Tumour vasculature targeted anti-cancer therapy

© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Tumour vasculature targeted anti-cancer therapy


ANGIOGENESIS
In general, there is an efficient vascular network that supplies blood to normal tissues. The hierarchal architecture and growth of blood vessels are maintained by the balance between pro-apoptotic and anti-apoptotic factors. This balance is controlled by the metabolic demands of the corresponding tissue. Lymphatic channels on the other hand, remove metabolic waste from the interstitium. Thus, the microstructure of the vascular network is capable of supplying adequate oxygen and nutrition to all associated cells [1] . During tumour progression, there is rapid proliferation of tumour tissue. When the tumour reaches a critical size (1~2 mm 3 ), tumour cells located further from the supplying blood vessel become starved of oxygen and nutrients, leading to the impairment of tumour growth by apoptosis or necrosis. In turn, this triggers angiogenesis, the formation of new blood vessels from existing ones [2] . Although tumour angiogenesis provides for tumour growth and a route for metastasis, it is not as tightly regulated as embryonic angiogenesis [2] .

DIFFERENCES BETWEEN BLOOD VESSELS OF NORMAL AND CANCER TISSUES
The growth of tumour blood vessels does not follow any hierarchy. It is typically heterogeneous, tortuous, branches irregularly, and is enlarged circumferentially [3][4][5] . The endothelial cells, pericytes (multifunctional mural cells that wrap around endothelial cells) and basement membrane of tumour blood vessels are all abnormal [3] : endothelial cells have abnormally loose intracellular associations and focal intercellular openings that are < 2 µm in diameter [6] while their association with multiple layers of the vascular basement membrane is also loose due to high interstitial pressure, leading to hyper-permeable tumour blood vessels and vascular leakage [7] .
Tumour blood vessels also have a reduced surface area: volume ratio. The high interstitial pressure, coupled with a reduced surface area, impairs the delivery of oxygen, nutrients, and removal of metabolites. As such, the tumour microenvironment is typically characterized by hypoxia and acidosis which in turn, selects for apoptosis-resistant and metastasis competent tumour cells [ Figure 1].

CELL SIGNALLING PATHWAYS IN HYPOXIA-INDUCED ANGIOGENESIS
Cell signaling pathways in hypoxia-induced angiogenesis is shown in Figure 2. HIF-1α is the founding member of the hypoxia-induced factor (HIF) family [8] . It regulates the genes associated with oxygen deprivation [9] . The HIF activity pathway is regulated by prolyl hydroxylase enzymes (PHD1-3) [10] . PHD acts as an oxygen sensor; in normoxia, PHD hydroxylates the proline residues of HIF-1α. The hydroxylated HIF-1α then binds to the von Hippel-Lindau E3 ubiquitin ligase complex leading to proteasomal degradation of HIF-1α [11,12] . Under hypoxic conditions, oxygen and cofactor 2-oxo-glutarate substrates are depleted [13] and PHD becomes inactivated, resulting in stabilization and intracellular accumulation of HIF-1α. HIF-1α is then translocated into the nucleus to bind with transcriptional factor Arnt (Aryl hydrocarbon nuclear translocator family protein) [14] . Subsequently, a transcriptional complex is formed with p300/ CBP which binds to HREs (hypoxia response elements) in the promoters and enhancers of target genes, leading to vasodilatation (for better delivery of oxygen), lowering of oxygen demand and upregulation of proangiogenic factors like fibroblast growth factor (FGF), insulin-like growth factor (IGF), and vascular endothelial growth factor (VEGF) [15] . Vasodilatation is also caused by the upregulation of inducible nitric oxide synthase leading to increased production of nitric oxide and relaxation of vascular smooth muscle cells [16] . Under hypoxic conditions, the demand for oxygen is lowered due to over expression of glucose transporter 1 enzyme (GLUT1). GLUT1 improves the uptake of glucose [17] and induces glycolytic enzymes such as phosphoglycerate kinase [18] . In turn, phosphoglycerate kinase is regulated by aldolase A and HIF-α. Aldolase A helps in better utilization of glycolysis, tumour epithelium mesenchymal cell proliferation [19] and upregulation of pyruvate dehydrogenase kinase (PKD1) which inhibits mitochondrial respiration [20] . HIF-1α helps in cancer cell proliferation [21] by regulating the expression of a number of proangiogenic genes like VEGF, Ang-1, Tie 2, platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), monocyte chemoattractant protein-1 (MCP-1), IGF and epidermal growth factor (EDGF). These HIF regulated factors bind to corresponding receptors on the cell membranes of pericytes and increase vascular permeability, endothelial cell proliferation, sprouting, migration, adhesion, and tube formation. The angiogenic factors, their corresponding receptors, and functions are shown in Table 1. Vascular permeability is increased due to overexpression of VEGF [22][23][24][25] . In endothelial cells and pericytes, Ang-1 (angiopoietin-1) is induced by hypoxia. It is a Tie-2 receptor agonist which recruits pericytes to mature vessels and promotes tumour angiogenesis [22] . Despite active angiogenesis, the tumour microenvironments still have hypoxic domains that lead to sustained stabilization of HIF-α. HIF-α then promotes cap-dependent translation of selective mRNAs for angiogenesis through up-regulation of translational factor eIF4E1. In contrast, 4E-BP1 is a translation initiation repressor that sequesters eIF4E1 and is thus a tumour supressor protein. The activity of translational factor eIF4E1 is also controlled by pathways such as Ras and PI3K/AKT. These pathways act by inhibiting 4E-BP1 and increasing the expression of eIF4E1.
The inducible enzyme cyclooxygenase-2 (COX-2) is also an important mediator of angiogenesis and tumor growth. It induces matrix metalloproteinases that have traditionally been associated with the degradation and turnover of most of the components of the extracellular matrix (ECM). Plasminogen activator inhibitor type 1 (PAI-1) though has the opposite effect of remodeling the ECM by regulating plasmin.

Spatial and temporal heterogeneities in blood supply
Vascular morphology and blood flow rate govern the movement of blood-borne particles through tumour vasculature. Depending on the tumour type, location and growth rate, the architecture of the tumour Cell signalling pathways of hypoxia-induced tumour angiogenesis. MNK: mitogen-activated protein kinase interacting protein kinases; EGFR: endothelial growth factor; VEGFR2: vascular endothelial growth factor receptor type 2; PDGFR: platelet derived growth factor receptor; VEGF: vascular endothelial growth factor; ECM: extracellular matrix; MMP: matrix metalloproteinase; mTOR: mammalian target of rapamycin; TCEB: transcription elongation factor B; FGFR: fibroblast growth factor receptor; IGFR: insulin-like growth factor receptor vasculature may vary [26] . Blood vessel distribution throughout the tumour mass is also not uniform and each region may have either peripheral or central vascularization. In other words, the central portion of some regions is well perfused whereas the periphery may have better perfusion elsewhere.
Microscopically, the tumour vasculature is highly heterogeneous. They are characterized by dilated, secular and tortuous blood vessels having tri-furcations, self-loops, and sprouts. The endothelial cell lining may even be absent. Blood flow is also chaotic and lacks a definite route between the arterial and venous systems. Therefore, in general, necrotic foci develop in a growing tumour. In turn, this decreases the average rate of perfusion.
Regions I and II have a low blood flow rate whereas in regions III and IV, flow is more variable but still higher than that of surrounding normal host tissue.
With tumour growth, the widths of regions I and II increase while that of III and IV remain unchanged, resulting in variation in vascular morphology at both VEGF: vascular endothelial growth factor; FGF: fibroblast growth factor; PDGFB: platelet-derived growth factor subunit B; PLGF: placental growth factor; THBS 1: thrombospondin 1; SDF1: stromal cellderived factor 1; DLL1-4: delta like1-4 (notch ligands); SCF: stem cell factor; Ang-1: angiopoietin-1; CXXR4: chemokine (C-X-C motif) receptor 4; VEGFR: vascular endothelial growth factor; PDGFR: platelet-derived growth factor receptor the macroscopic and microscopic levels. The resulting spatial and temporal heterogeneities in blood supply is thus responsible for non-uniform distribution of the therapeutic agent. Generally, the average uptake of a therapeutic agent decreases with an increase in tumour mass.
Poor extravasation and high interstitial fluid pressure limit transport across the microvascular wall Diffusion and convection are the main mechanisms behind the transport of drug molecules across the vascular wall. The concentration gradient of the therapeutic agent across the plasma (C p ) and interstitial fluid (C i ) is the driving force for the diffusion process. This mass transfer process is proportional to the surface area; the proportionality constant is known as vascular permeability P (cm/s). Transfer of therapeutic agents by convection is associated with the leakage of plasma/fluid across the vascular wall due to differences in hydrostatic pressure of fluid in the blood vessel and interstitial space. The associated experimental constant is known as hydraulic conductivity, L p (cm/mmHg-s). Similarly, the convection process is also proportional to the osmotic pressure difference between the blood vessel and the interstitial space [27] . This proportionality constant is known as the osmotic reflection coefficient (σ). These three experimental constants (P, L p , and σ) are used to describe the extent of transport of plasma content across tumour vessels. Tumour vessels have relatively high P and L p values [28,29] as they have wide endothelial junctions, a large number of fenestrae and trans-endothelial channels, discontinuous or absent basement membrane and significant spatial heterogeneities [30,31] . Although these physiological characteristics increase vascular permeability, tumours also have poor extravasation, which is a significant barrier to the delivery of therapeutic agents. This can be explained as follows: tumour vessels have sluggish blood flow. The hydrostatic fluid pressure in the blood vessel (P v ) is less than that of fluid in the interstitial space (P i ). Of note, the Pi in animal/human tumours is even higher than that of normal tissue [32] . Furthermore, it has been reported that Pi increases with the growth of a tumour. This is mainly due to high vascular permeability and poor, impaired lymphatic drainage [32][33][34][35] . Both tumour hyperplasia around a blood vessel and increased production of extracellular matrix components contribute to high interstitial fluid pressure (IFP). In normal tissue, IFP is 0 mmHg but in tumour blood vessel, the IFP varies from 10-40 mmHg [36] . The IFP is elevated throughout the mass of a tumour except at the periphery, where it becomes equal to normal physiological values. Therefore, intratumoral fluid may extravasate from the periphery of a tumour, resulting in nondelivery of a therapeutic agent. In different animal and human tumour models, it was found that 1%-14% of plasma entering the tumour leaked into the periphery [28,37,38] . Again, the tumour interstitial space has a higher concentration of endogenous plasma protein, leading to higher interstitial osmotic pressure. Thus, the transfer of therapeutic agents by diffusion is further limited.

Resistance to transport through the interstitial space and distribution into the tumour microenvironment
Diffusion and convection are the main mechanisms behind the movement of therapeutic agents that have extravasated into the interstitial space [39] . The concentration gradient is the driving force behind diffusion whereas fluid velocity determines the convection process. The interstitial diffusion coefficient (D) and hydraulic conductivity (K) [32] are the experimental constants used for quantitative measurements of therapeutic agent distribution in the interstitial space. The interstitial space of a tumour is located at the TME (tumour microenvironment) and composed largely of a collagen and elastic fibre network, filled with a hydrophilic gel made up of interstitial fluid and macromolecular constituents [40] . Its structural integrity is maintained by collagen and elastin whereas resistance to transport is provided by macromolecular constituents such as glycosaminoglycans and proteoglycans [40,41] . Compared to normal tissues, tumours have a higher collagen content but lower concentrations of hyaluronate and proteoglycans [32] due to increased activity of lytic enzymes such as hyaluronidase in the tumour interstitial space. Thus, the tumour interstitial space should provide lower resistance to the distribution of therapeutic agents, suggesting larger values of D and K. Paradoxically however, therapeutic agents are not distributed homogeneously in tumours. This can be explained as follows: the time constant for a molecule with diffusion coefficient, D is proportional to the diffusion path length, raised to a power of two. Therefore, if the diffusion path length is doubled, the required time will be increased by four times. In solid tumours, the exchange vessels are at a large distance apart (~200 µm) [42,43] . Therapeutic agents will need a prolonged transit time for homogenous distribution. High interstitial pressures also slow down the distribution process. Thus, low molecular weight (M r < 1000 D a ) anticancer drugs do not accumulate in the tumour because of their small size and hence, rapid clearance [44] . The drug distribution process in a tumour may be further limited by the high affinity of the drug molecule for proteins present in interstitial fluid.

Growth induced solid stress
A tumour mass consists of proliferating cancer cells and stromal cells (i.e., fibroblasts, immune, and perivascular cells) [45] . It is supplied by a dense ECM, and a tortuous and chaotic network of blood vessels [45] . During tumour growth, there is rapid proliferation of cancer cells in a limited space resulting in the generation of mechanical forces from different structural components such as cancer cells, various host cells, and the ECM. Thus, there is also a growth induced solid stress, which commonly ranges from 10 to 142 mmHg [46] , that can deform the vascular and lymphatic structures and cause limited perfusion and hypoxia throughout tumour tissue. This creates a barrier to the penetration of therapeutic agents [47] which restricts their flow to cells within the perivascular space, such that resistant cells in hypoxic regions are missed [45] . Shear stress can also induce vascular endothelial growth factor receptor type 2 (VEGFR2) expression and ligand-independent phosphorylation. This causes activation of MAPK, PI3K, and Akt signalling pathways that are involved in promoting angiogenesis [46] . Additionally, there is VEGFR2 membrane clustering and downstream signalling. Recently VEGFR3 has also been found to be a part of this mechanosensory complex. Depletion of VEGFR2 or VEGFR3 thus causes significant reduction in endothelial cell response to mechanical stress [46] .
Specific integrins can also contribute to tumour angiogenesis and tumour progression [46] . In endothelial cells, VEGF upregulate the expression of α1β1 and α2β1 integrins. The α5β1, αvβ3 and αvβ5 integrins are also expressed in angiogenic vasculature to facilitate the growth and survival of newly forming vessels [46] . Therefore, the general strategy to overcome the barriers to vascular and tumour tissue permeability is functionalization of the surface of nanoparticles with tissue and cell-penetrating peptides, such as the iRGD [48] . It interacts with αν integrins on the endothelium and stimulates proteolytic cleavage. The released CendR peptide subsequently binds with neuropilin-1 [45] to ensure the homing of and penetration of tumour tissue by nanoparticles.

TARGETED DELIVERY OF THERAPEUTIC AGENTS BY EXPLOITING TUMOUR VASCULATURE
A therapeutic agent is delivered to the target tissue via supplying arterioles to that particular tissue. As discussed in the previous sections, there are a number of barriers that hinder the distribution process of therapeutic agents in the tumour. First, the tumour vasculature is highly heterogeneous in distribution. Unlike the tight endothelium of normal blood vessels, the vascular endothelium in tumour microvessels is discontinuous and leaky. Elevated levels of growth factors such as VEGF and bFGF cause vasodilatation and enhancement of vascular permeability. Therefore, the gap sizes between endothelial cells can range from 100 to 780 nm, depending on the anatomic location of the tumour [49] . As such, low molecular weight anticancer drugs (M r < 1000 D a ) can easily enter the tumour microenvironment but at the same time, they can also be easily removed because of their small size. Consequently, when delivered as an aqueous solution, small-molecule chemotherapeutic agents like paclitaxel [50] , gemcitabine [51] , cisplatin [52] , etc. do not accumulate in the tumour at the desired concentration for an adequate duration. These potent anticancer drugs undergo unwanted bio-distribution, leading to unfavourable pharmacokinetics characterized by a large volume of distribution, high renal clearance and short half-life [53] . Furthermore, these cytotoxic agents can cause severe dose-dependent side effects such as myelosuppression, neurotoxicity, mucositis, nausea, vomiting, and alopecia that may become fatal for patients [54] , or even, the development of drug resistance and relapse of cancer [55] .
This problem can potentially be solved by delivering anticancer drugs encapsulated within nanoparticles [56,57] or as drugs conjugated to the nanoparticle's surface [58][59][60][61] . Due to their size range, nanoparticles are inherently able to permeate through leaky tumour microvessels but impaired lymphatic drainage of the solid tumour, together with a higher interstitial fluid pressure, hinders clearance of nanoparticles from the TME. Thus, retention of anticancer drugs is enhanced when delivered as nanomedicine. This mechanism of passively targeting a solid tumour is known as the enhanced permeation and retention (EPR) effect, which was first described by Matsumura and Maeda [62] in 1986.
The size of the tumour, degree of tumour vascularization, and angiogenesis are the main factors affecting EPR [63][64][65] . Thus, the stage of the disease is critical for drug targeting using the EPR effect [66] . Another factor is the challenge for the chosen delivery system to penetrate deep into tumour tissue due to the high interstitial fluid pressure at the centre of a tumour [67] . This results in initial tumour regression, followed eventually by recurrence from residual cells in the non-accessible regions of the tumour [68] . Therefore, the drug delivery system needs to be optimized for deep tumour penetration [69][70][71] . This can be achieved by (1) enhancing blood perfusion to a tumour; (2) modulating the structure of tumour vasculature; and (3) destroying the mass of cancer cells to increase passage of nanoparticles.

Enhancing blood perfusion to a tumour
As discussed earlier, tumour blood vessels have sluggish blood flow. The hydrostatic fluid pressure in a blood vessel (P v ) is less than that of fluid in the interstitial space (P i ). This limits the distribution of therapeutic agents in the TME. Therefore, an increased rate of blood flow in tumour vessels will enhance the distribution of nanoparticles in the TME because of higher extravasation. Strategically there are two ways to increase the rate of blood flow in tumour vessels. First, vasoconstrictors such as angiotensin can be parenterally administered [72] . This will constrict normal blood vessels but not tumour blood vessels which will remain unaffected because of their impaired muscular structure. As a result, more blood will be delivered to tumour blood vessels. Second, vasodilators like NO and CO should be delivered directly to tumour blood vessels without affecting blood vessels of normal tissue [73] .
In experimental rats with subcutaneously transplanted AH109A solid tumours, Suzuki et al. [74] found a 5.7 fold enhancement of blood flow in the tumour after intravenous administration of angiotensin II. This enhanced the chemotherapeutic effect of mitomycin C on the main tumour and metastatic foci in lymph nodes. Nagamitsu et al. [72] then successfully treated patients with SMANCS (neocarzinostatin, the anti-tumour antibiotics conjugated with a hydrophobic copolymer of styrene) under angiotensin induced hypertensive states. The induction of hypertension at ~15-30 mm Hg higher than normal blood pressure for 15-20 min resulted in remarkably enhanced and passively targeted delivery of neocarzinostatin to the tumour. This resulted in faster reduction of tumour size with the least toxicity to normal tissue.
Many research groups have developed nano-medicines that induce tumour-specific vasodilatation by releasing mediators such as NO [75,76] and CO [73] in situ. This helped in the accumulation of nanoparticles within the TME. Tahara et al. [77] incorporated NONOate, a typical NO donor, into PEGylated liposomes. Its retention in blood was similar to that of empty PEGylated liposomes but its accumulation within the tumour was doubled. Due to successful augmentation of the EPR effect, this liposome could be a potential vehicle for the targeted delivery of potent chemotherapeutic agents.
Wei et al. [78] then developed tumour vascular-targeted multifunctional hybrid polymeric micelles for the targeted delivery of doxorubicin [ Figure 3]. Poly (d,l-lactide) (PLA) and poly (ε-caprolactone) (PCL) formed the inner core to encapsulate doxorubicin. The poly (ethylene glycol) (PEG) was linked to PLA with disulphide linkages to form the outer surface of the particle. Copper ion-chelated porphyrin (PpIX-Cu) was then added to the end of the PEG segment, providing a catalytic function to decompose endogenous NO donors like S-nitroso-glutathione (GSNO), S-nitrosocysteine, and S-nitrosoalbumin. Since these endogenous NO donors are also present in human plasma and all tissue fluid, 2-propionic-3-methyl-maleic anhydride (CDM)-modified methoxy polyethylene glycol (mPEG) (mPEG-CDM) was linked to the PpIX-Cu component as a pH-sensitive protective layer, in order to mask the positive charges of the micelles and avoid copper ion-catalysed NO production in the general circulation. Copper catalysed NO production occured only in mildly acidic (pH 6.5) tumour tissue. Furthermore, cRGD grafted PCL-PEG-cRGD (PCE-cRGD) copolymer was added during the synthesis of micelles. The grafted cRGD peptide then effectively targeted the tumour vasculature and tumour cells, on which αvβ3 integrin is overexpressed. Once taken up by the cancer cell, doxorubicin was immediately released due to the high cytoplasmic level of GSH. Thus, this complex hybrid polymeric micelle structure was very effective in treating tumours in an animal model.
Fang et al. [79] reported augmentation of the EPR effect and efficacy of anticancer nanomedicine by CO generating agents. Haem oxygenase (HO) catalyses the degradation of haem to produce CO which causes vasodilatation similar to NO [80][81][82] . Pegylated hemin is the HO inducer whereas tricarbonyl-dichloro-ruthenium (II) dimer (CORM2) is the CO-releasing molecule [79] . The authors showed that in tumour-bearing mice, the accumulation of intravenously administered Evans blue-albumin complex (a macromolecule) in a tumour can be enhanced by the intradermal injection of recombinant haem oxygenase-1, intra-tumoral injection of tricarbonyl-dichloro-ruthenium (II) dimer (CORM2) and intravenous administration of PEGylated hemin. Thus CO plays a significant role in tumour uptake of macromolecular drugs by EPR [83] . They have also developed polymeric micelles of CORM2 copolymer and styrene maleic acid. It had a prolonged plasma half-life and was able to maintain a sustained release of CO. They used it for photodynamic therapy with pyropheophorbide-a [79] .

Modulating the structure of tumour vasculature
The balance between pro-angiogenic (e.g., VEGF, PDGFB, IGF, PDGFRB, FGF-2, and TIE2) and antiangiogenic factors (e.g., thrombospondin-1, angiostatin and endostatin) is responsible for the formation of normal tissue vasculature. This balance tips in favour of overexpression of pro-angiogenic factors in pathological conditions such as the progression of solid tumours [84] . The purpose of such is to meet the high demand for oxygen and nutrients of tumour cells. Therefore, restoring this balance of factors may restore tumour vasculature back to normal. This process involves the inhibition of pro-angiogenic factors at a different level of their cell signalling pathways [ Figure 2], which will reduce the diameter of tumour microvessels, prune immature vasculature, increase vasculature maturity with higher pericyte coverage, reduce tortuosity of microvessels, and decrease IFP. Although normalization of tumour vasculature is the rationale for inhibition of tumour growth [85] , it is not effective enough alone in clinical settings. Instead, it has been found in clinical trials that combinations of radiotherapy or chemotherapy together with antiangiogenic agents are very effective [84,86] . Ionizing radiation generates ROS that leads to DNA damage and cell death. Since the presence of oxygen helps in the generation of ROS, a well-vascularized and perfused tumour tissue would be more susceptible to radiotherapy [86] . It has also been shown that under low-dose irradiation, cancer cells are induced to express proangiogenic factors (e.g., VEGF, PIGF) at a level sufficient to stimulate endothelial cell migration and sprouting. This is known as the vascular rebound effect [87] , which can be overcome by combining anti-angiogenic agents with radiotherapy. In one clinical trial on advanced pancreatic cancer patients, a combination of optimal dosages of bevacizumab, capecitabine and radiotherapy was found to be very effective [88] . In another clinical study with rectal cancer patients, promising results were reported when radiotherapy was combined with bevacizumab, capecitabine, and oxaliplatin [89] . In cases of chemotherapy used in combination with anti-angiogenic agents, normalization of tumour vessels will not only reduce vascular permeability but at the same time, enhance the trans-capillary pressure gradient (due to lowering of IFP), resulting in better distribution of small molecule anticancer drugs and nanoparticles (< 60 nm) into the TME [84] .
Strategically, one may either block the pathways for synthesis of pro-angiogenic factors and their target receptor proteins, or neutralize the effects of these factors by inhibiting the corresponding target receptors with monoclonal antibodies. Such angiogenesis inhibitors can either target endothelial cells of the growing vasculature (known as direct inhibitors) or tumour cells and tumour-associated stromal cells (indirect inhibitors). Direct inhibitors like angiostatin [90] , endostatin [91] , arrestin [92] , canstatin [93] and tumastatin [94,95] bind with integrin receptor to prevent the proliferation and migration of endothelial cells in response to different pro-angiogenic factors. Indirect inhibitors prevent the expression of pro-angiogenic proteins (e.g., VEGF) expressed by tumour cells or block the expression of corresponding endothelial cell receptors (VEGFR). Many angiogenesis inhibitors have been approved by the FDA for cancer therapy including thalidomide [96] , bevacizumab [97] , pazopanib [98] and everolimus [99] amongst others. There are also many candidate anti-angiogenic drug molecules such as siRNA, shRNA, VEGF aptamer, KPQPRPLS-peptide currently under study.
Different types of nanomedicines such as polymeric nanoparticles, lipid nanoparticles, micelles, mesoporous silica particles, metal nanoparticles, noisomes, and liposomes have been developed for the delivery of anticancer drugs. Amongst them, liposomal delivery systems are mostly approved by the FDA for clinical use.
Therapeutic nucleic acids like small interfering RNA (siRNA) and short hairpin RNA (shRNA) are negatively charged and thus, frequently delivered with liposomes made up of cationic phospholipids.
Cai et al. [100] developed Bio-reducible fluorinated peptide dendrimers for efficient and safe delivery of VEGF siRNA. It improved physiological stability, serum resistance; promoted intratumoral enrichment, cellular internalization, as well as facilitated endosomal/lysosomal escape and reduction-triggered cytoplasm siRNA release. It had found to have excellent VEGF gene silencing efficacy (~65%) and a strong ability to inhibit HeLa cell proliferation. Upon intratumoral injection in mice with HeLa tumor xenografts, it significantly retarded tumour growth. Yang et al. [101] developed strategy for co-delivery of VEGF siRNA and docetaxel. This dual peptide modified liposome binds specifically to glioma cells, undergoes specific receptor-mediated endocytosis and deep tissue penetration. Once within target cells, the siRNA silences the VEGF gene to inhibit angiogenesis while docetaxel kills tumour cells.
Chen et al. [102] studied the effect of silencing the VEGF gene using siRNA for the treatment of breast cancer (MCF7 xenograft model) with doxorubicin. They prepared calcium phosphate/siRNA nanoparticles and further encapsulated it in a liposome. The liposome was injected intratumorally while doxorubicin was administered intraperitoneally. This combination therapy resulted in 91% tumour inhibition using only 60% of the standard dose of doxorubicin. In a more recent study, Zheng et al. [103] utilized mesoporous silica nanocarriers (148.5 nm) for the co-delivery of sorafenib (a multikinase inhibitor) and VEGF targeted siRNA to treat hepatocellular carcinoma. The particles were further coated with lactobionic acid to target asialoglycoprotein receptors that are overexpressed on cancer cells. Taking one step further, Shen et al. [104] co-delivered sorafenib and survivin shRNA with nano-complexes to reverse multidrug resistance in human hepatocellular carcinoma. Survivin is an angiogenesis promoting agent. Suppression of survivin with shRNA thus resulted in the reversal of drug resistance and promoted sensitization to sorafenib treatment, leading to cell cycle arrest and apoptosis.
While positively charged liposomes are best suited for the delivery of negatively charged RNA molecules, they undergo nonspecific electrostatic adsorption with blood components and are quickly recognized by the immune system, leading to rapid clearance from the blood by the reticuloendothelial system (RES). This limitation can be overcome by coating the positively charged liposomes with negatively charged anionic polymers, which would then prolong circulation of the nanoparticles in blood and enhance the accumulation of nanoparticles within the tumour due to the EPR effect. In a recent study, VEGF siRNA and etoposide were loaded in a cationic liposome that was further coated with PEGylated histidine-grafted-chitosan-lipoic acid (PHCL), a pH triggered charge-controllable and redox responsive polymer [ Figure 4] [105] .
In the TME, at low pH (6.5), protonation of the imidazole group in the histidine segment of PHCL causes a reversal of nanoparticle charge from negative to positive, leading to deep tumour penetration and enhancement of internalization of nanoparticles. The positive charge is further enhanced in the lower pH of endo-lysosomes, where the disulphide bond of the lipoic acid segment in PHCL-liposomes undergo GSH induced redox-activated breakage, leading to the release of cargo within the liposome [ Figure 4].
The antiangiogenic agent bevacizumab is a humanized monoclonal antibody that inhibits tumour growth and metastasis. When combined with a cytotoxic anticancer agent such as paclitaxel, therapeutic efficacy was significantly improved because of the targeted accumulation of paclitaxel within tumours [106] . In a preclinical study using the MX-1 human breast cancer xenograft model, different doses of paclitaxel were administered in combination with 5 mg/kg bevacizumab. 30 mg/kg paclitaxel in combination with bevacizumab was as effective as 100 mg/kg single dose of paclitaxel in inhibiting the growth of a tumour. This observation can be attributed to treatment with bevacizumab, which significantly enhances the effective concentration of paclitaxel within the tumour.
Gold nanoparticles have also been used for the targeted delivery of anti-angiogenic agents, either alone or in combination with an anticancer drug. Bartczak et al. [107] synthesized gold nanoparticles of ~15 nm and capped them with mono-carboxy (1-Mercaptoundec-11-yl) hexa (ethylene glycol). These particles were then further functionalized through surface coating with a peptide (KATWLPPR) that specifically binds to neuropilin-1 receptor to inhibit angiogenesis. In an in vitro study using human endothelial cells, it was found that this peptide coated gold nanosphere could block capillary formation by endothelial cells without causing toxicity. Patra et al. [108] then used gold nanoparticles for targeted co-delivery of cetuximab and gemcitabine. Cetuximab has been approved for the treatment of EGFR positive colorectal cancer whereas gemcitabine is used for pancreatic carcinoma. "2 in 1" nanoconjugates containing both cetuximab and gemcitabine on a single gold nanoparticle core were synthesized. Physically, this was more stable than a gold nanoparticle-containing either of the agents. This nanoconjugate could target metastatic EGFR expressing cells and inhibited 80% tumour growth and was significantly better than all other non-targeted groups.
EGFR tyrosine kinase inhibitors like cetuximab, lapatinib, afatinib, gefitinib, erlotinib, fedratinib are well studied for anticancer therapy when used in combination with different chemotherapeutic agents including doxorubicin, gemcitabine, paclitaxel, and carboplatin. They help in the normalization of tumour vasculature and sensitize tumour cells to cytotoxic drugs. Additionally, monoclonal antibodies such as cetuximab have been used as a targeting agent. Lin et al. [109] conjugated both paclitaxel and cetuximab on the surface of carbon nano-diamond particles of 3-5 nm diameter. This was found to enhance the mitotic catastrophe and tumour inhibition in the drug resistance of colorectal carcinoma in vitro and in vivo.
Among the other inhibitors, lapatinib also inhibits human epidermal growth factor receptor 2 (HER2) tyrosine kinases and ATP-binding cassette transporters, thereby sensitizing multidrug-resistant (MDR) cancer cells to chemotherapeutic agents. Lapatinib was clinically approved by the US FDA in 2007 for anticancer therapy. There have been many studies since where lapatinib has been used in combination with paclitaxel, and liposomes and polymeric micelles used as drug delivery vehicles. Li et al. [110] developed stealth polymeric micelles using an amphiphilic diblock copolymer named poly (ethylene glycol) -blockpoly (2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol) which formed a core-shell structure by self-assembly. Hydrophobic molecules like paclitaxel, lapatinib are loaded into the hydrophobic core while the hydrophilic shell of PEG prevents their aggregation, restricts plasma protein adsorption, prevents recognition by the RES, and minimizes rapid elimination from the bloodstream. This ~60 nm particle successfully overcame multidrug resistance in an athymic nude mouse xenograft model established with DU145-TXT MDR prostate cancer cells. The strategies of tumour-targeted drug delivery exploiting tumour vasculature aresummarised in Table 2. The FDA-approved anti-angiogenic agents for the treatment of cancer is summarized in Table 3.

Enhancement of vasculature permeability by physical treatment
EPR is a highly heterogeneous phenomenon. It is variable, even amongst different regions of the same tumour. In fact, within a single tumour, not all blood vessels are permeable to the same extent. Moreover, in many clinical settings, it has been found that tumours do not have a sufficient level of EPR to ensure the accumulation of nanomedicines. This is mainly because of the insufficient permeability of the vascular endothelium of tumour blood vessels. This problem can be addressed by local application of physical treatments such as sonoporation, hyperthermia, and radiotherapy that enhance tumour vasculature permeability, and aid in extravasation of nanomedicines uniformly throughout the TME. Sonoporation Sonoporation involves the application of ultrasonic sound to increase the gap between vascular endothelial cells. The mechanical effects can be further augmented with microbubbles and nanobubbles. The acoustic waves generate acoustic radiation force that causes bulk streaming and microstreaming. Bulk streaming is the movement of localized fluid current in the direction of propagation of ultrasonic sound while microstreaming involves localized eddies that are generated next to cavitating bodies. All these mechanical outputs may result in the release of drugs from carriers and the associated movement of drug molecules into targeted tissues. The efficiency of drug release is controlled by acoustic parameters like ultrasound frequency, power density, and pulse duration. Gas-filled micro-bubbles and nano-bubbles undergo violent collapse under large acoustic pressures. This phenomenon is known as inertial cavitation and is responsible for the generation of micro-streaming [167,168] , shock waves [169][170][171][172][173][174] , and jetting which are all responsible for enhancing the effect of EPR. The stability of bubbles is mainly affected by the transport properties of core gas. Air, and biologically inert heavy gases like sulphur hexafluoride, perfluorocarbon are used mainly. Though microbubbles are more responsive to ultrasonic radiation and undergo large changes in volume for the induction of EPR, they cannot escape the capillaries. In contrast, nanobubbles can easily penetrate the tumour via EPR. High-frequency ultrasound is thus suitable for targeted delivery of therapeutic agents to small and superficial tumours, whereas low-frequency ultrasound is beneficial for the treatment of large and deeply located ones.
In another study, Yan et al. [176] attached paclitaxel encapsulated liposomes to the lipid shell of microbubbles via avidin-biotin linkage. They achieved high encapsulation efficiency of doxorubicin and upon application of ultrasonic sound of optimized intensity for the optimal period of time, there was significant enhancement in the uptake of drug molecules in 4T1 breast tumours by EPR.
As an alternative approach, Meng et al. [177] developed a doxorubicin loaded nanobubble [ Figure 5]. It consisted of a core of a polymeric network where doxorubicin is dispersed. This core was encapsulated in a perfluoropropane gas bubble, the lipid shell of which was further stabilized with pluronic molecules. When delivered intravenously in combination with therapeutic ultrasonication, this ~170 nm diameter nanobubble showed higher accumulation and better distribution of doxorubicin in tumours, leading to significantly higher intracellular uptake and therapeutic efficacy.

Hyperthermia
In response to temperatures of 41-45 °C, there is increased tissue perfusion to dissipate heat. For healthy tissues like muscle and skin, this increase in perfusion can be as high as 10-and 15-fold respectively. In tumour tissue, perfusion rates are increased by 1.5-2 folds only [178,179] . Due to this insufficient perfusion, the temperature of tumour tissues raises further. This causes shut down of local blood flow due to (1) endothelial denaturation; (2) vasoconstriction in large pre-existing arterioles at the tumour periphery; and (3) increase in flow resistance because of high viscosity due to the formation of thrombus and fibrinogen gel. Ultimately, tumour cells are killed due to heat only.
Controlled, local heating of tumour tissue with radiofrequency [180] , microwave or ultrasound to temperatures between 40-45 °C has the following effects: (1) dilatation of tumour vessels leading to enhanced blood flow; (2) enhancement in microvascular permeability to macromolecules [181] and nanomedicine [181,182] . This further increases the EPR effect; and (3) triggering the release of cargo molecules (therapeutic agents) from thermoresponsive nanomedicine [179] .
Again, to control drug release at mild hyperthermia, leucine zipper peptide was incorporated into the liposome [24] . At ~42 °C, the leucine zipper gate dissociated to release the drug precisely.
The thermo-responsive bubble generating liposomes [24] was also developed [ Figure 6]. It consists of an ammonium bicarbonate loaded core, which generates CO 2 upon application of hyperthermia (42 °C) and increases the permeability of the liposome bilayer by triggering the release of the drug.
Gold nanoparticles coated with thermo-responsive hydrogel was developed for cancer therapy [198,199] . Local hyperthermia enhances the accumulation of nanoparticles within the tumour [200] . The gold nanoparticle has strong plasmon absorption, resulting in the generation of heat and removal of the polymeric shell. Thus, the gold nanoparticle acts as an anticancer agent [201,202] .
Sato et al. [203] successfully applied threefold strategies to chemotherapy with Fe (Salen) nanoparticle. After intravenous injection, this magnetic nanoparticle was guided to the tumour site for delivery in a rabbit toung tumour model. The nanoparticle, at the target site, was heated with an alternating magnetic field for the local induction of hyperthermia that helped in further distribution of the nanoparticle into the TME due to the EPR effect.
Hyperthermia by NIR laser irradiation causes shrinkage of blood vessels and tumour ablation. Combining hyperthermia and chemotherapy could be an efficient treatment approach. This is known as photothermal chemotherapy [204] . Docetaxel loaded polypyrrole and hyaluronic acid-modified phospholipid nanoparticle were used for photothermal chemotherapy [205] . There was complete inhibition of tumours in 4T1 tumourbearing mice.
Whole-body hyperthermia at the mild fever range (39.5 °C, for 4-6 h) was found to help in the therapeutic efficacy of doxorubicin-loaded liposome in syngeneic CT26 colorectal mice carcinoma [206] . There was a threefold increase in drug uptake in the tumour. It was also reported to be associated with decreased IFP and an increased fraction of perfused microvessels [207] .

CONCLUDING REMARKS
Hypoxia-induced formation of new blood vessels is the key factor in the progression of tumours. Tumour vasculature is heterogeneous, tortuous, irregularly branched, and hyperpermeable. Due to poor lymphatic Figure 6. Schematic diagram showing the structure and function of thermoresponsive, bubble-generating liposomes and the mechanism of localized extracellular drug release triggered by heat. A: drug release mechanism upon application of hyperthermia; B: internalization of the released drug by the target cell B A drainage, the TME has high IFP. This heterogeneity of the vasculature, high IFP, poor extravasation due to sluggish blood flow, and larger distance between exchange vessels are all potential barriers to the delivery of therapeutic agents to tumours. A rationally designed delivery system should overcome all these barriers to reach deep tumour tissue. As the endothelial cells of tumour vasculature have longer gaps, and the IFP is high, nanoparticles of proper size can inherently be accumulated in the tumour due to the EPR effect. This is known as passive targeting. The surface of nanocarriers can also be coated with monoclonal antibodies against receptor proteins overexpressed in proangiogenic tumour cells for active targeted drug delivery. The vascular barrier can be further reduced by enhancing blood perfusion in the tumour and normalization of tumour vasculature. Local delivery of mediators such as NO and CO enhance blood perfusion whereas inhibition of proangiogenic pathways and the use of antiangiogenic agents help in the accumulation of anticancer drugs loaded nanocarriers deep within tumour tissues. Furthermore, the use of sonoporation and hyperthermia boosts nanocarrier mediated tumour-targeted drug delivery.