Strategies of Overcoming the Physiological Barriers for Tumo

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Current Pharmaceutical Design, 2015, 21, 6236-6245

Strategies of Overcoming the Physiological Barriers for Tumor-Targeted Nano-Sized Drug Delivery Systems

Yufang Pi 1, Jinge Zhou1, Jing Wang1, Jian Zhong2, Lin Zhang3, Yiting Wang1, Lei Yu1,* and Zhiqiang Yan1,*

Institute of Biomedical Engineering and Technology, Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal university, Shanghai 200062, P.R. China; 2College of Food Science & Technology, Shanghai Ocean University, Shanghai 201306, Shanghai 200241, P.R. China; 3Department of Pharmacy, Shaoxing People’s Hospital, Shaox-ing Hospital of ZheJiang University, Shaoxing 312000, P.R. China

Abstract: Nano drug delivery systems (NDDSs) have been widely used in tumor-targeted therapy since they can effectively reduce the side effects of traditional antitumor drugs and improve the anti-tumor effect. We divided the in vivo process of tumor-targeted NDDSs into seven steps: blood circulation, tumor accumulation, tumor tissue penetration, target cells internalization, lysosome escape, drug release and drug response. In each step, NDDSs will encounter different types of barriers preventing their effective delivery or response. The researchers have been making efforts to find different strategies of overcoming the corresponding barriers for NDDSs. Hence, we here

reviewed the recent progress of NDDSs in breaking the physiological barriers for more effective in vivo anti-tumor effect, in order to shed a new perspective on the development of tumor-targeted NDDSs.

1

Keywords: Nano drug delivery systems, tumor-targeted nanomedicines, pathophysiological basis, physiological barriers. INTRODUCTION

Cancer is one of the leading causes of death in the world. Cur-rently, the main treatments in clinic for cancer are surgery, chemo-therapy and radiation therapy. Although traditional chemotherapy can effectively control the growth of tumor, it could cause severe toxic effects on healthy tissues. Compared with traditional che-motherapeutic drugs, NDDSs have demonstrated great superiority due to their ability of improving the efficacy of chemotherapy and reducing the side effects [1-3].

The most common types of NDDSs include nanoparticles, liposomes, micelles, dendrimers, etc. [4]. According to the tumor targeting mechanism, the NDDSs can be divided into passive tar-geting and active targeting NDDSs. The passive targeting mecha-nism mainly involves the “Enhanced permeability and retention (EPR) effect” arisen from the abnormal, leaky and highly heteroge-neous microvasculature and the dysfunction of lymphatic drainage in tumor tissues [5]. The active targeting mechanism refers to the interaction of specific receptors overexpressed on target cells in tumor tissues and the targeting moieties conjugated to the surface of nanocarriers [6, 7]. Large amounts of reports on NDDSs have proved the effectiveness of the passive targeting and active target-ing mechanism on improving the therapeutic outcomes of NDDSs and reducing the side effect to normal tissues [8-12].

The NDDSs need to overcome a number of anatomical and physiological barriers before they can effectively arrive at target site and release drugs in tumor. We divide the systemic journey of tumor-targeted NDDSs into seven steps: blood circulation, tumor accumulation, tumor tissue penetration, target cell internalization, lysosome escape, drug release and drug response. In each step, there are different types of barriers which prevent the effective delivery or response of NDDSs. In order to improve the therapeutic outcomes, researchers have attempted many strategies on NDDSs design to overcome the physiological barriers in each step of the

*Address correspondence to these authors at the NO. 3663 Zhongshan Road, Shanghai 200062, P.R. China; E-mails: zqyan@sat.ecnu.edu.cn; yulei@nbic.ecnu.edu.cn

systemic journey. Hence, based on the analysis of physiological basis of tumor targeting, we here reviewed the different physiologi-cal barriers which may impede the systemic transport and response of tumor-targeted NDDSs, and the corresponding strategies of overcoming these barriers reported recently.

PATHOPHYSIOLOGICAL BASIS OF TUMOR TARGET-ING

Tumor grows and progresses in an intricate and complicated tumor microenvironment (TME). The TME contains a variety of components including vascular systems such as blood and lym-phatic vascular endothelial cells, dense extracellular matrix (includ-ing hyaluronic acid, smooth muscle actin, collagen fibers and pro-teolytic enzymes), hypoxia and oxidosis environment, a large num-ber of tumor cells and cancer stem cells (CSCs) [13]. As a unique microenvironment, it has the properties of leaky vasculatures, low pH, and high interstitial fluid pressure within the tumor, which have important influences on tumor proliferation, metastasis and deterio-ration [14]. The pathophysiological features of tumor tissue is the basis of designing the tumor-targeted NDDSs [15].

Tumor Cells

Different from normal cells, tumor cells allow themselves to grow out of control and become invasive. In order to meet the un-limited proliferation of tumor cells, a variety of receptors become over-expressed on the cell surface, such as transferrin receptors, folate receptors, low density lipoprotein receptors and tumor necro-sis factor receptor family [16]. Besides, there is also over-expressed P-glycoprotein (P-gp) on the surface of tumor cells. It can efflux antitumor drugs outside the cell, causing the decrease of intracellu-lar drug concentration and further the failure of chemotherapy, which is called tumor multidrug resistance (MDR) [17]. Almost all human tumor cells express different levels of P-glycoprotein, and those with insensitivity to chemotherapy tend to have higher ex-pression levels.

Vasculature

Tumor angiogenesis is the main source of nutrients and oxygen of tumor cells. When the tumor grows to a diameter of 1 to 2mm,

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Overcoming Physiological Barriers of Tumor-targeted Nanomedicines the rapid and defective angiogenesis was induced in order to meet the demand of unlimited proliferation [18]. It requires tumor cells or stromal cells to secret a variety of stimulus factors, for example VEGF(vascular endothelial growth factor), to cause the prolifera-tion of endothelial cells and degradation of basement membrane and extracellular matrix, then the new capillaries founded with the form of migration and ‘sprouting’ of endothelial cells [19]. In con-trast to normal tissues, the vascular structure of tumor tissues is tortuous and aberrant, which is marked by a heterogeneous distribu-tion of vessel sizes and shapes [20]. The leaky blood vessels and the abnormally impaired lymphatic drainage make nanomedicines eas-ily accumulated and retention in the tumor tissues, which is so-called enhanced permeability and retention (EPR) effect. The EPR effect is the most important pharmacokinetic principle when de-signing tumor-targeted NDDSs [21].

Tumor Extracellular Matrix

Tumor extracellular matrix (ECM) is the structural support of the entire tumor, composed of hyaluronic acid (HA), tumor-associated fibroblasts (TAFs), smooth muscle actin (SMA), and collagen fibers, etc. [22]. The ECM of tumor is denser than normal tissue, causing a high intra-tumoral fluidic pressure, providing a significant transport barriers for nanomedicines penetrating into the tumor tissues [23]. In the process of tumor invasion and metastasis, the ECM is subjected to degradation and remodeling in order to degrade the basement membrane and the majority of proteins in the extracellular matrix [24]. This process is mainly regulated by many extracellular proteinases [25], such as matrix metalloproteinases (MMPs), a type of frequently used cleavable enzyme associated with tumor [26, 27].

Hypoxia and Oxidosis

Hypoxia and oxidosis are two basic characteristics of tumor tissues due to the abnormal nutrient metabolism. Intra-tumoral hy-poxia is generated as a result of oxygen consumption by the rapidly proliferating tumor cells, insufficient blood supply and poor lymph drainage. It has an extensive effect on a number of cellular path-ways and is one of the major contributors to drug resistance of nanomedicines [26, 28]. Besides, the anaerobic glycolysis in the hypoxia tissues can produce lactic acid. Due to the lack of tumor vascular systems, lactic acid cannot be fully discharged, thereby causing the tumor oxidosis [29]. Hypoxia and oxidosis are impor-tant events in tumor because they are related to a more aggressive phenotype with enhanced proliferation and metastasis formation, and poorer survival [30].

Cancer Stem Cells

According to the theory of tumor classification, there are two types of cells in tumor tissue: the first type is common tumor cells, which does not have the ability of tumor genesis and can be treated by chemotherapy; another is CSCs, which have the characteristics of self-renewal, proliferation and high tumorigenicity and show high resistance to drug treatment [31]. Many researches have proved that the CSCs is the main reason for tumor genesis, devel-opment, recurrence and metastasis, even if there are very few CSCs in tumor tissues. The insensitivity of CSCs to radiotherapy and chemotherapy may be resulted from that most of CSCs stay in sta-tionary phase, express a variety of ATP binding cassette transport-ers, and have DNA repair functions. This may be the key reason why tumors cannot be completely eliminated by the current clinical treatments [32].

PHYSIOLOGICAL BARRIERS OF NDDSs AND THE SOLV-ING STRATEGIES

As we mentioned above, there are seven steps in the systemic journey of tumor-targeted NDDSs before the drugs take effect. Each step is corresponding to one type of physiological barrier for the transport or response of NDDSs. Researchers have been finding

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solutions of overcoming these barriers and achieving more potent anti-tumor effect. We describe each physiological barrier and the corresponding strategies as follows See Fig. (1) and Table 1. Blood Circulation

NDDSs have the characteristics of nano-size, large specific surface area and high surface activity. Thus, after intravenous injec-tion into the blood, NDDSs could adsorb a variety of ingredients on its surface such as apolipoproteins and complement proteins, form-ing what is known as the protein corona [33]. Protein corona can be easily identified by phagocytic cells, causing the opsonization and leading themselves to be involved in phagocytosis and rapidly cleared from the blood. It is essential for NDDSs to either bypass electrostatic interaction with charged proteins in the blood or avoid phagocytosis by special functional material modifications. Several surface modification platforms have been built in order to overcome limitations of NDDSs in the blood circulation. First, the presence of poly(ethylene glycol) (PEG) at the surface of nanocarriers has showed to clearly extend circulation lifetime in blood [34]. The extended circulation lifetime that PEG affords is attributed to the repulsive interaction with plasma components resulted from the hydrophilic shell surrounding the NDDSs [35]. Large amount of studies have been carried out on PEGylated NDDSs. For example, for brain tumor-targeted therapy, the PEG modified magnetic iron oxide nanoparticles (PEG-MNPs) achieved long circulating life-time, reduced concentrations in liver and spleen and increased drug accumulation in the brain compared with non-modified magnetic iron oxide nanoparticles (MNPs) [36, 37]. Other PEGylated NDDSs including PEG modified liposomes, PEG grafted copolymers, PEG coated polymer micelles and PEGylated dendrimers also showed prolonged circulation time for tumor therapy [35, 38-42]. Besides, other hydrophilic polymers such as hyaluronic acid (HA), Pluronic copolymer L61 and polyglycerol (PG) have also been proved to be able to prolong the blood circulation time of NDDSs [43-46].

Despite the effectiveness of PEGylation, PEG has been exten-sively observed as the cause of an unexpected immunogenic re-sponse known as the “accelerated blood clearance (ABC) phe-nomenon” [45]. Accordingly, researchers tried to use endogenous materials, for instance red blood cells (RBC) membrane, to enable active immune evasion of NDDSs [47]. Liangfang Zhang et al. prepared the RBC membrane coated PLGA(poly(lactic-co-glycolic acid) nanoparticles, which showed significantly longer retention time in the blood 72h post injection compared with PEGylated PLGA nanoparticles [48]. The RBC membrane obtained the long circulation time through the immunomodulatory proteins such as CD47 on the surface [49]. RBC membrane-cloaked polymeric nanoparticles represent a promising nanocarrier platform with ex-tended circulation time in vivo [50, 51].

Tumor Accumulation

Inefficiency of drug accumulation in tumor tissue is one of the obstacles in the process of successful treatment of tumors. The ac-cumulation of NDDSs is mainly achieved by exploiting the en-hanced permeability and retention (EPR) effect of tumor caused by the leaky vasculature and dysfunctional lymphatic drain [52]. How-ever, the heterogeneities of vascular permeability among different tumor type or location make the NDDSs accumulation unstable [53]. Thus nanomedicines should be further optimized into smaller scale in order to achieve both efficient blood delivery and high tumor site accumulation through the EPR effect [54].

Angiogenesis, induced by a range of angiogenesis factors, is one of the hallmarks of tumor [55]. As tumor vasculature could be directly accessible for NDDSs by specific receptors overexpressed on the endothelial cells, tumor vasculature targeting is becoming an effective strategy for tumor accumulation [56]. The expression of specific receptors on the surface of endothelial cells enables their recognition by ligand-modified nanomedicines, thus increased con-

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Fig. (1). Illustration of the biological barriers for tumor targeted NDDSs and the corresponding strategies of overcoming these barriers.

centration of nanomedicines accumulating on the tumor site. A large number of studies have focused on NDDSs modified by ligand which can specifically bind with the over-expressed recep-tors on endothelial cells. For example, since vascular endothelial growth factor receptor-2 (VEGFR2) is a overexpressed receptor on tumor endothelial cells, anti-VEGFR2 antibodies conjugated multi-stage nano-vectors (MSV) was developed and displayed a 4-fold increased targeting efficiency towards VEGFR2 expressing cells, whereas exhibiting minimal adherence to control cells [52]. Be-sides, as NGR(Asn-Gly-Arg) tripeptide ligand possess high affinity to CD13, a receptor up-regulated on the tumor vasculature and perivascular cells, NGR-functionalized nanoparticles were formu-

lated and showed high accumulation and deepest penetration at the glioma sites [55, 56]. Therefore, a growing body of evidence sug-gests that tumor accumulation of NDDSs by targeting receptors on the tumor vasculature is an effective strategy for tumor targeted drug delivery.

Tumor Tissue Penetration

Following penetrating tumor vessel walls and entering tumor tissue, NDDSs must penetrate through a thick tumor ECM to arrive at deep tumor tissue and be exposed to tumor cells. Solid tumors are characterized by an unusually dense and complex ECM composed of fibroblasts, hyaluronic acid (HA), and endothelial cells, etc. [57].

Overcoming Physiological Barriers of Tumor-targeted Nanomedicines The ECM often generates high interstitial fluid pressures (IFPs) and presents substantial barriers for NDDSs penetration, causing most NDDSs to be retained near the blood vessels and leaving most of tumor cells in deep tissue be exposed to few drugs [58]. Research-ers have attempted several strategies to increase the tumor tissue penetration of NDDSs, such as breaking up components of tumor stroma (such as TAFs, HA, etc.) and penetrating the ECM by tu-mor-penetrating peptide modification.

HA provides the primary barrier for transport of NDDSs in tumor stroma. In order to overcome this barrier, hyaluronidase agents have been used to loose the intercellular connective matrix of hyaluronic acid and ablate stroma from the dense ECM [57, 59]. Co-administration of hyaluronidase and antitumor drugs can cause ECM degradation and facilitate drug penetration into tumor tissues [59-61]. Besides, TAFs or myofibroblasts are a type of cells that plays a key role in the deposition of ECMs and constitutes a major portion of tumor stroma [62]. These fibroblasts present substantial barriers for NDDSs transport, especially in pancreatic cancer and bladder carcinomas [60, 62]. In order to penetrate the ECM of blad-der carcinomas, Leaf Huang group developed complex nanoparti-cles(ComboNP) which were able to break up the thick ECM, and showed a significantly enhanced anti-tumor effect [63].

Recently, tumor penetrating peptides (TPPs) have displayed their special advantages on the mediation of NDDSs penetration in tumor tissue. TPPs usually contain a key sequence motif R/KXXR/K at the C terminal, which allows their binding to the neuropilin-1 receptor overexpressed on tumor cells and tumor endo-thelial cells [64]. Many reports have demonstrated that TPP modifi-cation or co-administration can effectively increase the tumor pene-tration of NDDSs [14, 65-67]. For instance, co-administration of iRGD, a TPP, signi??cantly improved the therapeutic index of doxorubicin and doxorubicin liposomes on tumor [68]. We previ-ously prepared RGERPPR or RPARPAR peptide conjugated liposomes, which could penetrate into the whole tumor tissue, whereas plain liposome could only stay at the vicinity of tumor vessels [66, 69]. Therefore, TPP modification is an effective strat-egy for increasing tumor tissue penetration of NDDSs.

Target Cell Internalization

Following arriving at deep tumor tissues, NDDSs must to be internalized by tumor cells in order to play a good anti-tumor effect. There are mainly four strategies developed to facilitate NDDSs internalization: 1) cell penetrating peptide decorated NDDSs; 2) receptor-targeted NDDSs; 3) fusogenic NDDSs; 4) co-administration of P-gp inhibitors with NDDSs [70-73].

Cell-penetrating peptides (CPPs) are a type of peptides typically with a high relative abundance of positively charged amino acids or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar/hydrophobic amino acids [74, 75]. Large numbers of traditional cell-penetrating peptides such as TAT pep-tide (AYGRKKRRQRRR), and R8 (RRRRRRRR) could be able to transport different molecules or anti-miRNAs through micelles, liposomes, complexes or other nanocarriers across biological barri-ers to be taken up by various cell lines [76-79]. Nowadays, re-searchers have developed a series of cell-penetrating peptides for intracellular cargo delivery. For example, RLW(RLWMRWYSPR TRAYG) linked nanoparticles (RNPs) showed a significantly en-hanced delivery in A549 lung cancer xenografts [71]. VG-21(VTPHHVLDEYTGEWVDSQFK), a CPP derived from vesicu-lar stomatitis virus glycoprotein(G), was conjugated to gold nanoparticles to enhance the cellular internalization, being suitable for various biomedical applications [80]. CPPs can efficiently de-liver hydrophilic and macromolecular cargos inside cells [81]. Another method is to induce cell membrane fusion with fu-sogenic NDDSs, which contain fusogenic molecules on the surface [82]. For instance, dioleoyl phosphatidylethanolamine (DOPE) is widely used as a fusogenic lipid to prepare fusogenic liposomes,

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which usually showed increased cell internalization [83]. Besides, fusogenic liposomes can also be prepared by mixing the lipid com-ponents and complexes which generally have a net positive charge to facilitates their interaction with the negatively charged cellular membrane [84]. The main components of liposomes, analogous series of cationic lipids such as DODAP(1,2-dioleoyl-3-dimethyl-aminopropane), DSDMA(1,2-distearyloxy-N,N-dimethyl-3-amino-propane), DODMA(1,2-dioleyloxy-NN,-dimethyl-3-aminopro-pane), DLinDMA(1,2-dilinoleyloxy-NN,-dimethyl-3-aminopro-pane), DLenDMA(1,2-dilinolenyloxy-NN,-dimethyl-3-aminopro-pane), would affect the fusogenicity and efficiency of cell internali-zation [85]. Besides, some fusogenic peptides such as HA2 and

KALA functionalized nanoparticles for gene transduction were proved to be able to effectively enhance cellular uptake and bio-logical effects [86, 87]. Therefore, fusogenic NDDSs is an effective strategy of overcoming the tumor cell internalizing barrier.

However, a significant drawback of CPPs and fusogenic vesi-cles is their poor selectivity, which hampered their applications in delivering drugs into specific tissues systemically [71]. Thus, the active targeting molecules with cell selectivity like monoclonal antibody, peptide and aptamer may be better choices for tumor-targeted NDDSs [76, 88]. For example, since iron can effectively cross the blood–brain barrier by transferrin receptors, a transferrin-bearing generation 3-polypropylenimine dendrimer allowed the transport of plasmid DNA to the brain at least 3-fold higher than the unmodified dendrimers [73]. The folate receptor (FR), which is absent in most normal tissues but at a high level in human malig-nancies, is an attractive tumor-selective receptor and has been used on folate-derivatized adenoviruses, cationic polymers, liposomes, etc. [89, 90]. Peptide such as angiopep-2, a ligand of LRP receptor, was also used to modify nanoparticles for successfully treatment of glioma [89]. Through specific binding of receptors on tumor tis-sues, active targeting could effectively enhance drug internalization and limit the adverse side effects of tumor chemotherapy on healthy organs [91, 92].

As mentioned above, P-glycoprotein (P-gp) is a highly ex-pressed multidrug transporter which is often responsible for multidrug resistance (MDR), causing the failure of many chemical treatments against tumor [17]. Recently, some excipients which have a modulating effect on P-gp have been used on NDDSs [93]. For example, an P-gp inhibitor TPGS(d-??-tocopheryl polyethylene glycol 1000 succinate) loaded docetaxel micelles exhibited signifi-cantly higher antitumor activity on resistant KBv tumors [70]. Be-sides, both chitosan and anti-P-gp antibody conjugated PLGA nanoparticles showed increased cell internalization compared with chitosan conjugated PLGA nanoparticles [93, 94]. Owing to P-gp modulation from the inhibitors, P-gp targeting NDDSs provide an effective method for overcoming drug resistance and increasing drug internalization.

Endosomal Escape

Endolysosomal confinement is a major barrier for efficient transport of NDDSs [95]. It is necessary to perform further func-tionalization of NDDSs to enhance their escape ability from the endosome and avoid possible degradation of drugs in the endosome and lysosome. Currently, nanoparticles can escape from the en-dosome by two different mechanisms as follows: Fusion with Endosomal Membrane

Membrane fusion plays an important role not only in endocyto-sis but also in endosomal escape. Endosomal escape can be caused by the destabilization of the endosomal membrane through a series of fusogenic NDDSs [96, 97]. For example, fusogenic lipid DOPE is the most commonly used material in the preparation of NDDSs for lipid membrane fusion, because it could rapidly fuse with en-dosomal membrane based on its phase transition behavior [85]. DOPE-containing fusogenic liposomes or DOPE/PLGA nanoparti-cles have been formulated and proved to be able to facilitate

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Table 1. The strategies of overcoming the transport barriers of tumor-targeted NDDSs.

Physiological basis 1) 2)

The mononuclear

phagocyte system (MPS) Electrostatic interaction

1) 2)

Strategies Hydrophilic polymer nanoparticles

RBC cloak nanoparticles

2) 1)

Examples

PEG-MNPs, PEG modified nanoparticles, HA, Pluronic and polyglycerol copolymer nanoparticles

RBC encapsulating PLGA nanoparticles Smaller and compacted scale nanoparticles MSV conjugated with anti-VEGFR2 antibodies

NGR-functionalized nanoparticles Chitosan-coated liposome containing siVEGF, chemically modified heparin LHT7 nanoparticles, cRGD-linked polymeric micelles, chitosan-graft-PEI-candesartan conjugate

Complex nanoparticles(ComboNP), MMP-2 cleavable peptide inserted nanoparticles, functionalizing with hyaluronidase agent nanoparticles

Neuropilin-1-targeted liposomes, TPP functionalized PEG-PLA nanoparticles, angiopep-2 decorated gold nanoparticles, TPP fused EGFR single-domain antibody P-gp inhibitor loading micelles, anti-P-gp antibody PLGA nanoparticles, chitosan-modified or anti-P-glycoproteins conjugated PLGA nanoparticles

TAT peptide or R8 nanoparticles, RLW or VG-21 conjugated to gold nanoparticles Fusogenic liposome, HA2 and KALA functionalized gene nanoparticles Transferrin , folate or LRP receptor mediated nanoparticles

DOPE-containing fusogenic liposomes or DOPE/PLGA nanoparticles, fusogenic peptides(HA2 ,diINF-7, KALA and GALA) complexes

Polyethylenimine (PEI)-coated

mesoporous silica nanoparticles of siRNA and PAMAM dendrimer–siRNA

complexation, PAAs and PPAA as non-viral vectors

Chitosan enclosed mesoporous silica

nanoparticles(MSN), P(St-co-DMAEMA) complexes, MPEG-b-PMaIPG

nanoparticles, PELG/PEI/CAD complexes Gelatin-DOX conjugates, cationic gelatin combined polyGCeDOX, cathepsin B and cathepsin D responsive drug delivery vehicles

Delivery barriers 1)

Blood circulation

2)

Drug accumulations in tumor tissues

1) 2) 3) 4)

ECM

Vascular endothelial barrier Thick stroma

Proteolytic enzymes in the tumor

1) 2) 3)

EPR effect

Vascular targeting for accumulation

Antivascular targeting by inhibiting tumor angiogenesis

1) 2) 3) 4)

3)

Tumor tissue penetration

1) 2) 3) 4)

Low pH Tissue hypoxia EPR effect

High interstitial fluid pressure(HA, TAF, smooth muscle actin, collagen fibers)

Electric interaction need to stride over the cell

membrane to enter inside the cells

1)

Breaking up components of tumor stroma (TAFs, HA, etc.)

TPP functionalized nanoparticles

1)

2)

2)

4)

Drugs internalization into the targeted cells

1) 1) 2)

P-gp inhibitors combining nanoparticles

Tumor homing cell penetrating peptide

decorated nanomedicines Encapsulated cargo

internalized by fusogenic vesicles

Receptor-targeted nanocarriers

Fusion in the endosomal membrane

The proton sponge effect

1)

2) 3) 4)

3)

4)

5) Endosomal escape 1) 2)

Cellular uptake Endosomal escape

1) 2)

1)

2)

6)

Drug release 1)

Acidic environment and specific enzymes present in CSCs in the tumor

1) 2)

PH-sensitive nanoparticles Enzymes sensitive nanparticles

1)

2)

Overcoming Physiological Barriers of Tumor-targeted Nanomedicines Current Pharmaceutical Design, 2015, Vol. 21, No. 42 6241

(Table 1) Contd….

Delivery barriers 7)

Drug response

1)

Physiological basis Based on summarized pathophysiological basis

1) 2) 3)

Strategies Directly target and kill CSCs

Synergistic combination of two or more drugs Multi-functional targeted delivery

3) 1)

Examples

NPDAC combined with NPDOX, SAL-SWNTCHI-HA complexes, phenformin-loaded polymeric micelles

Combination of different chemotherapy and combination of chemotherapy and gene therapy

Liposome system functionalized with PEG, RGD and TAT, DGlueNP/PTX nanoparticles, octa-functional PLGA nanoparticles

2)

endosomal escape and enhance the biological response of various drugs [82, 98-101]. Besides, based on the fusion process occurring between the viral envelope and the endosomal membrane of host cell, some influenza-derived fusogenic peptides such as HA2 and diINF-7 have been used to increase the endosomal escape of siRNA and further the gene silencing efficiency [84]. Other synthetic fu-sogenic peptides such as KALA and GALA, capable of forming ??-helical structure for destabilization of endosomal membrane, have also been used to increase the endosomal escape of NDDSs [102, 103].

The Proton Sponge Effect

Unlike fusion systems that relied on the fusogenic property of the lipid or peptide to mediate endosomal escape, some nanocarri-ers, usually composed of cationic polymers and drugs, are supposed to use the so-called “proton sponge” effect to enhance the endoso-mal escape. Proton sponge effect is mainly caused by cationic polymers coated on the NDDSs that promote endosome osmotic swelling, rupture of the endosome membrane and intracellular re-lease of loaded drugs. Based on the mechanism, the cationic poly-mers with ‘proton-sponge’ nature such as polyethylenimine(PEI) and Polyamidoamine(PAMAM) dendrimers have been extensively studied as plasmid DNA (pDNA) delivery vehicles [104]. Cationic polymers containing NDDSs, such as PEI-coated and siRNA-loaded mesoporous silica nanoparticles and PAMAM dendrimer-siRNA complexation, were proved to possess the capability of initi-ating effectively endosomal escape before the degradation of the packaged siRNA in endolysosomes [95, 105]. Other chemical agents such as poly(amido amine)s (PAAs) and poly(propylacrylic acid) (PPAA) also have the function of endosomal escape and have been proved to improve the transfection efficiency of genetic drugs [97, 106]. Therefore, “proton sponge” agents are a promising can-didate for cytosolic drug delivery.

Drugs Release

As the last line of defense, an optimal drug release profile can be realized through biological stimuli-responsive NDDSs based on the acidic environment and specific enzymes present in tumor tissue or endolysosomes, thereby enhancing the killing effect on tumor cells [107].

pH-sensitive materials are more and more used to promote drug release in tumor stroma or tumor cells based on the low pH in tu-mor tissues(~6.5) or endolysosomes (4.5-6.5) compared with nor-mal tissues (pH7.4) [108, 109]. Thus various pH-responsive nano-carriers such as liposomes, nanoparticles, nanogels, polymer-drug conjugates and micelles have been extensively designed and re-ported [110]. As the conformational change can be exploited to trigger the drug release, a pH-sensitive charge-conversion system including micelles and polycarboxylates nanospheres was recently designed and the drug could be released in a pH-dependent manner

[109, 110]. Beside, both inorganic nanoscale materials like chitosan enclosed mesoporous silica nanoparticles and hybrid organic nano-materials such as P(St-co-DMAEMA) complexes(poly(styrene-co-N,N'-dimethylaminoethyl methacrylate) nanoparticles) and MPEG-b-PMaIPG(methoxy-polyethylene glycols (PEG)-b-poly (d-galactopyranose)nanoparticles have been proved to show a sensitive response to narrow pH changes and a good drug release behavior, exhibiting a high antitumor activity [109, 111-113].

Enzyme-responsive NDDSs were another effective system de-veloped to promote the drug release based on the specific enzymes present in tumor. For example, gelatin can be easily hydrolyzed into its sub-compounds by gelatinase, which is an endogenous prote-olytic enzyme and usually over-expressed in tumor tissues but not in normal tissues [114]. Gelatin-DOX (gelatin-doxorubicin) conju-gates or cationic gelatin/polyGC-DOX complexes can be specifi-cally digested when exposed to gelatinase and release doxorubicin [114, 115]. Some lysosomal cysteine proteases such as cathepsin B and cathepsin D, which play important roles in tumor progression and metastasis, also provided the possibility of designing enzyme-responsive drug delivery vehicles [116, 117]. Appropriate substrate which effectively degraded upon exposure to these cysteine prote-ases had been used as the linker of bioconjugates of antitumor drugs and polymers, permitting intra-lysosomal drug release after endocy-tosis [116, 117]. Nowadays, some esterases are found to be highly expressed in the intracellular compartments, and their substrates have been used as responsive self-immolative linkers in polymeric drug conjugates to trigger the drug release once taken up by tumor cells. For example, cholesterol esterase is highly expressed in en-dosome and lysosome, and the poly(ethylene carbonate), which can be degraded when exposed to cholesterol esterase, enabled release of bovine serum albumin (BSA) in an enzyme-responsive manner [118, 119].

Drug Response

Synergistic Antitumor Effect of Two or More Drugs

The use of single chemotherapeutic drug has shown limitations in anti-tumor treatment, such as development of drug resistance, high toxicity and low therapeutic index [120]. Thus combined ad-ministration of different drugs is emerging as a promising approach to achieve synergistic therapeutic efficacy [121]. Combinations of drugs with different mechanisms or targeting sites have been ex-plored to exploit either additive or synergistic effects. Some exam-ples are combination of different chemotherapy, and combination of chemotherapy and gene therapy [122]. These drugs can be simulta-neously loaded into nanocarriers such as functional polymeric mixed micelles, polyamidoamine dendrimer and copolymers, which have been proved to be helpful inhibiting tumor growth compared to single drug therapy [121, 123-125].

6242 Current Pharmaceutical Design, 2015, Vol. 21, No. 42 Antivascular Treatment

Inhibition of tumor angiogenesis or vascular system has also been proved to be a promising strategy for tumor therapy. Many reports have proved that the elimination of endothelial cells can remarkably inhibit tumor cell growth [38]. Some gene nanomedici-nes such as chitosan-coated liposome containing siVEGF and che-mically modified heparin LHT7 nanoparticles, have been demon-strated to significantly inhibit tumor angiogenesis, and further the tumor growth [126, 127]. Doxorubicin loaded TPP(RGERPPR) modified liposomes were able to destroy the tumor vessels while killing the tumor cells, which would signi??cantly amplify the anti-tumor effect [66]. Furthermore, the destruction of tumor vessels is considered to improve the inhibition of not only primary tumors but also metastatic solid tumors. This is a great advantage since hema-togenous metastasis is the most important way for tumor metastasis [38].

CSC Targeted System

Conventional tumor chemotherapy often fails as most anti-tumor drugs are not effective against drug-resistant CSCs [128]. As we mentioned above, although accounting for only a small part of the bulk tumors, CSCs have the ability of self-renewing, proliferat-ing, differentiating and producing new tumor bulks [129, 130]. Specific markers on CSCs could be used as the targets for CSCs targeted NDDSs [129, 131]. In general, combination of traditional chemotherapy with CSC targeted therapy could produce a synergis-tic antitumor effect [129, 132]. For example, best inhibitory effect for breast cancer was achieved by combining low-dose DAC (decit-abine) loaded nanoparticles and doxorubicin loaded nanoparticles with the lowest proportion of ALDHhi CSCs and the highest pro-portion of apoptotic tumor cells [133]. Due to the innate drug resis-tance of CSCs, effective killing of CSCs has been believed to be a key factor in eradicating tumor by NDDSs [134]. Multi-Functional Targeting Delivery System

At present, the most promising application of NDDSs is the multifunctional targeting delivery systems, which are formed via various modifications in order to overcome several barriers at the same time. For example, a multistage liposome system functional-ized with PEG, RGD and TAT has been developed and demon-strated its synergistic antitumor effect from the following three aspects: 1) increase blood circulation from PEG, 2) active malig-nant tumor targeting from RGD for integrin binding, and 3) effi-cient internalization from TAT peptide [76]. Based on the facilita-tive glucose transporter (GLUT) over-expression on both blood-brain barrier (BBB) and glioma cells, 2-deoxy-D-glucose modi??ed poly(ethylene glycol)-co-poly(trimethylene carbonate) nanoparti-cles (DGlueNP) were recently developed as a dual-targeted drug delivery system for enhancing the BBB penetration and improving the drug accumulation in the glioma via GLUT-mediated endocyto-sis [135, 136].

In order to enhance the antitumor effect of NDDSs, combined administration, anti-vascular treatment, CSCs targeted delivery system, and multi-functional targeting delivery system are more and more used for achieving additive or synergistic effects, representing a promising direction of tumor targeted therapy.

CONCLUSION AND FUTURE PERSPECTIVES

In this review, we divided the in vivo journey of NDDSs for tumor therapy into seven steps. In each step, we analyzed the trans-port barriers and reviewed the recently reported corresponding strategies of overcoming these barriers. It is essential for NDDSs to go through all the barriers to achieve a successful and effective anti-tumor therapy. A problem in any step will lead to the failure of the tumor treatment, which is in accordance with the \effect. Therefore, NDDSs should be rationally designed based on their whole systemic process in order to break up the seven barriers as we mentioned above. However, we should also keep in mind that

Pi et al.

the multi-functionalization is guaranteed to increase the complexity and decrease the druggability of NDDSs. There is still a long way to go to effectively kill tumors using rationally designed NDDSs. It requires the joint efforts from researchers of oncology, molecular biology, materials science, pharmaceutics and other related disci-plines.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

This work was supported by National Basic Research Program of China (2013CB932500), National Natural Science Foundation of China (81202471, 51203024), Shanghai Innovation Fund for Small Technology based Firms (1402H234200), Natural Science Founda-tion of Shanghai (15ZR1430200) and the Zhejiang Provincial Natu-ral Science Foundation of China (Y14H300005). REFERENCES

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Received: August 14, 2015 Accepted: October 26, 2015

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