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Abstract

  1. Top of page
  2. Abstract
  3. Relation of vascular permeability to infection and cancer: Inflammatory mediators in bacterial infection and cancer
  4. Endogenous free radical generation in infection: A significant cause of tissue damage by inline image and mutation
  5. Vascular permeability leading to tumor-targeted drug delivery, lymphotropism, and mechanism of the EPR effect of macromolecules
  6. Influences on and augmentation of the EPR effect
  7. Enhanced permeability and retention (EPR) effect in tumor imaging
  8. Drug uptake by tumor cells and drug release from nanomedicines
  9. Conclusion
  10. Acknowledgments
  11. Disclosure Statement
  12. References

This review focuses primarily on my own research, including pathogenic mechanisms of microbial infection, vascular permeability in infection and tumors, and effects of nitric oxide (NO), superoxide anion radical (inline image), and 8-nitroguanosine in the enhanced permeability and retention (EPR) effect for the tumor-selective delivery of macromolecular agents (nanomedicines). Infection-induced vascular permeability is mediated by activation of the kinin-generating protease cascade (kallikrein–kinin) triggered by exogenous microbial proteases. A similar mechanism operates in cancer tissues and in carcinomatosis of the pleural and peritoneal cavities. Infection also stimulates inline image generation via activation of xanthine oxidase while generating NO by inducing NO synthase. These chemicals function in mutation and carcinogenesis and promote inflammation, in which peroxynitrite (a product of inline image and NO) activates MMP, damages DNA and RNA, and regenerates 8-nitroguanosine and 8-oxoguanosine. We showed vascular permeability by using macromolecular drugs, which are not simply extravasated through the vascular wall into the tumor interstitium but remain there for prolonged periods. We thus discovered the EPR effect, which led to the rational development of tumor-selective delivery of polymer conjugates, micellar and liposomal drugs, and genes. Our styrene–maleic acid copolymer conjugated with neocarzinostatin was the first agent of its kind used to treat hepatoma. The EPR effect occurs not only because of defective vascular architecture but also through the generation of various vascular mediators such as kinin, NO, and vascular endothelial growth factor. Although most solid tumors, including human tumors, show the EPR effect, heterogeneity of tumor tissue may impede drug delivery. This review describes the barriers and countermeasures for improved drug delivery to tumors by using nanomedicines.

As the recipient of the Tomizo Yoshida Award of the Japanese Cancer Association in 2011, I herein describe my personal recollections of my research. This review covers the pathogenic mechanisms of microbial infection and generation of endogenous bradykinin (also called kinin) and free radicals inline image and NO in bacterial and viral infections. This review also discusses selective delivery of anticancer macromolecular agents (now called nanomedicines) to tumor tissue, which take advantage of the vascular permeability of tumor tissue (analogous to inflamed tissue). Projects involving the antitumor antibiotic protein neocarzinostatin (NCS, 13 kDa) were pursued in my laboratory at the Department of Microbiology, Kumamoto University School of Medicine (Kumamoto, Japan) for more than two decades,[1-3] and this research led me to the field of nanomedicine.

Around 1978, my research focused on developing an antitumor agent that would target metastatic tumors, or more specifically, to target to the lymphatic system. For this purpose, I had to design an anticancer agent with lipophilic and macromolecular characteristics, because these features would favor preferential recovery of the agent through the lymphatic system from lymphatic networks in the interstitial space after extravasation from blood vessel lumina. In 1978, therefore, I prepared a conjugate of NCS with SMA, in which SMA conferred lipophilic and polymeric characteristics.[1-3] This agent was the first polymer-conjugated macromolecular anticancer drug, named SMANCS, and was reported as the first macromolecular anticancer agent having antimetastatic activity.[4-8] SMANCS possessed many advantages including lipid formulation using Lipiodol®,[9] as described later, and also effective to the multidrug resistance of tumor cells.[8-11]

Thereafter, we carried out more detailed pharmacological studies of SMANCS and other macromolecular drugs, including plasma proteins and synthetic polymers, which led to the discovery of the unique pharmacokinetics of polymeric drugs and, more importantly, the EPR effect.[5-8, 12-15] We then investigated the EPR effect in greater detail and found that many vascular mediators, such as bradykinin (also called kinin), NO, ONOO, prostaglandins, vascular endothelial growth factor, collagenase, and more recently, carbon monoxide, are responsible for this effect (Table 1).[15-22] Our earlier studies of the kinin-generating cascade triggered by microbial proteases and biological generation of free radicals in infection were quite useful for understanding these vascular mediators and the EPR effect and for developing methods to further augment cancer drug delivery based on the EPR effect. The ultimate outcome of these studies can provide theoretical mechanism, and extend to the application of nanomedicines for tumor selective delivery to treat cancer in the future.

Table 1. Factors affecting the enhanced permeability and retention (EPR) effect of macromolecular drug delivery to solid tumors
MediatorsResponsible enzymes and mechanismsReferences
  1. †Extensive production of vascular mediators that facilitate extravasation from normal and tumor vessels. ‡Enzymes or mechanisms involved in each process (see text for details). iNOS, inducible form of nitric oxide synthase; NO, nitric oxide; VEGF, vascular endothelial growth factor; VPF, vascular permeability factor.

BradykininKallikrein/protease [16, 19, 32, 33]
NOiNOS [17, 18, 96]
VPF/VEGFInvolved in NO generation [76, 77]
ProstaglandinsCyclooxygenase-1 [76, 77, 95]
Collagenase (proMMPs [RIGHTWARDS ARROW] MMP)Activated from proMMPs by peroxynitrite or proteases [20]
Peroxynitrite (ONOO)NO + inline image [RIGHTWARDS ARROW] ONOO [RIGHTWARDS ARROW] proMMP [RIGHTWARDS ARROW] MMP [20]
Carbon monoxideHeme oxygenase-1 [97]
Induced hypertensionUsing angiotensin II [70, 77, 89]
Inflammatory cells and H2O2Neutrophil/NADPH oxidase, etc. 
Transforming growth factor-β inhibitor  [114]
Vascular endothelial cell growth factor (VEGF)Also involves NO [114]
Tumor necrosis factor-α  [115]
Anticancer agents
Heat  [116]

Relation of vascular permeability to infection and cancer: Inflammatory mediators in bacterial infection and cancer

  1. Top of page
  2. Abstract
  3. Relation of vascular permeability to infection and cancer: Inflammatory mediators in bacterial infection and cancer
  4. Endogenous free radical generation in infection: A significant cause of tissue damage by inline image and mutation
  5. Vascular permeability leading to tumor-targeted drug delivery, lymphotropism, and mechanism of the EPR effect of macromolecules
  6. Influences on and augmentation of the EPR effect
  7. Enhanced permeability and retention (EPR) effect in tumor imaging
  8. Drug uptake by tumor cells and drug release from nanomedicines
  9. Conclusion
  10. Acknowledgments
  11. Disclosure Statement
  12. References

We first discovered that kinin, the most potent pain-inducing endogenous peptide hormone, was generated by exogenous bacterial proteases through the activation of one or more steps in the kinin-generating cascade, which begins with stimulation of Hageman factor, or factor XII (which would lead to the blood coagulation cascade), and continues to involve prekallikrein to kallikrein (Fig. 1), and then generation of kinin from high- and low-molecular-weight kininogen in blood plasma. All pathogenic bacterial and fungal infections induce inflammation,[23-25] in which extravasation of plasma proteins is a hallmark event and causes edema and in which bradykinin is a key mediator. Similar such events were later found to occur in solid tumors. We have discovered no effective inhibitor in the body against all microbial proteases, including fungal protease, that we tested.[26-31]

image

Figure 1. Kinin-generating cascade activated by proteases, which begins with activation of Hageman factor (factor XII), continues to prekallikrein and kallikrein, then yields kinin (blue). Kinin stimulates vascular leakage (permeability). Tumor tissue has a more active cascade, which thus produces excessive kinin. Corn trypsin inhibitor, soybean trypsin inhibitor, and carboxypeptidase N inhibitor (CPNI) can inhibit this cascade. ACEI, angiotensin-converting-enzyme inhibitor; MW, molecular weight.

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With regard to cancer, we found a significantly high concentration of kinin, which was generated by the above-mentioned mechanism, in blood plasma and in peritoneal or pleural carcinomatosis in tumor-bearing hosts including humans.[32-34] Kinin generation was effectively inhibited by inhibitors of kallikrein such as soybean trypsin inhibiter or by kinin antagonists.[32-34]

Various tumor cells highly express bradykinin receptors, which lead to growth stimulation, angiogenesis, metastasis, and release of MMPs involving NO, prostaglandins, and various other cytokine signaling pathways.[35-38] We focused our attention, however, on vascular permeability in tumors and delivery of macromolecular drugs that was given i.v., which led to discovery of the EPR effect.

Wu and others in our department showed that the kinin antagonist HOE 140 (Hoechst, Frankfurt, Germany) or kallikrein inhibitors, such as soybean trypsin inhibitor, can block this extravasation of plasma proteins in cancer tissue.[19, 20] Soybean trypsin inhibitor and other serine protease inhibitors (such as PMSF) effectively suppressed formation of peritoneal and pleural ascites,[32-34] in which the kinin concentration was quite high, and facilitated fluid accumulation.[16, 32, 33] Kinin antagonists were later found to suppress tumor growth, thus they may be good therapeutic targets in cancer therapy and pain control.[37, 38] However, the involvement of bradykinin in tumor biology and pathogenesis warrants further investigation.

My experiences with protein anticancer drugs, that is, NCS and SMANCS, therefore made me realize, in the early 1980s, the potential use of the enhanced vascular permeability of solid tumors for delivery of macromolecular anticancer drugs.[36-38]

Endogenous free radical generation in infection: A significant cause of tissue damage by inline image and mutation

  1. Top of page
  2. Abstract
  3. Relation of vascular permeability to infection and cancer: Inflammatory mediators in bacterial infection and cancer
  4. Endogenous free radical generation in infection: A significant cause of tissue damage by inline image and mutation
  5. Vascular permeability leading to tumor-targeted drug delivery, lymphotropism, and mechanism of the EPR effect of macromolecules
  6. Influences on and augmentation of the EPR effect
  7. Enhanced permeability and retention (EPR) effect in tumor imaging
  8. Drug uptake by tumor cells and drug release from nanomedicines
  9. Conclusion
  10. Acknowledgments
  11. Disclosure Statement
  12. References

We focused on two research areas of infectious diseases: one was exogenous bacterial proteases, which involve the kinin cascade and degenerative tissue damage; and the other was biological free radicals, that is, ROS and RNS, as derived from NO. We first found that inline image was generated in excess during respiratory infection with influenza virus in mice.[39, 40] Our initial hypothesis, that Nox of infiltrated leukocytes would play a major role, was determined to be untrue. Instead, the major source of inline image generation was excessive activation of XO to about 400-fold from xanthine dehydrogenase. This enzyme is involved in the latter part of adenosine catabolism: adenosine [RIGHTWARDS ARROW] inosine [RIGHTWARDS ARROW] hypoxanthine [RIGHTWARDS ARROW] xanthine [RIGHTWARDS ARROW] uric acid + 2 × inline image; in this cascade, adenosine deaminase activity increased approximately 170-fold,[39-42] and Nox was second in importance.

Our discovery of excessive generation of inline image in viral infection was an unprecedented finding that was proved by means of three completely different methods. The first used pyran copolymer-conjugated SOD, which removed inline image and markedly improved the survival rate (Fig. 2a). The polymer–SOD conjugate was designed to have a plasma half-life more than 100 times longer than that of native SOD, which is cleared from the blood circulation in mice within a few minutes.[39, 40] This conjugate was likely the first polymer-conjugated therapeutic enzyme, the idea for which derived from our experience with SMANCS, as described in more detail later.

image

Figure 2. Pathological and molecular events in influenza virus-infected mice. (a) Treatment of influenza virus-infected mice with native superoxide dismutase (SOD) (○), polymer (pyran)-conjugated SOD (△), or no drug (control) (●). (b) Time course of virus yield (○), consolidation score (●), and mortality (▲). All these events occurred separately. (c–e) Activity of the inducible form of nitric oxide synthase (iNOS) and superoxide (inline image). (c) iNOS activity. (d) Induction of PCR-detectable iNOS mRNA. (e) Amount of inline image generated after virus infection. (f) Effect of mutant virus formation in wild-type iNOS+ B6 mice (red bars) and iNOS/B6 transgenic mice (green bars). More mutant virus was formed in NO-generating wild-type mice, which indicated a need for NO for viral mutation. BALF, bronchoalveolar lavage fluid.

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The second method used the XO inhibitor allopurinol, which improved the survival rate of virus infected mice in a dose-dependent manner. The third was injection of adenosine, which generates inosine and then hypoxanthine (a substrate of XO) and inline image, which itself facilitated a pathogenic effect and resulted in severe exacerbation of this viral infection and an accelerated death rate of mice.[39-41]

During influenza virus infection, the amount of virus (i.e. virus yield), consolidation score of the lung, and mortality rate of infected mice increased, but not simultaneously. Figure 2(b) illustrates that the amount of virus in the alveolar compartment, as determined by bronchoalveolar lavage, was highest on day 4 after infection then decreased to undetectable levels on day 10, whereas pathological manifestations as indicated by the consolidation score and inline image generation started to increase on day 6 and reached a maximum on day 10, when the mice started to die and virus was no longer detectable.[39-41] Thus, all three events occurred separately, not simultaneously, and I coined the phrase “virus disease in the absence of virus,” for situations in which the amount of virus did not completely correspond to pathological severity.[42]

As another important finding, the data showed that oxygen radicals (ROS) can be generated by a truly biological event as a host response in vivo, not just by intense UV irradiation, X-ray, or γ-ray exposure in vitro. That the generation of free radicals is clearly an etiological principle in infectious diseases can also be stated. In other words, this pathological mechanism extends beyond the boundary of Robert Koch's postulate or paradigm.[42] This mechanism will also offer alternative therapeutic options against infectious diseases by using scavengers of inline image (Fig. 2a) or by inhibiting ROS-generating systems that will reduce the pathogenic potential and cure diseases.

We subsequently found that another biological free radical, NO, is excessively generated under conditions similar to those of influenza virus infection (Fig. 2c–e), which also possesses a pathogenic potential.[43-48] That inline image and NO react rapidly and form ONOO, which is an agent with extremely potent oxidizing and nitrating activities occurring simultaneously, is well known. ONOO would therefore modify DNA and RNA by nitration or oxidation, or by strand breaks (Fig. 2f).[43-50] This increased mutation by ROS and RNS was also shown in bacteria[49, 50] as well as influenza and Sendai viruses,[44, 47, 48] and antioxidant scavengers suppressed mutagenesis and carcinogenesis.[49-52] These data support the idea that carcinogenesis induced by infection and inflammation would proceed through free radical generation or oxidative stress.[51, 52]

Furthermore, a finding relevant to free radical-induced carcinogenesis is that 8-nitroguanosine becomes a substrate for NOS. Also, the NADPH reductase, cytochrome b5 reductase, and reductase domain of NOS similarly generate inline image (Fig. 3a).[53-57] In separate but related studies, we showed that various heterocyclic amines (all 11 kinds) and NCS effectively produced inline image. All these chemicals are mutagens in the presence of NADPH and cytochrome P450 reductase (Fig. 3b).[53-56] Also, the mutation rate of Salmonella typhimurium (Ames strains) paralleled to the amount of inline image produced.[57]

image

Figure 3. Generation of free radicals in infection and cancer. (a) Nitric oxide synthase (NOS) can generate nitric oxide (NO) and superoxide (inline image), and then peroxynitrite (ONOO) can nitrate guanine ([RIGHTWARDS ARROW] 8-nitroguanine), and 8-nitroguanine (NitroGuo) can become a substrate of NOS or cytochrome c reductase, thereby generating inline image. The total system thus works as a progressive reaction, with a stoichiometry of greater than 1:1. (b) Generation of inline image from heterocyclic amine (HCA) in the presence of cytochrome (Cyt.) P450 reductase and NADPH, which results in DNA damage or cleavage and mutation.[53-55, 57] (c) Heme oxygenase (HO)-1 can generate carbon monoxide (CO), which results in the enhanced permeability and retention (EPR) effect. HO-1 is usually upregulated in most tumors. (d) Enhancement of the EPR effect by application of nitroglycerin. FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; fp(ox), flavoprotein oxidized form; fp(red), flavoprotein reduced form.[90, 96]

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We collaborated with Dr. Tatematsu of Aichi Cancer Center Research Institute (Nagoya, Japan) in studies of our novel phenolic antioxidant compound named canolol, which suppressed Helicobacter pylori-induced inflammation, ONOO cytotoxicity, and carcinogenic potential, whereas canolol did not affect the number (colony-forming units) of H. pylori bacilli.[49-51] These data support the proposal that free radical generation is a key process in the mediation of inflammation and chemical carcinogenesis, as previously expected.[52] Additional details about the chemical damage to nucleic acids by ONOO have been provided by Niles et al.,[58] Sawa and Ohshima,[59] and others.

In view of the relation among inflammation, oxidative damage, and carcinogenesis, Okada, Kobayashi, Tanaka, Cao, Shimizu, Muto, Surh, and many others showed the potent cancer-promoting effects resulting from inflammation, in which the oxygen burst caused by Nox and the NO generation induced by iNOS derived from infiltrated leukocytes[51, 60-67] were crucial requirements. This concept has now became a textbook example of an essential component of carcinogenesis, including that in humans.[68]

Vascular permeability leading to tumor-targeted drug delivery, lymphotropism, and mechanism of the EPR effect of macromolecules

  1. Top of page
  2. Abstract
  3. Relation of vascular permeability to infection and cancer: Inflammatory mediators in bacterial infection and cancer
  4. Endogenous free radical generation in infection: A significant cause of tissue damage by inline image and mutation
  5. Vascular permeability leading to tumor-targeted drug delivery, lymphotropism, and mechanism of the EPR effect of macromolecules
  6. Influences on and augmentation of the EPR effect
  7. Enhanced permeability and retention (EPR) effect in tumor imaging
  8. Drug uptake by tumor cells and drug release from nanomedicines
  9. Conclusion
  10. Acknowledgments
  11. Disclosure Statement
  12. References

Uniqueness of tumor vasculature, and extravasation of macromolecules given i.v

As mentioned earlier, we simultaneously carried out multiple research projects in my laboratory. The anticancer agent composed of a conjugate of a polymer (SMA) and a protein (NCS) (i.e. SMANCS) showed considerable lymphotropic accumulation, consequently it became effective against metastatic tumors in rats.[2-4, 6-8] Another important finding was the markedly high accumulation of SMANCS in tumor tissues, as we expected,[5-8, 69, 75-78] which was approximately 10–100 times greater than that in normal tissues (Fig. 4). We also found that the plasma concentration (or AUC) of SMANCS at 24 h after i.v. inoculation in both mice and patients was more than 20 times greater than that of the parent drug NCS.

image

Figure 4. Enhanced permeability and retention (EPR) effect. (a) A S180 tumor on the skin of a mouse. The tumor shows relatively homogeneous uptake of Evans blue/albumin, but normal skin in the background contains no blue color.[5, 76-79] (b) Heterogeneity of the EPR effect. Only the tumor periphery took up Evans blue/albumin. (c) Blood vessels in normal liver had no leakage of polymer resin. (d) Metastatic tumor nodule (N) in liver, approximately 200 μm in diameter, showed distinct extravasation of polymer resin in small nodules (T). (e) Computed tomography of a patient that shows selective uptake of Lipiodol in a tumor (white area) in the liver that was metastatic (met.) from gastric cancer (ca.): two tumors (arrows) are intensely stained (white) by Lipiodol. Styrene–maleic acid copolymer conjugated with neocarzinostatin/Lipiodol was infused into the hepatic artery under angiotensin II-induced hypertension (see text, and refs [79] and [89]. 60 M, patient, 60 yr old male; SX i.a. AT, SMANCS given via ia route under angiotensin II induced hypertension. (e′) Computed tomography of the same patient approximately 1 month later, showing a considerably reduced tumor size (arrows). Drug retention lasted for more than 1 month. (f) Relationship between radiolabeled polymers of N-(2-hydroxypropyl) methacrylamide (P-HPMA) various molecular sizes and their uptake by tumor, kidney, and liver. The EPR effect depended on time (6 h vs 5 min is shown). conc, concentration. (g) Relationship between the molecular size of drugs and tumor uptake of drug (○), urinary clearance (CL, ●), and area under the concentration versus time curve (AUC) of plasma (▲).[12, 15, 77]

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To understand the mechanism underlying this tumoritropic behavior of biocompatible macromolecules and SMANCS, we carried out additional investigations with biocompatible macromolecules such as albumin (68 kDa), transferrin (90 kDa), and IgG (160 kDa) as well as small proteins (NCS, 13 kDa, and ovomucoids, 29 kDa).[5, 6] The results showed a progressive increase in accumulation of large proteins in solid tumors over time. Concentrations of these proteins in most tumors strikingly exceeded their concentrations in blood. Also, drug accumulation in tumors paralleled the AUC for macromolecules.[5, 12-15] SMANCS (16 kDa), which binds with plasma albumin (resulting in a size of 90 kDa) manifested tumoritropic characteristics, and we proposed a new concept of tumor-selective drug delivery based on the EPR effect (Fig. 4).[5, 6, 15, 69] In this concept, tumor uptake of drugs is not transitory, as observed by angiography for low-molecular-weight contrast agents; instead, tumor tissues show persistent retention of macromolecules for a very long time, for example, many weeks.

To elucidate the EPR effect, we collaborated with Duncan, Ulbrich, and others and used the well-characterized biocompatible P-HPMA, whose size ranged from 4.5 kDa to 800 kDa and had a neutral charge.[12-15] As a putative macromolecular drug, P-HPMA showed progressive accumulation in tumor issue (Fig. 4f). The EPR effect, therefore, results in little delivery of macromolecular drugs to normal tissues and thus fewer systemic toxic effects compared to the delivery of low-molecular-weight drugs,[69, 70] which have no tumor selectivity. Kimura et al. reported that i.v.-injected Bifidobacterium bifidum, with a size >1 μm, accumulated preferably in tumor tissue compared with other normal tissues.[71] Subsequently, we and Skinner et al. showed that polymer resin of acrylamide that was given i.v. extravasated into interstitial tissue of solid tumor.[72-75]

In addition, we observed the EPR effect in small (200-μm diameter) tumor nodules in liver that were metastatic from colon cancer (Fig. 4d).[72-74] Numerous laboratories have now reported on the EPR effect, and as of 2012 more than 12 000 published reports cited our own papers on this matter.[76-79]

Clinical demonstration of vascular permeability and the EPR effect after arterial infusion of SMANCS/Lipiodol

The classic example of the clinical demonstration of the EPR effect may be gallium scintigraphy, in which radioactive 67Ga citrate is injected i.v.[80] We now interpret this finding to indicate that the gallium ion forms a complex with the plasma protein transferrin, thereby becoming a 90-kDa macromolecule in vivo. Consequently, because of the EPR effect, the tumor-selective accumulation of radioactive 67Ga would produce a distinct tumor image in a day or two after i.v. injection, as seen by radioscintigraphy.

We also found that CT obtained striking tumor images after a lipidic radiocontrast agent, Lipiodol® (a product of Laboratoire Guerbet, France, which is iodinated poppy seed oil ester), was injected into the tumor-feeding artery, as discussed blow (Fig. 4e, e′). This result is clearly based on the EPR effect.[1, 75, 77, 82-86]

Shortly after biochemical, physical, and preclinical characterizations of SMANCS were completed, our colleagues at Kumamoto University were quite fascinated with its tumor selectivity and high antitumor potency. With Toshimitsu Konno, M.D., a surgeon, we developed one of the most effective tumor-targeting methods, arterial infusion of SMANCS dissolved in Lipiodol. Because of its high lipophilicity, despite it being a macromolecule, SMANCS could be dissolved in Lipiodol and a homogeneous solution could be obtained. We believed that SMANCS/Lipiodol would penetrate the interstitial tumor tissue directly through the tumor's vascular walls after arterial infusion given into the tumor-feeding artery. This technique was the first theranostic approach (see later), in that the uptake of Lipiodol by the tumor tissue allowed highly sensitive X-ray visualization of the tumor, preferably CT.[75, 81-90]

Quantitative evaluation of uptake by the tumor of 14C-labeled Lipiodol that we synthesized showed the extremely high tumor selectivity of this approach: the tumor Lipiodol concentration was 2000 times higher than that of blood at 15 min after, and more than 3000 times higher at 3 days after intra-arterial infusion.[75, 81] The imaging potential with an X-ray system was thus clear.[82-85] These results were then applied to difficult-to-treat human tumors, specifically hepatoma and other abdominal and renal cancers, with or without angiotensin II-induced hypertension, which augmented drug delivery.[84, 87-89] Tumors such as metastatic liver cancer and cancers of the gallbladder, pancreas, liver, and kidney responded quite well to this treatment.[89] A marked therapeutic effect and diagnostic value, even early detection, were obtained. Also, as a unique result, estimation of the level of drug (SMANCS/Lipiodol) delivered to the tumor using CT became possible (Fig. 4e,e′). This protocol produced very few adverse effects such as bone marrow suppression or anorexia. Also, the prolonged retention of drug in the tumor meant less frequent drug administration (once in 3–4 months) was required, so that patient compliance was quite good.

As an extension of this method, bronchoarterial infusion of ISDN (Nitrol®, Eisai, Tokyo. Japan), which also enhanced tumor blood flow and drug delivery, followed by intra-arterial infusion of SMANCS/Lipiodol for advanced lung cancer gave very encouraging results.[90] Several reports provide descriptions of these results.[1, 22, 76, 77]

Influences on and augmentation of the EPR effect

  1. Top of page
  2. Abstract
  3. Relation of vascular permeability to infection and cancer: Inflammatory mediators in bacterial infection and cancer
  4. Endogenous free radical generation in infection: A significant cause of tissue damage by inline image and mutation
  5. Vascular permeability leading to tumor-targeted drug delivery, lymphotropism, and mechanism of the EPR effect of macromolecules
  6. Influences on and augmentation of the EPR effect
  7. Enhanced permeability and retention (EPR) effect in tumor imaging
  8. Drug uptake by tumor cells and drug release from nanomedicines
  9. Conclusion
  10. Acknowledgments
  11. Disclosure Statement
  12. References

Architectural differences in tumor vasculature

The enhanced vascular permeability of solid tumors depends on two features. One is the microanatomical architecture of tumor blood vessels, which was observed by electron microscopy.[72-74, 91, 92] The tumor vasculature was extremely irregular, for example: the vascular network branched and stretched; endothelial cell–cell junctions had large gaps between them, with pores as large as 4 μm rather than the <10-nm pores of normal vessels[92]; the vascular diameter was larger, with a uniquely irregular shape, and frequently missing pericytes or the smooth muscular layer that surrounds blood vessels; and leakage of acrylic polymer resin occurred, similar to leakage of albumin into the interstitial space, as seen in Figure 4(a,b).[72-74, 76, 77] The second feature concerns vascular mediators, as described below.

Factors that facilitate the EPR effect and artificial augmentation of the effect

Pharmacological factors that facilitate the EPR effect

As described earlier, we began our study of vascular permeability in the bacterial infection and activation of the kallikrein cascade, which resulted in the generation of kinin (Fig. 1).[15-22] This same mechanism was found to occur in cancer tissues.[15, 16, 18, 32-34] We subsequently determined that NO, ONOO, carbon monoxide, prostaglandins, and collagenases among others, are mediators that facilitate the EPR effect (Fig. 3a,c,d),[17, 19, 21, 94] as summarized in Table 1. Recent reviews give good accounts on these issues.[21, 76-79]

Augmentation of the EPR effect

With the above-described knowledge in hand, we continued to develop methods of augmenting the EPR effect for drug delivery to tumors. We first infused angiotensin II i.v.,[12] during the arterial infusion of SMANCS/Lipiodol, to raise the blood pressure, as discussed earlier.[70, 89] This strategy takes advantage of the architectural defects of tumor vessels to make drugs more permeable. The second method used NO-releasing agents such as nitroglycerin, ISDN, and others, which are known to be quite safe.[77, 90, 96] Frequently, the tissue of many tumors is hypoxic compared with normal tissues, similar to infarcted heart tissue. This situation means that denitrase is involved in NO formation by reducing nitrite to NO, as Figure 3(d) shows. This process of NO generation is preferred by hypoxic tissues such as metastatic cancers and other hypoxic cancers of the prostate and pancreas. Yasuda, Jordan, and Mitchell and their colleagues also showed the use of NO-releasing agents to be beneficial in conventional cancer chemotherapy in terms of redox modulation.[98-102]

The third method uses bradykinin-potentiating agents such as inhibitors of angiotensin I-converting enzyme to inhibit kinin degradation in tumor tissue, which would result in a higher kinin level at the site of kinin generation (tumor) (Fig. 1).[19-21, 34, 103] All these methods enhanced the EPR effect and thereby drug delivery by two- to threefold. The limited clinical applications have indicated the potential for delivery of SMANCS/Lipiodol to tumors, as noted in descriptions of methods using elevated blood pressure[89] or ISDN,[90] which warrants further exploration.

Heterogeneity of the EPR effect

The heterogeneity of the EPR effect poses a problem in that some areas of tumor tissue resist the uptake of drugs (of nanomedicines) for both chemotherapy and tumor imaging (see below), and drugs have great difficulty reaching the tumor interstitium.[76-79] However, we demonstrated that augmentation of the EPR effect led to result in a more uniform and enhanced drug delivery.[76-79, 89] Large necrotic areas of tumor (as seen in Fig. 4b vs 4a) did not show uptake of Evans blue/albumin, whereas the EPR effect was more prominent at the tumor periphery, where tumor growth is rapid.[76-79, 89] Angiography revealed that pancreatic and prostate cancers are hypovascular (so, less uptake of the contrast agent occurs). However, even in these hypovascular tumors, angiotensin II-induced hypertension seemed to improve drug delivery to tumors (Fig. 4e,e′).[70, 77, 88]

Enhanced permeability and retention (EPR) effect in tumor imaging

  1. Top of page
  2. Abstract
  3. Relation of vascular permeability to infection and cancer: Inflammatory mediators in bacterial infection and cancer
  4. Endogenous free radical generation in infection: A significant cause of tissue damage by inline image and mutation
  5. Vascular permeability leading to tumor-targeted drug delivery, lymphotropism, and mechanism of the EPR effect of macromolecules
  6. Influences on and augmentation of the EPR effect
  7. Enhanced permeability and retention (EPR) effect in tumor imaging
  8. Drug uptake by tumor cells and drug release from nanomedicines
  9. Conclusion
  10. Acknowledgments
  11. Disclosure Statement
  12. References

We first demonstrated the use of the EPR effect in tumor imaging by injecting Evans blue dye i.v., so that the blue tumor could be visualized (Fig. 4a).[5, 15, 21, 22, 76-79] To make tumor detection more sensitive, we recently developed fluorescent-labeled macromolecules, named fluorescent nanoprobes.[79, 104]

Figure 5(a) compares detection using low-molecular-weight free ZnPP and macromolecular HPMA polymer conjugated with ZnPP. The free low-molecular-weight fluorophore (ZnPP, molecular size 626.0) does not show tumor-selective uptake (Fig. 5a′), whereas the polymer-conjugated ZnPP showed marked tumor-selective uptake and remained in the tumor even after 48 h (Fig. 5a).[104] Another example involves free rhodamine B (molecular size 479.0) versus rhodamine isothiocyanate-conjugated albumin (67 kDa). Here again, the EPR effect-based tumor uptake was demonstrated and was unique for macromolecular probes but not for free rhodamine B or ZnPP (Fig. 5b vs 5b′).[79] These tumor images were obtained by using the in vivo fluorescence detection system IVIS XR (Caliper Life Sciences, Hopkinton, MA, USA) with intact animals. This finding suggests that fluorescent endoscopy for detecting human tumors should be possible. Treatment with the NO-releasing agent nitroglycerin in this model produced a more uniform uptake in the tumor and elevated and prolonged plasma drug concentration, which favor a greater EPR effect (Fig. 5c vs 5c′).

image

Figure 5. (a,b) Staining of tumors (T) with fluorescent nanoprobes and free low-molecular-weight probes. (a) Polymer N-(2-hydroxypropyl) methacrylamide (P-HPMA)-conjugated zinc protoporphyrin ZnPP (micelles). (b) Rhodamine-conjugated BSA. These drugs were given i.v. and show clear tumor-selective fluorescence. The low-molecular-weight fluorescent counterparts, free ZnPP and free rhodamine B (images (a') and (b'), respectively), manifested no tumor-selective fluorescent staining. EPR; enhanced permeability and retention effect. (c) Fluorescence after surgical organ removal, only tumor (T) and blood plasma (P) showed fluorescent staining. (c′) is same as (c) except that this mouse was treated with nitroglycerin (NG). Results here show a more uniform tumor delivery (T) and higher plasma level of the nanoprobes than seen in (c). H, heart; K, kidney; Li, liver; Lu, lung; S, spleen.

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A unique property of ZnPP is that it not only emits fluorescence during endoscopic imaging with xenon light irradiation, it also generates singlet oxygen (1O2), which has cytocidal effects on tumor cells, the result being significant tumor regression and cure in an in vivo model.[104] This theranostic approach was confirmed with ZnPP–SMA micelles in autochthonous breast cancer in Sprague–Dawley rats in vivo.[105]

The term theranostic was coined by Funkhouser in 2002[106] and is becoming quite popular. A recent comprehensive review of this topic can be found in ref. [107].

Drug uptake by tumor cells and drug release from nanomedicines

  1. Top of page
  2. Abstract
  3. Relation of vascular permeability to infection and cancer: Inflammatory mediators in bacterial infection and cancer
  4. Endogenous free radical generation in infection: A significant cause of tissue damage by inline image and mutation
  5. Vascular permeability leading to tumor-targeted drug delivery, lymphotropism, and mechanism of the EPR effect of macromolecules
  6. Influences on and augmentation of the EPR effect
  7. Enhanced permeability and retention (EPR) effect in tumor imaging
  8. Drug uptake by tumor cells and drug release from nanomedicines
  9. Conclusion
  10. Acknowledgments
  11. Disclosure Statement
  12. References

Interactions with the cell surface: Influence of charge and hydrophobicity

The most desirable anticancer agents must ultimately possess properties to overcome various barriers, as given in Figure 6.[79] The first, most crucial barrier is the vascular wall, and the EPR effect plays a key role here. To take advantage of the EPR effect, drugs must have macromolecular characteristics (or nanomedicines), which permit selective extravasation into tumor tissues but not normal tissues (Figs 4, 5).

image

Figure 6. Barriers to targeting of drugs to tumors before the target molecules in tumor cells are reached, from the vascular level to the molecular target at the subcellular level. EPR, enhanced permeability and retention.

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Among other critical steps, cellular internalization of drugs is indispensable and can be a great barrier to therapeutic effectiveness (Fig. 6). A contradictory issue exists in the interaction between nanoparticles and cell surfaces of normal as well as tumor cells. Requisites for the EPR effect include a sustained high concentration of nanomedicines in plasma during circulation that requires less interaction of nanomedicines with surfaces of cells such as vascular endothelial cells, and escape of nanomedicines from clearance by phagocytic cells. In this respect, the “stealth” characteristic of PEGylated and HPMA-polymer conjugates is now known as a favorable feature. Hatakeyama et al. reported, however, that such stealth nanoparticles are poorly taken up by cancer cells.[108] In our laboratory, we compared HPMA-, PEG-, and SMA-conjugated micelles, and found that among these, SMA conjugates had the highest cellular uptake, whereas both PEG- and HPMA-polymer conjugates had much less efficient cellular uptake.[109]

In our earlier studies of SMANCS, we showed that conjugation of SMA conferred far greater cellular uptake, which corresponded to cytotoxicity.[11, 110, 111] That is, more efficient cellular uptake (50 to 100-fold) occurred with the hydrophobic SMA-polymer conjugate (SMANCS) than with the parental NCS, and more potent cytotoxicity (20 to 100-fold) was observed in a weakly acidic environment, as in tumors, than in the neutral pH of normal tissues. In the weakly acidic setting, the protonated (COOH) form of the maleyl residue in SMA has stronger hydrophobicity and a higher affinity for cell membranes than does the ionized COO form of SMA (see Fig. 7b vs 7b′).[11, 110, 111] Also, many cell surfaces are negatively charged, so that interaction with negatively charged nanoparticles is repelled, which results in less cellular uptake. Maleyl carboxyl residues would therefore provide a pH-sensing property in the tumor environment.

image

Figure 7. Chemical structure of styrene–maleic acid copolymer conjugated-neocarzinostatin (SMANCS), and the styrene–maleic acid copolymer (SMA) residue as a pH sensor and lipophilicity enhancer. (a) Chemical structure of SMANCS, which consists of a protein portion of neocarzinostatin (NCS) and two chains of SMA copolymers linked at the N-terminal alanine and at lysine 20. (b,b′) Close-up views of the SMA unit with styrene and maleyl residues, in which the maleyl carboxyl group has the role of a pH sensor. In acidic pH (b′), the R-COOH of maleyl residues becomes to COOH, which possesses higher lipophilicity than does the COO form. SMANCS would thus have greater cell-binding affinity, a more than 10 to 100-fold higher cellular uptake in weakly acidic pH, with cytotoxicity increasing in parallel. (b) At neutral or higher pH of normal tissues, deprotonation occurs, with formation of the negatively charged R-COO and more hydrophilicity of SMANCS.[109-112] Cell interaction is thus impeded and internalization into cells is lower. buSMA indicates the n-butylated ester form of maleyl residues in SMA, in which approximately 37 mol% maleyl residues of SMA are replaced for proton and the remaining carboxyl residues are free.

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These results show that hydrophobicity and charge are important for the cell-binding property. However, this hydrophobic feature should be carefully controlled, or hemolysis or cell lysis may be induced.[112] This hydrophobic property of cell lysis and strong anionic nature would also cause rapid uptake by the liver or spleen. These effects may be another drawback, but they can be controlled by proper modification of the carboxyl group (Tsukigawa K and Maeda H, unpublished data).

Drug release from drug complexes or carriers

Release of drugs from nanoparticles is another critical step for tumor-selective drug delivery. We found that HPMA–ZnPP and SMA–ZnPP micelles, for example, disintegrated during endocytosis, not during circulation, and that disintegration in the cell made the drug more accessible to the target molecules.[109] A similar phenomenon of disruption of micelles was seen after treatment with lecithin or detergent.[109] Many researchers conjugated active ingredients to the polymers using specific protease-cleavable peptides with preferred amino acid sequences, or ester or other chemical bonds.[113] For example, SMA was conjugated to NCS by amide bonds in SMANCS, and the maleyl amide underwent spontaneous hydrolysis in acidic pH. In addition, the hydrazone linker bond between the polymer and ZnPP spontaneously released ZnPP in the weakly acidic pH of tumor tissues (Nakamura H, Subr V, Ulbrich K, Maeda H, unpublished data, 2013). We also found disruption of an SMA–cisplatin complex on endocytosis or incubation at weakly acidic pH was 6–7-fold faster than that at neutral pH (Saisyo A, Maeda H, and Nakamura H, unpublished data, 2013). Also, micelles or liposomes should be stable during circulation but release the drug as it arrives at the tumor site is needed. Many improved ways to control the release of drugs from conjugates or complexes as based on the condition of the tumor environment are thus anticipated.

Conclusion

  1. Top of page
  2. Abstract
  3. Relation of vascular permeability to infection and cancer: Inflammatory mediators in bacterial infection and cancer
  4. Endogenous free radical generation in infection: A significant cause of tissue damage by inline image and mutation
  5. Vascular permeability leading to tumor-targeted drug delivery, lymphotropism, and mechanism of the EPR effect of macromolecules
  6. Influences on and augmentation of the EPR effect
  7. Enhanced permeability and retention (EPR) effect in tumor imaging
  8. Drug uptake by tumor cells and drug release from nanomedicines
  9. Conclusion
  10. Acknowledgments
  11. Disclosure Statement
  12. References

The vascular permeability of infected, inflamed, and tumor tissues results from multiple factors such as vascular mediators listed in Table 1, and architectural defects in tumor vessels, as described earlier. This phenomenon occurs especially with macromolecules and nanoparticles, but tumor tissue manifests great differences in this phenomenon, in that it tends to retain macromolecules in the tissue interstitium for far longer than does normal inflamed tissue. We named this phenomenon the EPR effect of macromolecules in cancer. SMANCS (Fig. 7a) was the first polymer conjugate that we developed that possesses the EPR effect.[69, 77, 79, 84, 89]

Different mechanisms participate in the generation of ROS and RNS in infected, inflamed, and cancer tissues. These mechanisms include ONOO, a product of inline imageand NO, which is one of the most potent oxidizing, nitrating, and DNA/RNA-cleaving molecules that is involved in mutagenesis, drug resistance, carcinogenesis, vascular permeability, and tumor metastasis (Fig. 3).

We previously demonstrated the EPR effect using Evans blue (Fig. 4a,b), but it can also be visualized by using fluorescent probe-labeled macromolecules for tumor imaging in vivo (Fig. 5). However, the EPR effect as seen with Evans blue albumin staining is heterogeneous (Fig. 4b), which may impede uniform macromolecular drug delivery. This poor drug delivery to an area of a tumor with an apparently low EPR effect may be augmented by raising systemic blood pressure using slow infusion of angiotensin II or prodrugs of vascular mediators such as nitroglycerin.

To achieve efficient nanomedicine drug delivery to tumors on the basis of the EPR effect, a number of barriers, such as molecular size, surface charge, hydrophobicity, and drug release, must be overcome (Fig. 6). Differences between the environments of tumor tissues and normal tissues can be exploited to achieve greater tumor-selective drug release at the cellular level; examples include the weakly acidic pH of tumor tissues by using the hydrazone bond, and by using tumor-secreted proteases such as cathepsin or collagenases to cleave linker peptides. All these effects are more important in vivo than in cell-free systems or at the molecular level.

We are now working on endoscopic detection of tumors at a very early stage by using light irradiation of tumors to generate ROS, that is, phonon-generated singlet oxygen, as the active principle. We have developed fluorescent nanoprobes such as polymer-bound ZnPP that show tumor-selective accumulation and that, together with tumor-selective generation of ROS, will kill tumor cells in situ.[104] Such a theranostic approach will provide a highly tumor-selective therapeutic method to achieve the least invasive and most patient-friendly cancer treatment.

Abbreviations
AUC

area under the concentration versus time curve

CT

computed tomography

EPR

enhanced permeability and retention

HPMA

N-(2-hydroxypropyl) methacrylamide

ISDN

isosorbide dinitrate

NCS

neocarzinostatin

NO

nitric oxide

NOS

nitric oxide synthase

Nox

NADPH oxidase

inline image

superoxide anion radical

ONOO

peroxynitrite

P-HPMA

polymer of N-(2-hydroxypropyl) methacrylamide

Pyran copolymer

copolymer of divinylether-maleic acid

RNS

reactive nitrogen species

ROS

reactive oxygen species

SMA

styrene–maleic acid copolymer

SMANCS

styrene–maleic acid copolymer conjugated with neocarzinostatin

SOD

superoxide dismutase

XO

xanthine oxidase

ZnPP

zinc protoporphyrin

References

  1. Top of page
  2. Abstract
  3. Relation of vascular permeability to infection and cancer: Inflammatory mediators in bacterial infection and cancer
  4. Endogenous free radical generation in infection: A significant cause of tissue damage by inline image and mutation
  5. Vascular permeability leading to tumor-targeted drug delivery, lymphotropism, and mechanism of the EPR effect of macromolecules
  6. Influences on and augmentation of the EPR effect
  7. Enhanced permeability and retention (EPR) effect in tumor imaging
  8. Drug uptake by tumor cells and drug release from nanomedicines
  9. Conclusion
  10. Acknowledgments
  11. Disclosure Statement
  12. References
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