation and accumulation of body fluids in inflammatory tissues (edema).89,90 Kinin is known to activate endothelial cell-
derived NO synthase,91 which ultimately leads to an increase in NO, an important mediator of tumor vascular permeability. Prostaglandins (PGs) are lipid compounds that are derived enzymatically from arachidonic acid by means of cyclooxygenases (COXs).92,93 Similar to bradykinin, PGs are important mediators in inflammation and can be upregulated by inflammatory cytokines as well as kinin.94,95 Among the various PGs, PGE1 and PGI2 exhibit effects similar to those of NO, i.e., preventing platelet aggregation, leukocyte adhesion, and thrombosis formation and facilitating extravasation and the EPR effect. Injection of a stable analogue of PGI2, beraprost sodium (Dorner), which has a long plasma half-life in humans, resulted in increased extravasation of the Evans blue/albumin complex from 2- to 3-fold.96 Ansiaux et al. have shown that local administration of Botulinum neurotoxin type A increases tumor oxygenation and perfusion, leading to improvement in the tumor response to radiotherapy and chemotherapy. This is the result of interference with neurotransmitter release at the perivascular sympathetic varicositities, leading to the inhibition of the neurogenic contractions of tumor vessels and improvement of tumor perfusion and oxygenation.97 Modulating the Tumor Vasculature and Stroma. Use of promoter drugs or methods that induce selective enhancement of the permeability of the tumor endothelial barrier is another strategy to improve uptake and distribution of drugs. In fact, despite areas of leakiness and increased permeability, the endothelial barrier represents a major limiting factor for drug delivery. It may appear paradoxical that two maneuvers that lead to opposite effects, i.e., reduction of the vascular permeability through normalization of tumor blood vessels and enhancement of the permeability of tumor blood vessels, allow for increased uptake and penetration of drugs. The crucial point, however, for drugs that modulate tumor blood flow, is that both maneuvers temporarily lead to an increased transcapillary pressure gradient, which results in enhanced tumor uptake of drugs.31 Since the ECM is an additional key determinant of solid stress, it also hinders interstitial drug distribution within tumor tissue. A tumor priming strategy to alleviate stroma-mediated solid stress by targeting the ECM (directly by degradation or indirectly by inhibiting its synthesis) may be useful to improve chemotherapeutic efficacy. Enhancement of the Permeability of Tumor Blood Vessels. Sonoporation. Sonoporation combines ultrasound (US) and microbubbles (MB) to induce stable and inertial cavitation effects, which permealizes cell membranes and opens tight junctions in vascular endothelium. Sonoporation has been reported to enhance the ability of nanosized drugs to extravasate out of blood vessels into the tumor interstitium, suggesting that sonoporation may be a useful strategy for improving EPR-mediated drug targeting to tumors.98,99 However, while it might be tempting to damage endothelial cells in an attempt to increase permeability, this can only be achieved at the risk of decreasing or even eliminating blood flow to the tumor due to thrombosis. Moreover, sonoporation is untargeted, so both tumor and surrounding normal tissue may be damaged by the drug. Growth Factors. Initially, tumors are dependent on the vasculature of the surrounding host tissues for their blood supply. However, as they grow further, they switch into an angiogenic state in order to meet their increasing metabolic demands.100 Tumors also show increased levels of growth F
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ment in the delivery of drugs has also been reported to be short-lived so that accurate timing between normalization and administration of the nanodrug is mandatory.123 Decrement of Interstitial Fluid Pressure. High IFP in tumors is a direct consequence of angiogenesis and limits nanosized drug extravasation. Targeting angiogenesis is a simple approach to circumvent this barrier.124,125 Paclitaxel treatment has been shown to be effective in reducing IFP values in the clinic.126 A VEGF blockade to inhibit angiogenesis is another promising strategy to aid in drug penetration against the pressure gradient.127 Treatment with Imatinib, a PDGF receptor-β inhibitor, led to decreased VEGF expression and subsequently decreased IFP.128 Similarly, Dickson et al. have shown that pretreatment with Bevacizumab, an anti-VEGF monoclonal antibody, helped to improve the antitumor efficacy of systemically administered topotecan in a murine neuroblastoma model.129 Vascular disrupting agents such as combretastatin and ZD6126, a tubulin-binding agent, have also been used to successfully reduce IFP.130,131 Modulating the Stroma. Direct Extracellular Matrix Degradation. Abnormal ECM composition and structure in solid tumors are the major obstacles for penetration of anticancer drugs, especially in desmoplastic tumors. In solid tumors, penetration of macromolecular therapeutic agents is particularly affected by interstitial stromal barriers such as collagen networks.132 Numerous studies have shown that the ECM-degrading enzyme collagenase can improve the distribution of macromolecules in solid tumors.133,134 The hormone relaxin has been reported to also be effective in increasing drug delivery by modifying collagen structure.44 The collagen content in tumors showed correlation with the IgG diffusion coefficient measured in situ with fluorescence, and IgG penetration into tumor interstitial tissue increased after collagenase treatment.135 Similar to collagen, hyaluronan, also called hyaluronic acid (HA), is a key matrix element that increases IFP and vascular collapse. Hyaluronidase has been reported to induce HA degradation, thus improving vascular perfusion and facilitating efficient penetration of chemotherapeutic agents.42,136 However, this only will be effective where it is injected and to that extent is nontargeted. Reduction of Extracellular Matrix Synthesis by Inhibition of CAFs. CAFs are key regulators of tumorigenesis. In tumor cells, which are genetically unstable and mutate frequently, the presence of genetically stable fibroblasts in the tumor-stromal compartment makes them an optimal target for cancer immunotherapy. Loeffler et al. proposed the use of an oral DNA vaccine targeting fibroblast activation protein (FAP), which is specifically overexpressed by fibroblasts in the tumor stroma. Through CD8+ T cell-mediated killing of CAFs, this vaccine successfully suppressed primary tumor cell growth and metastasis of multidrug-resistant murine colon and breast carcinoma. Furthermore, tumor tissue of FAP-vaccinated mice revealed markedly decreased collagen type I expression and up to 70% greater uptake of chemotherapeutic drugs.56 In addition to killing CAF cells, ECM deregulation can be inhibited by blocking the growth factors involved in CAF stimulation. Hence, inhibiting collagen synthesis has been accomplished either by using anti-TGF-β antibodies or agents that have antifibrotic activity by causing TGF-β signaling blockade, and resulted in an enhanced delivery of therapeutic agents.137 Losartan, a well-known antihypertensive agent with antifibrotic effects, has also been reported to be effective in reducing the tumor tissue collagen content and successfully
factors like vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) among others.101,102 These growth factors significantly increase permeability of macromolecules by modulating the subendothelial structures.103 However, the vascular permeability of tumors is also very heterogeneous in its distribution due to this abnormal angiogenesis, which can negate the effects of EPR. Application of exogenous VEGF was found to increase the pore size of human colon carcinoma xenografts, allowing for the enhanced extravasation of albumin (7 nm) as well as PEGylated liposomes (100−400 nm).104 There is also some reluctance to use growth factors such as VEGF as they might have an unpredictable effect on tumor growth, to the extent that receptors are found not only on endothelial cells but also on tumor cells. Tumor Necrosis Factor-α. Tumor necrosis factor-α (TNF) is an inflammatory cytokine that causes hemorrhagic tumor necrosis in mice.105 Studies showed high response rates where high-dose TNF in combination with chemotherapeutic drugs was administered by isolated-limb perfusion to patients with melanoma or sarcoma of the extremities.106−109 Clear evidence was obtained in these studies for TNF as a promoter drug that increases tumor uptake and penetration of chemotherapeutic effector drugs. However, in humans the drug is very toxic and functions only in a narrow range of doses before causing severe systemic side effects. Hyperthermia. Kong et al. reported that, with hyperthermia, a 100 nm liposome experienced the largest relative increase in extravasation from tumor vasculature; moreover, hyperthermia did not enable extravasation of 100 nm liposomes from normal vasculature, potentially allowing for tumor-specific delivery.110 Li et al. have shown that local hyperthermia was able to increase the vascular permeability up to particle diameters of 10 μm in a variety of tumor models.111 This allowed for increased liposomal extravasation not seen with normothermia. Similarly, Liu et al. were able to demonstrate that the thermally induced extravasation of liposomes led to their increased accumulation in murine mammary carcinomas.112 Tumor-specific hyperthermia induced by high intensity focused ultrasound has also been reported to improve drug delivery to tumors.113−117 Once again, it is difficult to contain hyperthermia just to the tumor, and therefore, this method can increase drug delivery to normal tissue at the periphery of the tumor causing off-target side effects. Reduction of the Vascular Permeability. Vascular Normalization. Abnormalities in tumor vasculature and lymphatic system create an unfavorable tumor microenvironment for molecular movement, which ultimately prevents adequate and homogeneous distribution of anticancer drugs (either small molecules or macromolecules, including nanosized drugs and antibodies) in solid tumors.118,119 Vascular normalization repairs not only abnormal structures, but also the functions of tumor vasculature by correcting rapid angiogenic signaling. Anti-angiogenic treatment reestablishes the balance between pro- and anti-angiogenic agents, and the blood vessels become more stable and uniform in structure and function.120 Hence, remarkable improvements have been demonstrated in the delivery as well as efficacy of anticancer therapeutics.121,122 However, normalized vessels also tend to reduce the size of fenestrations which will hinder EPR-based delivery of large nanosized drugs to tumor tissue. Therefore, vascular normalization tends to improve tumor penetration only in the case of smaller nanosized drugs (
