The link between infection and cancer: tumor vasculature, free radicals, and drug delivery to tumors via the EPR effect

Abstract

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 (O₂⁻), 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 O₂⁻ 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 O₂⁻ 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.

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.

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 (${\mathrm{O}}_2^{-}$). (c) iNOS activity. (d) Induction of PCR‐detectable iNOS mRNA. (e) Amount of ${\mathrm{O}}_2^{-}$ 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.

Figure 3

Generation of free radicals in infection and cancer. (a) Nitric oxide synthase (NOS) can generate nitric oxide (NO) and superoxide (${\mathrm{O}}_2^{-}$), and then peroxynitrite (ONOO ⁻) can nitrate guanine (→ 8‐nitroguanine), and 8‐nitroguanine (NitroGuo) can become a substrate of NOS or cytochrome c reductase, thereby generating ${\mathrm{O}}_2^{-}$. The total system thus works as a progressive reaction, with a stoichiometry of greater than 1:1. (b) Generation of ${\mathrm{O}}_2^{-}$ from heterocyclic amine (HCA) in the presence of cytochrome (Cyt.) P450 reductase and NADPH, which results in DNA damage or cleavage and mutation.53, 54, 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

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, 77, 78, 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

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.

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.

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, 110, 111, 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.