The Enhanced Permeability and Retention (EPR) Effect: The Significance of the Concept and Methods to Enhance Its Application. — TEI-tagged view

Jun WupersName (2021date). “The Enhanced Permeability and Retention (EPR) Effect: The Significance of the Concept and Methods to Enhance Its Application..” Journal of Personalized Medicinetitle·j.

Object of study — the pathophysiology of solid tumours that governs drug deliveryterm·key

Research question — why the EPR effectterm·dom matters and how it can be harnessed better in nanomedicineterm·key

Contribution — places clinical failures on the pegylationterm of liposomal drugs rather than on the EPR effectterm·dom

Full text — tagged

The Enhanced Permeability and Retention (EPR) Effect: The Significance of the Concept and Methods to Enhance Its Application.

Abstract Chemotherapy for human solid tumorsterm·key in clinical practice is far from satisfactory. Despite the discovery and synthesis of hundreds of thousands of anticancer compounds targeting various crucial units in cancer cell proliferation and metabolism, the fundamental problem is the lack of targeting delivery of these compounds selectively into solid tumorterm·key tissue to maintain an effective concentration level for a certain length of time for drug-tumor interaction to execute anticancer activities. The enhanced permeability and retention effectterm·dom (EPR effectterm·dom) describes a universal pathophysiological phenomenon and mechanism in which macromolecular compounds such as albumin and other polymer-conjugated drugs beyond certain sizes (above 40 kDa) can progressively accumulate in the tumor vascularized area and thus achieve targeting delivery and retention of anticancer compounds into solid tumorterm·key tissue. Targeting therapy via the EPR effectterm·dom in clinical practice is not always successful since the strength of the EPR effectterm·dom varies depending on the type and location of tumors, status of blood perfusion in tumors, and the physical-chemical properties of macromolecular anticancer agents. This reviewterm·mth highlights the significance of the concept and mechanism of the EPR effectterm·dom and discusses methods for better utilizing the EPR effectterm·dom in developing smarter macromolecular nanomedicineterm·key to achieve a satisfactory outcome in clinical applications.

Keywords: EPR effectterm·dom, nanomedicineterm·key, drug deliveryterm·key, arterial infusionterm·key, canine cancerterm·key 1. Introduction It is crucial to understand the pathophysiological characteristics of solid tumorterm·key growth, especially the compound transportation regulation of the tumor vasculature, in order to achieve selective drug deliveryterm·key and therapeutic effects for cancer chemotherapy. It has been well observed that tumor vessels are highly permeable to macromolecular compounds. After entering tumor tissue, these macromolecular compounds are trapped inside the tumor tissue for a prolonged period of time. In 1986, Hiroshi Maeda and his colleagues from Kumamoto University School of Medicine coined the term enhanced permeability and retention effectterm·dom (the EPR effectterm·dom) to describe the unique pathophysiological phenomenon of the solid tumorterm·key vasculature [ 1 ]. Since this theory is very important for understanding tumor vessel transportation regulation, the EPR effectterm·dom has been well accepted as one of the universal pathophysiological characteristics of solid tumorsterm·key, and acts as a fundamental principle for designing and developing tumor-targeting delivery of anticancer drugs [ 2 , 3 ]. However, the development of nanomedicineterm·key has been frustrated for decades in achieving satisfactory therapeutic benefits in clinical practice. Therefore, the existence and intensity of the EPR effectterm·dom in human solid tumorterm·key circumstances has been debated [ 4 , 5 ]. For example, it is considered that the EPR effectterm·dom is more significant in experimental small animal tumor models than in human tumors. The delivery efficiency of nanoparticles into human tumor tissue is very low compared to that in animal tumor models. The extravasation mechanism for nanoparticles into tumors is not only via the gaps between endothelial cells in the tumor vasculature, but also via the transcellular pathways by vesiculo-vacuolar organelles (VVOs) [ 6 ]. Therefore, it is crucial to recognize the significance of the EPR effectsterm·dom, its pathophysiological mechanism, its pitfalls, and strategies for better harnessing this concept in drug development and clinical application.

2. The EPR Effectterm·dom: The Universal Pathophysiological Phenomena in Rodents, Other Mammalian Animals and Human Solid Tumorsterm·key The EPR effectterm·dom has been well observed and documented in solid tumorsterm·key of rodents, rabbits, canines, and human patients [ 1 , 2 , 3 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. It is based on several pathophysiological characteristics of solid tumorsterm·key: (1) Massive irregular neovascularization in tumors with structural and functional abnormalities in tumor blood vessels. To meet urgent demands for nutrient and oxygen supplies, the tumor vasculature is very dense and tortuous, with deficient basement membranes and fenestrated structures of endothelial tubes in some immature vessels. The pericytes and smooth muscle cells surrounding tumor blood vessels are either deficient or malfunctional in smooth muscle alpha actin when responding to blood pressure regulation stimuli [ 14 , 15 , 16 ]. Recent studies have found that the gaps between endothelial cells in tumor vessels are at low frequency, and the transendothelial pathways are the dominant mechanism of nanoparticle extravasation in tumors [ 17 ]. This is consistent with the previous observation that macromoleculesterm·key are highly permeable in the mature veins or venules, constructed by a continuous endothelium with closed interendothelial cell junctions [ 18 ]. These structures render them highly permeable to nutrients, especially macromoleculesterm·key, to be extravasated from tumor blood vessels into the interstitial space of tumor tissue.

(2) Elevated expression of inflammatory factors such as prostaglandins, bradykinin, nitric oxide, peroxynitrite, interleukin 1 beta, interleukin 2, interleukin 6, proteases, interferon gamma, VEGF and HIF−1 alpha. All these factors coordinate in solid tumorterm·key tissues and sustain the EPR effectterm·dom [ 7 , 19 , 20 , 21 ].

(3) Lack of efficient drainage of lymphatic systems in solid tumorterm·key tissue. This deficiency results in the retention of extravasated macromoleculesterm·key in tumor tissues, which provides the opportunity for passive targeting delivery of macromolecular anticancer drugs [ 1 , 7 , 22 ].

3. The Significance and Challenges in Concept and Application of the EPR Effectterm·dom in Human Cancer Therapy One of the arguments for the EPR effectterm·dom concerns the roles of interstitial fluid pressureterm·dom [ 23 , 24 ] and solid stressterm·dom [ 25 ] in solid tumorterm·key tissue. The interstitial fluid pressureterm·dom and solid stressterm·dom exist due to the expansion of the tumor mass against surrounding normal tissue. Different from interstitial fluid pressureterm·dom, the solid stressterm·dom in tumors is considered to be residual stress that compresses blood vessels in tumor tissues, causing hypoxia and impeding drug deliveryterm·key [ 26 ]. It is believed that interstitial fluid pressureterm·dom and solid stressterm·dom are the major obstacles preventing efficient delivery of macromoleculesterm·key into tumor tissue [ 27 ]. However, we have seen tremendous evidence that macromoleculesterm·key do accumulate in both rodent and human solid tumorterm·key tissues in size-dependent and time-dependent manners. Interstitial fluid pressureterm·dom or solid stressterm·dom hinders drug penetration into the center of tumor tissue, but it does not prevent the macromolecular agents from extravasating and accumulating in the peripheral area of tumor tissue. The EPR effectterm·dom occurs primarily in the peritumoral area [ 28 ]. Interstitial fluid pressureterm·dom and solid stressterm·dom provide the mechanism for the retention effect because under such pressure or stress the formation of functional lymphatic vessels is prevented due to their collapse from the pressure [ 22 ]. The interstitial fluid pressureterm·dom and heterogeneous blood supply are both observed in rodent and human solid tumorsterm·key; therefore, these are not valid reasons for rebuttals that assume the EPR effectterm·dom won’t function in human solid tumorterm·key tissue.

It is critical to understand and remember that the peripheral highly vascularized area is the most vigorously growing zone of tumors. The center of tumor tissues lacks blood flowterm·key and is necrotic or seminecrotic [ 28 ]. When suppressing the growing activities in peritumoral zones, tumors are restricted or eliminated. It is not necessary for an anticancer drug to penetrate into the center of a solid tumorterm·key to execute anticancer activities. For example, trastuzumab (Herceptin) is an antibody that is successful in treating Her2 positive breast cancer growth, and penetrates only into the vascularized area [ 29 ].

There are limited examples of successful nanomedicineterm·key treatments in cancer therapy. This is one of the biggest challenges for the application of the EPR effectterm·dom in nanomedicineterm·key design and clinical practice. Some doubt the usefulness of the EPR effectterm·dom in clinical practice by using the example of Doxil, a polyethylene glycol (PEG) modified-liposome formulation of doxorubicin, which does not appear to significantly improve the benefits of solid tumorterm·key treatment compared with parental free drug doxorubicin [ 4 , 5 ]. Pegylated liposomal doxorubicin does have a significantly longer half-life in blood circulation in patients and achieves about a 10~15-fold higher concentration in tumor tissues compared with surrounding normal tissues [ 30 ], which indicates that pegylated liposomal doxorubicin does accumulate into tumor tissue by the EPR effectterm·dom. In a Phase III clinical trial, an overall response rate of 45.9% was achieved [ 31 ]. Pegylated liposomal doxorubicin exhibits better therapeutic benefit than that of free doxorubicin; however, the overall therapeutic outcome is still not satisfactory. The problem of therapeutic efficacy is probably due to compromised tumor cell killing properties by the pegylationterm of liposomes. An in vitro study found that pegylated liposomal doxorubicin had almost no cytotoxicity effect in the first 24 h, and only achieved about 12% cytotoxicity at 48 h in a colon cancer cell line HT29 [ 32 ]. Pegylationterm of liposomes significantly reduced the drug release rateterm, and also significantly reduced the cytotoxicity potency (25% vs. 75%) of anastrozole when compared with the free drug at the 72 h time point [ 33 ]. In another similar case, pegylationterm of liposomal cisplatin drastically decreased cytotoxic potency by increasing cytotoxicity IC50 from 2 µg/mL (free cisplatin) to 40 µg/mL (pegylated liposomal cisplatin) when compared to that at the 48 h time point [ 34 ]. Therefore, it is not the EPR effectterm·dom that failed in clinical trials but the pegylationterm of liposomal chemo drugs that failed to achieve satisfactory cytotoxicity efficacy within 48-h period in clinical application.

The abnormality of tumor blood vessels obstructs the blood flowterm·key into tumor tissue. Since the tumor vascular formation is mainly attributed to the effects of vascular endothelial growth factor (VEGF), the application of VEGF antibodies antagonizes the effect of VEGF and temporarily normalizes tumor vasculature [ 35 ]. The tumor vascular normalization increased the uptake and penetration of fluorescent-labeled bovine serum albumin into tumor tissue, indicating that normalization of the tumor vasculature could increase the uptake of small particles (less than 20 nm in diameter) into tumor tissue, but hindered the uptake of larger particles above 125 nm in diameter [ 36 ]. The procedure of normalization could hamper the EPR effectterm·dom because it decreases the permeability of large particles crossing the tumor vessel by reducing the gaps between endothelial cells in the tumor vessels. Many permeability factors like bradykinin, nitric oxide, prostaglandins are produced by infiltrating inflammatory cells and these factors may not be “corrected” by the anti-VEGF normalizing strategy. Thus, when attempting to normalize tumor vessels to improve the delivery of nanomedicineterm·key, the size of nanoparticles, the timing order of drug administration and vascular normalization could be critical to achieving the desired delivery results [ 36 ]. The normalization effect is therefore transient, limited, and highly heterogeneous in various tumor types or tumor locations. It should be combined with other modulation approaches such as hyperthermia, radiotherapy and sonoporation to enhance the EPR effectterm·dom when applying vascular normalization [ 37 ].

4. Potential Solutions for Improving EPR Effect-Based Nanomedicineterm·key in Human Cancer Therapy 4.1. Better Design of Drug and Combination with EPR Effectterm·dom Enhancing Modulators The extent of the EPR effectterm·dom varies between small animal tumors and human tumors by types and locations. To better utilize the EPR effectterm·dom for human tumor therapy, the design of nanomedicineterm·key should be improved.

Size and physical-chemical properties such as surface charge and spatial configuration are crucial for a drug to achieve the EPR effectterm·dom. Studies using serial molecular sizes of HPMA copolymers in solid tumorterm·key animal models indicated that the threshold of macromolecular molecules (drugs) to be retained and accumulated in tumor tissue is above 40 kDa [ 38 ]. Nanoparticles in the range of 100~200 nm are considered the optimal size for achieving the EPR effectterm·dom in solid tumorsterm·key while escaping the filtration traps of the liver and spleen [ 39 ]. Negative or neutral surface charges are also important for achieving excellent plasma half-lives longer than several hours in circulation in order to be accumulated in tumor tissue. Particles in worm-like shapes such as ellipsoidal, cylindrical and discoidal shapes, or filomicelles, can achieve better accumulation results within tumors [ 40 ]. Deformability and degradability are also important for a smarter drug to be released in the right environmental condition to execute a tumor-killing effect once it enters the tumor tissue [ 39 , 40 ]. As an example, HPMA copolymer-conjugated pirarubicin achieved very promising clinical therapeutic results in a patient with stage IV prostate cancer with extensive metastasis [ 41 ].

On the other hand, the delivery of macromolecular drugs can be enhanced by temporarily modulating the EPR effectterm·dom in the targeted tumor tissues, such as applying adjuvants like nitric oxide donors to enhance the EPR effectterm·dom to facilitate the drug deliveryterm·key into tumor tissue. As mentioned before, many other inflammatory factors involved in the EPR effectterm·dom can be utilized to modulate the intensity of this effect to facilitate drug extravasation, accumulation, and penetration into tumor tissue [ 42 , 43 , 44 , 45 , 46 ]. The EPR effectterm·dom can also be markedly enhanced by photo-immunotherapy with antibody-photosensitizer conjugate pretreatment to achieve up to a 24-fold greater accumulation of nanomedicinesterm·key in tumors. Such significant enhancement has been termed the super-enhanced permeability and retention effect [ 47 ].

4.2. Improving EPR Effect-Based Nanomedicineterm·key by Enhancing Blood Flowterm·key in Solid Tumorterm·key during Drug Administration Blood flowterm·key in solid tumorsterm·key is critical for the success of nanomedicineterm·key delivery via the EPR effectterm·dom [ 44 , 46 , 48 , 49 , 50 , 51 ]. However, it is usually overlooked. One of the major differences between rodent solid tumorsterm·key and human solid tumorsterm·key is blood flowterm·key rate. Generally, the blood flowterm·key rate is about 800-fold higher in human normal organs than in mouse normal organs. For example, the normal flow rate for a mouse normal liver is about 1.8 mL/100 g/min, but it is about 1450 mL/100 g/mL in a typical human normal liver. The blood flowterm·key in mouse muscle is about 0.91 mL/100 g/mL, but it is about 750 mL/100 g/mL in a typical human muscle [ 52 ]. In 6 C3 HED lymphosarcoma in C3 H mice, the blood flowterm·key in large tumors was about 5.4 mL/100 g/min [ 51 ] while in human breast tumors, the mean blood flowterm·key was about 30~64.8 mL/100 g/min [ 53 ]. Higher blood flowterm·key means higher shear force in blood vessels and quicker wash off. The difference in blood flowterm·key rate between normal tissue and tumor tissue in rodents and humans may be important to explain why nanomedicinesterm·key can accumulate better in rodent solid tumorsterm·key than in human solid tumorsterm·key.

The application of angiotensin IIterm could be very efficient for drug deliveryterm·key via increasing the blood flowterm·key into stagnated tumor blood vessels. Hori et al., demonstrated that when applying angiotensin IIterm in rodent or human subjects, the blood flow in tumorsterm was selectively increased up to 5.7-fold without increasing the blood flowterm·key in normal tissue [ 15 ]. This is because the systemic blood pressure increased but the tumor blood vessels remain relaxed due to a lack of response to angiotensin IIterm. Such increases in blood flow in tumorterm tissue greatly improved the perfusion of blood into tumor tissue to achieve a higher magnitude of delivery of anticancer drugs into the solid tumorterm·key tissue [ 15 , 16 ]. Hori and his colleagues further demonstrated that tumor blood flowterm·key fluctuates due to circadian regulation. Tumor blood flowterm·key is increased during the night and the tumor doubling rate is also higher during the night. When they administered anticancer drugs during the night, the therapeutic efficacy was significantly improved [ 54 ]. Such brilliant discoveries have yet to be broadly recognized and applied in clinic settings by the nanomedicineterm·key drug developers and clinical oncologists.

4.3. Improving EPR Effect-Based Nanomedicineterm·key Therapeutic Effect by Arterial Infusionterm·key via Tumor Feeding Artery Administration of nanomedicinesterm·key via intravenous infusion could be problematic to achieve the desired amount of drug into tumor tissue because of high shear force to the endothelial wall brought by fast blood flowterm·key, as we discussed above. However, when administering nanomedicinesterm·key via the tumor feeding artery, the strong blood flowterm·key brings more nanomedicineterm·key into tumor tissues if the size and stickiness of the nanomedicineterm·key are right for blood vessels in tumors. There are tremendous successful reports about using lipiodol for delivering SMANCS and other anticancer drugs [ 12 , 55 , 56 ] by infusion via the tumor feeding artery. However, very few other nanomedicinesterm·key are designed for arterial infusionterm·key to solid tumorsterm·key. It should be noted that nowadays imaging-guided catheter interventional therapy for solid tumorsterm·key is very popular in clinics around the world. Radiologists are very skillful in performing interventional therapy via a tumor-feeding artery; however, the optimal nanomedicineterm·key fit for such arterial infusionterm·key is rarely available. Current low-molecular-weight drugs such as cisplatin for tumor arterial infusionterm·key are simply suspended in lipid vehicle solution. The drugs diffuse and wash out like they would in common intravenous infusion. They cannot achieve the retention effect in tumor tissue, which results in low therapeutic efficacy.

4.4. Improving EPR Effect-Based Nanomedicineterm·key Preclinical Development by Using Large Animal Tumor Models The last but not least critical issue is the selection of better animal models for the preclinical development of nanomedicineterm·key. Most products of nanomedicineterm·key are developed in small rodent tumor models. The tumors in mice are either induced by carcinogens or created by genetic engineering such as knocking in or knocking out certain genes that are related to the tumor initiation. Transplantation of a tumor from donor to recipient is another major way to create syngeneic tumor models, or xenograft tumor models, by established cell lines or fresh tumor tissues. Patient-derived xenograft (PDX) models have featured in preclinical studies in recent years. However, there is a vast difference in size between mice and humans, and thus the drug absorption, metabolism, distribution and exclusion profiles, as well as the pharmacokinetic and pharmacodynamic properties of the drugs in tumor tissue, would be very different in mice and humans. The tumors induced by cell lines or tumor tissue might also result in extreme growth behavior, such as an extraordinarily large size or an abnormally fast-growing pattern that is rarely seen in human tumors. Although rodent solid tumorsterm·key exhibit excellent results of the EPR effectterm·dom, the strength of the EPR effectterm·dom could be very different compared to that in solid tumorsterm·key of human patients. Thus, the EPR effectterm·dom of a nanomedicineterm·key candidate measured in rodent tumor models might not be correctly estimated for translating into human clinical application. Canine cancersterm·key are naturally occurring with full-spectrum heterogeneity of tumor cell populations, bona fide tumor microenvironments and spatial structures that faithfully reflect the intrinsic status of blood flowterm·key and interstitial pressures of tumors. Studies show that copper−64 liposomes exhibited excellent permeability and retention (tumor uptake levels at 24 h after injection achieved 3.68-fold higher than at 1 h after injection) in different canine cancersterm·key at various locations such as mammary glands, neck muscle, front paw and intranasal regions [ 13 ]. Using larger animals such as canine cancerterm·key models is, therefore, better for guiding the preclinical development of nanomedicineterm·key. Unfortunately, there are few publications featuring utilization of canine cancerterm·key models for nanomedicineterm·key development. Companion animals as translational models can provide more accurate measurements of therapeutic efficacy based on the EPR effectterm·dom and, therefore, should be considered as major animal models for the development of nanomedicineterm·key.

5. Conclusions The EPR effectterm·dom is the fundamental pathophysiological phenomenon of solid tumorsterm·key universally observed in solid tumorsterm·key in rodents and humans, as well as in other mammalian species. It is also the guiding principle for developing nanomedicineterm·key (including polymer-conjugated macromolecular anticancer drugs) aimed at passive and progressive drug deliveryterm·key and retention inside the tumor tissue to achieve selective and highly efficient anticancer outcomes. However due to the heterogeneous strength in various microenvironment situations, the EPR effectterm·dom has been challenged for its existence and importance in nanomedicineterm·key design and application. As a matter of fact, the EPR effectterm·dom has been observed in human tumor tissues for various macromolecular compounds. When discussing the heterogeneity of the EPR effectterm·dom, it should be clear that the heterogeneity is confined to the strength of the accumulation and retention of the EPR effectterm·dom that varies in different types of solid tumorsterm·key under various tissue environments.

The real challenge is how to utilize the EPR effectterm·dom in designing and improving the therapeutic efficacy of nanomedicineterm·key. There are several enhancing strategies to improve delivery and accumulation efficacies, such as optimal size and surface charge, smarter mechanism for drug release and administration kinetics. Due to their rigid structure property, the accumulation performance of nanoparticles in tumor tissues may not necessarily be the same as with other macromoleculesterm·key such as linear polymers and biological macromoleculesterm·key such as albumins. Therefore, further modification of nanoparticles with polymers to improve the affinity of the nanoparticles with tumor related endothelial cells may be necessary.

Blood flowterm·key plays a critical role in delivering nanomedicinesterm·key into tumor tissues. Arterial infusionterm·key via a tumor-feeding artery, and the timing of using tumor blood flowterm·key enhancers or EPR effectterm·dom modulators should be applied to nanomedicineterm·key to achieve better therapeutic effects. As the strength of the EPR effectterm·dom is quite different between small animal tumor models and human tumors, the selection of big animal models is also critical for guiding the design of nanomedicineterm·key by properly estimating the efficacy of tumor-targeting delivery via the EPR effectterm·dom. Companion animal tumor models such as canine cancerterm·key should be utilized to guide the development of nanomedicineterm·key.

When considering the EPR effectterm·dom, retention efficacy is crucial because for a drug to execute anticancer activities it should maintain above a certain effective concentration level for a certain length of time in the tumor tissue to achieve a satisfactory result.

A better future of nanomedicineterm·key via the EPR effectterm·dom is yet to come.

Acknowledgments The author thanks Jim Isbell and Yingyan Wu.

Funding This research received no external funding.

Institutional Reviewterm·mth Board Statement Not applicable.

Informed Consent Statement Not applicable.

Data Availability Statement Not applicable.

Conflicts of Interest The authors declare no conflict of interest.

Footnotes References Associated Data Data Availability Statement Not applicable.