Dual and multi-targeted nanoparticles for site-specific brain drug delivery

Journal Pre-proof Dual and multi-targeted nanoparticles for site-specific brain drug delivery

Yan Luo, Hang Yang, Yi-Fan Zhou, Bo Hu PII:

S0168-3659(19)30706-0

DOI:

https://doi.org/10.1016/j.jconrel.2019.11.037

Reference:

COREL 10042

To appear in:

Journal of Controlled Release

Received date:

27 September 2019

Revised date:

27 November 2019

Accepted date:

28 November 2019

Please cite this article as: Y. Luo, H. Yang, Y.-F. Zhou, et al., Dual and multi-targeted nanoparticles for site-specific brain drug delivery, Journal of Controlled Release (2019), https://doi.org/10.1016/j.jconrel.2019.11.037

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© 2019 Published by Elsevier.

Journal Pre-proof Dual and multi-targeted nanoparticles for site-specific brain drug delivery Yan Luoa,1 , Hang Yanga,1 , Yi-Fan Zhoua,* , Bo Hua,* a

Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of

Science and Technology, Wuhan 430022, China

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Short title: Dual and multi-targeted nanomedicines for the brain

* Correspondence to:

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Bo Hu

Union Hospital, Tongji Medical College

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Department of Neurology

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Huazhong University of Science and Technology, Wuhan 430022, China.

Yifan Zhou

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[email protected]

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Fax: +86-27-85726028;

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Tel:+86-13707114863;

Department of Neurology

Union Hospital, Tongji Medical College Huazhong University of Science and Technology, Wuhan 430022, China. Tel:+86-13971604910; Fax: +86-27-85726028; [email protected]

1

Yan Luo and Hang Yang contributed equally to this work.

No conflicts of interest were declared.

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ABSTRACT

In recent years, nanomedicines have emerged as a promising method for central nervous system drug delivery, enabling the drugs to overcome the blood-brain barrier and accumulate preferentially in the brain. Despite the current success of brain-targeted nanomedicines, limitations still exist in terms of

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the targeting specificity. Based on the molecular mechanism, the exact cell populations and

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subcellular organelles where the injury occurs and the drugs take effect have been increasingly

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accepted as a more specific target for the next generation of nanomedicines. Dual and multi-targeted

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nanoparticles integrate different targeting functionalities and have provided a paradigm for precisely

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delivering the drug to the pathological site inside the brain. The targeting process often involves the sequential or synchronized navigation of the targeting moieties, which allows highly controlled drug

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delivery compared to conventional targeting strategies. Herein, we focus on the up-to-date design of

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pathological site-specific nanoparticles for brain drug delivery, highlighting the dual and

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multi-targeting strategies that were employed and their impact on improving targeting specificity and therapeutic effects. Furthermore, the background discussion of the basic properties of a brain-targeted nanoparticle and the common lesion features classified by neurological pathology are systematically summarized.

Keywords: Nanomedicine; Brain drug delivery; Dual-targeted; Multi-targeted; Stimuli-responsive; Multifunctional nanoparticles.

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1. Introduction

With an increasing prevalence since 1990, neurological diseases have exacted a heavy toll on the population, especially due to the global aging trend.[1] The current lack of effective medical solutions highlights the pressing need to further develop novel approaches for neurological health management. [2] Since the dawn of this century, the emergence of nanomedicines has aroused a

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veritable explosion of interest in the field of brain-targeted therapy. [3,4] Nanocarriers such as lipid-based, polymeric and inorganic nanomaterials have been engineered to deliver therapeutics to

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the brain by bypassing or crossing the blood-brain barrier (BBB).[5] The capability of

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nanomedicines to enter the brain is highly dependent on their physicochemical properties and surface

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modifications.[6] Among them, modifications with ligands that bind to the brain endothelial cells

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allow nanoparticles to traverse the vessel wall into brain tissues.[7,8] Transferrin and folic acid are

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classic examples of ligands that mediate efficient brain targeting process. Successfully, these nanomedicines have enabled the in vivo application of potential agents and has enhanced the

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therapeutic effects of a number of conventional agents.[7,9–11] The previous decade has been memorable regarding the rapid development of nanomedicines for the neurological disorders, as the nanoplatforms have gone far beyond the vehicles for drug delivery through the BBB but become controllable systems to deliver the pharmaceuticals to their active sites. At the most basic level, the exact cell populations and subcellular organelles where the injury occurs and the drugs take effect have been increasingly accepted as a more specific target for the next generation of nanomedicines.[12–14] Based on the fact that cells in a pathological state often exhibit altered expression profiles, the upregulated molecules characteristic of certain diseases have been identified as markers of the abnormal cell population.[15,16] Nanoparticles that can specifically

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interact with the molecular signatures have a preference for the pathological sites over normal regions.[17,18] For upregulated receptors and transporters, such as the transferrin (Tf) receptors on the glioma cells, ligands and substrates have been conjugated to the surface of nanoparticles to specifically bind the diseased cells overexpressing the targets and trigger endocytosis.[19] For environmental factors such as the spatial variations in the redox potential, nanoparticles have also

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been established with stimulus-sensitive bonds to precisely release their cargo in pathological

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sites.[20] Though different in nature, both of these strategies contribute to drug accumulation within

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the pathological site and thus site specificity of nanomedicines.

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Addressing both the processes of entering the brain and targeting to the lesion can further improve

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the therapeutic effects. The need promotes the application of dual and multi-targeting strategies in establishing pathological site-specific nanomedicines. As noted above, the site-specific targeting

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strategies hold great promise for improving drug efficacy, depending on the availability of molecular

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signatures, the specificity of targeting components and the exact properties of loaded drugs.[19]

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However, due to the hindrance of the functional BBB, target molecules expressed in the abnormal brain tissues seem to be inaccessible for nanoparticles retained within the circulation, which may limit the overall therapeutic effect of pathological site-specific nanomedicines.[21] By integrating both the BBB-targeting group and the pathological site-specific moieties, dual and multi-targeted nanoparticles have been constructed to tackle this problem. The brain glioma cascade delivery system functionalized with the BBB-targeting TGN peptide and the cancer cell-specific aptamer AS1411 provides a typical example.[22] Similarly, nanoparticles developed with the EGF peptide and two types of bioresponsive bonds are featured by their acidic and redox-responsive quality and high vascular permeability.[23] These nanoparticles were endowed with the capability to distinguish

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between targets and non-targets from the system level to the tissue and cellular level and overcome the biological barriers selectively, which can be fairly valuable for brain drug delivery.[9,24] With the growing pursuit of therapeutic effect, targeting elements for subcellular organelle delivery have received increasing interest and have also been adopted as part of a site-specific nanoparticle. For certain therapeutics that influence the cellular contents inside certain subcellular compartments,

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transportation into the compartments allows the maximization of drug efficacy, such as delivering

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reactive oxide species (ROS)-producing drugs into the mitochondria, and DNA-binding therapeutics

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into the nuclei.[25–27] These nanoparticles are known to provide effective treatments for major

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neurological disorders including stroke, glioma, Alzheimer's disease (AD) and epilepsy, as described

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below.

In general, to optimize control over the biological process of nanomedicines, the proper design of the

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dual or multi-targeted nanoparticles is essential, with several key points along the way to the target

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being the focus of attention: (1) overcoming the BBB; (2) specifically targeting the lesion and

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triggering internalization by target cells; (3) enabling the endo/lysosomal escape and navigation to the target organelle; (4) releasing the drugs in a controlled manner. Usually, some or all of these points should be taken into consideration when designing a targeted nanosystem, depending on the conditions under which the drug will demonstrate the maximum in vivo potency (Fig. 1).[28–30] Many excellent reviews have been published previously on the topic of brain targeted nanomedicine, laying the foundation for countless novel pharmaceuticals.[10,31–38] However, the previous works mainly focus on the process of overcoming the blood-brain barrier, a review that comprehensively describes the targeting strategies for drug delivery to the pathological sites, abnormal cells and their subcellular compartments is still lacking. In this review, we focus on the up-to-date design of

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pathological site-specific nanoparticles for brain drug delivery. A systematic perspective is presented, elucidating the multiple aspects that should be taken into consideration when designing a site-specific nanoparticle for the brain. The representative molecular signatures classified by neurological pathology and the corresponding targeting strategies are summarized in the present review. Furthermore, the state-of-the-art developments of dual and multi-targeted therapeutics

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combating neurological disorders are highlighted. Our objective is to inspire new site-specific

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nanomedicines built on previous efforts that have been made in dual and multi-targeting strategies

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for brain drug delivery.

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2. Multiple aspects of designing a site-specific nanoparticle for the brain

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It is well-accepted that a large variety of properties can produce profound effects on the distribution

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of nanoparticles, such as particle size causing an enhanced permeability and retention (EPR) effect,

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ligands mediating selective cellular uptake and biosensitive groups responding to the microenvironment. The nanoparticles should be tailored in multiple aspects for optimal in vivo performance according to the need. In order to obtain an improved therapeutic effect, it is necessary to optimize the relevant properties and targeting mechanisms.[39]

2.1. Physicochemical factors Among various physicochemical parameters that influence brain accumulation, the size, surface charge and shape are primary. The particle size has an impact on the biodistribution of nanoparticles. Typically, a particle size smaller than 100 nm is considered suitable for brain drug delivery. [40,41] Particles smaller than 10

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nm in diameter can be rapidly removed from the bloodstream through renal clearance and as the diameter increases to exceed the upper limit of 200 nm, nanoparticles often accumulate in the liver, the spleen and the bone marrow.[42] On the other hand, the penetration of the nanoparticles within the extracellular matrix is inversely correlated with the particle size, while the impact of size on cellular intake is diverse in different cell types.[42]

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Similarly, the effects of surface charge on drug delivery into the brain have been studied. Cationic

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nanoparticles can induce interaction and intake by the negatively charged brain capillary

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endothelium, but are also prone to rapid clearance by the mononuclear phagocyte system (MPS).

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Moreover, a positive charge on the surface is believed to result in systemic adverse effects, including

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hemolysis and platelet aggregation. In contrast, negatively charged and neutral nanoparticles have a longer circulation half‐ life and rather less adverse effects.[43,44]

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The shape of a nanoparticle also influences important qualities in relation to its biodistribution: the

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flow properties, cellular uptake and extravasation from the vasculature. In synthesized nanoparticles

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larger than 100 nm, rod-shaped particles showed an advantage in cellular uptake over spheres, cylinders, and cubes.[45] Conversely, for sub-100-nm nanoparticles, spheres were associated with the highest cellular intake among the tested shapes.[46] The flow properties of non-spherical particles are highly complex. Compared to spherical particles, some exhibit more significant lateral drifting in the bloodstream, as well as stronger attachment to the endothelium, probably as a result of their higher surface-to-volume ratio.[43]

2.2. Ligand-based targeting strategy The conjugation of targeting ligands to nanoparticles can increase drug delivery to an intended site

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while decreasing off-target effects, which is a most commonly studied form of active targeting. [47] In contrast to the force exerted on nanoparticles to prompt their accumulation in the intended site, ligand-receptor interactions are limited to a fairly short range and cannot produce an attractive force to nanoparticles over large distances and can therefore hardly increase the chance that particles reach the pathological site.[47] Indeed, conflicting results have been presented regarding the issue of how

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targeting groups enhance the therapeutic efficacy of active-targeted nanomedicines.[48,49] Presently,

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the dominant theory describes an enhancement of the cellular internalization of nanoparticles in the

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presence of targeting groups, probably due to stronger binding to the target cells and a faster rate of

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endocytosis via ligand-receptor interaction.[50] As a result, drug removal from the extracellular

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matrix is accelerated, increasing the diffusion gradient between the serum and the matrix, subsequently driving the nanoparticles into the tissue.[51]

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As noted above, targeting groups allow enhanced drug endocytosis and retention at the pathological

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site only when they are in close contact with the cells. On their way to the target cells within the

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brain, ligand-conjugated nanomedicines actually face the same challenges as the passive-targeted ones do in terms of biological barriers (e.g., the BBB, cell membranes).[52] Nanoparticles for brain drug delivery can be generally divided into two categories according to their target accessibility: vascular-targeted, towards the endothelium and the deep-tissue-targeted, into the brain tissues beyond the endothelium. Vascular targeting confers easier access to the target, as well as enhanced retention in the endothelial cells and the intercellular matrix. In contrast, deep-tissue targeting triggers the internalization of nanoparticles by the parenchymal cells. However, direct contact with the target cells as a precondition is highly dependent on transvascular extravasation and sometimes interstitial transport through the extracellular spaces.[53]

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2.3. Stimuli-responsive drug release One emerging trend with a high potential to change traditional delivery protocols is drug delivery with bioresponsive nanomaterials. Unlike the commonly referred to “active targeting strategy”, bioresponsive nanomedicines are developed with components that exhibit no binding affinity to a

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particular target molecule, but have responsive properties to certain stimuli, such as enzymes, redox, pH, temperature, etc.[54] When exposed to stimuli, functional components within the structure

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experience a physicochemical change (e.g., protonation, deprotonation, breakage or degradation),

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which leads to the structural transitions of nanoparticles (e.g., swelling, collapse, or disaggregation)

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and subsequent drug release or light emission.[55–57] These stimulus-sensitive systems are believed to remain stable under physiological conditions but release their cargo rapidly upon arrival at the

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pathological site, thus ensuring delivery specificity.[20]

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While the internal stimulus-sensitive nanomaterials enable self-controlled drug release regulated by a

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certain environmental factor, the external stimulus-responsive nanoparticles allow for more deliberate control over drug release which could be set at will.[58] As a special type of targeting group, bioresponsive components can also be utilized in more complex systems. Actually, they are particularly versatile in constructing nanoscale drug delivery systems given the small size of stimulus-sensitive bonds as the typical functional groups. For example, used as a linker integrating polyethylene glycol (PEG) to nonspecific targeting moieties (e.g., cell-penetrating peptides), stimulus-sensitive bonds help to form a mask to shelter targeting moieties from contact with cells until arrival at the target site. When the mask is removed in response to environmental signals, the interaction with cells is restored.

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2.4. Cell-mediated transport In response to tissue damage, various cell types (e.g. immune cells and stem cells) migrate specifically towards the pathological site. The capability of these cell types to traverse the BBB and accumulate in the brain parenchyma makes them potential carriers for brain drug delivery.

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Stem cells are believed to possess intrinsic regenerative capabilities in vivo.[59] After transplantation,

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they migrate to the injured site and differentiate into target cells to fulfill their therapeutic

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functions.[60] Their active secretion also influences a variety of biological processes including

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tumorigenesis and neurogenesis.[61] Interestingly, stem cells serving as “Trojan horses” can be

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regulated by the cargoes and display different therapeutic attributes.[62] For example, superparamagnetic iron oxide nanoparticles (SPIONs) can improve the homing of the carrier stem

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cells to the injured brain by upregulating the expression of CXCR4, a homing‐ related chemokine

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receptor. [63] Based on these properties, stem cells mediated transport of nanomedicines has

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attracted widespread attention and will be able to display special functions in regenerative medicine. The inflammatory response occurs in multiple pathological scenarios of the brain. Leukocytes recruited by proinflammatory factors can also be exploited to carry nanomedicines to the pathological site. For example, after systemic injection, drug-loaded neutrophils have successfully trafficked to the surgical margin of the brain tumor and released extracellular traps containing drugs or contrast agents to prevent and monitor tumor recurrence after surgery.[64,65] Carrier cells can be loaded with nanoparticles in vitro or in vivo. Certain cell type can be isolated before entrapping nanoparticles. They are loaded with drugs in vitro through cellular uptake or surface conjugation.[35] On the other hand, nanoparticles can be engineered to actively target the

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surface receptors on carrier cells in vivo, thereby triggering subsequent internalization and cell-mediated delivery. [66] In addition to the living cell-based targeting strategy, nanoparticles derived from natural membranes, such as plasma membranes, subcellular organelle membranes or extracellular vesicles, are also endowed with targeting potential to some extent.[67] For more detailed discussion, the readers are

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referred to some excellent reviews.[68–71]

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3. Molecular signature of the lesion as a dynamic factor for site -specific targeting

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As noted above, targeting to an intended site requires the specific interaction between targeting moieties and the target molecules.[17,18] The pathological site, as well as the diseased cell

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populations, can be distinguished from normal tissues by the altered expression profile of certain

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molecules, denoted as their molecular signatures.[72] Among the molecular signatures, those

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elevated on the cell membranes or in the extracellular matrix offer easier accessibility and comprise the typical target molecules for pathological site-specific nanoparticles.[73] With a growing number of molecular targets identified, targeting strategies towards cells expressing these molecules have become an area of interest, constituting an intersection of the medical and materials disciplines. The selection of target molecules and corresponding targeting moieties are highly flexible, dependent on the disease, subtype, its state and clinical course, making this the most dynamic factor to consider when constructing the site-specific nanoparticles. [74–78] It is anticipated that these molecular recognition-based nanoparticles can not only enhance drug accumulation inside the pathological tissues, but also be individualized to distinguish patients of different response to the

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drug and fulfill the mission of precision therapy.[79–81] The overexpressed molecules representative of a particular brain disorder often display an enhanced function related to certain pathological processes of the disease, as is the case with the vascular endothelial growth factor receptor (VEGFR) in glioblastoma (GBM), which regulates the pathological angiogenesis for tumor progression.[82] Due to an overlap of pathological processes

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among different diseases, a number of molecules are known to play a role in more than one disease,

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such as ROS which plays a pathological role in various neurological diseases including strokes, AD

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and Parkinson's disease (PD). Although these diseases have distinct phenotypic consequences, they

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can be accessed with nanoparticles utilizing common targeting strategies for the molecule, as

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summarized below in Table 1. Theoretically, this possibility may extend the applications of the previously constructed targeted nanoparticles. However, the same possibility may also lead to

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3.1. Apoptosis and tissue injury

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off-target effect under complex pathological situations when applied in vivo.

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Apoptosis is a genetically controlled form of cell death in which a sequence of energy-requiring events leads to the elimination of damaged cells.[83] Triggered by multiple factors, pathological apoptosis has an extensive influence on human diseases including ischemic damage, autoimmune disorders, neurodegenerative diseases and cancers.[84] The balance between apoptosis and survival signals regulates the survival of cells. Similar impact factors may induce tissue injury as well. Nanomedicines can be used to target the molecular features of apoptosis and tissue injury that occur in different brain disorders. For example, ischemic stroke increases the expression of HSP70 heat shock proteins in the penumbra, which regulate the protective response helping to refold denatured proteins.[85] Anti-HSP72 vectorized liposomes for diagnostic and therapeutic properties have been

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developed and confirmed with a higher degree of accumulation in the peri-infarct area compared with the control group.[86] Cancers are characterized by their capability for uncontrolled proliferation and ability to escape apoptosis. Interleukin 13 receptor α2 (IL-13Rα2) is a decoy receptor facilitating apoptosis escape through binding IL-13 with a higher affinity than the ubiquitously expressed IL13Rα1.[87] As the

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targeting molecule, IL-13Rα2 has unique properties in terms of mediating endocytosis without

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causing the activation of downstream signaling.[41,88] Fn14, a member of the tumor necrosis factor

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receptor superfamily, is known to increase in response to tissue repair. Despite the largely unknown

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role of Fn14 in GBM, the temporally and spatially specific expression patterns render its antibody

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ITEM4 useful as the targeting group in surface modifications of nanosystems. [89,90] Nanomedicines functionalized with IL-13Rα2 or ITEM4 have shown high specificity for GBM, indicating a potential

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3.2. Oxidative stress

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use of these systems in the treatment of brain tumors.

Oxidative stress is a common pathophysiological process that occurs in a variety of brain disorders, including strokes, AD, PD and several psychiatric illnesses.[91] While a moderate level is considered critical for neuronal growth and function, high ROS concentrations are believed to reduce synaptic signaling and influence synaptic plasticity.[92] When the production of ROS and free radicals overwhelms the capacity of the antioxidant system, cells can suffer from a series of stress responses, including protein, lipid and DNA damage and cell function disorders. An elevated level of ROS and similar molecules in the lesion has been widely exploited to trigger site-specific drug release through the oxidization and destabilization of sensitive components

Journal Pre-proof incorporated into the nanoparticles. In the treatment of AD, a β-cyclodextrin (β-CD) and ferrocene (Fc) host-guest interaction has been tested as a H2 O2 sensitive controller of drug release, as well as arylboronic esters.[93,94] Similarly, α-lipoic acid showed great potential as a ROS-responsive switch of the prodrugs in the treatment of GBM.[95] The concept of ROS/ H2 O2 sensitivity has also been incorporated into nanosystems developed for

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imaging purposes. The Lipo@HRP&ABTS nanoprobe, a liposome loaded with horseradish

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peroxidase (HRP) and its substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),

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was fabricated for the photoacoustic imaging of inflammation and cancer. In the presence of H2 O2,

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the HRP catalyzes the oxidization of ABTS into its green oxidized form, creating strong

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near-infrared (NIR) absorbance of the nanoprobe.[96] In another case, ROS detection was established as a novel way to identify infarct regions in the brain. Once the fluorescein- labeled

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hyaluronic acids (HA) immobilized onto the surface of gold nanoparticles are cleaved by ROS and

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HAdase, fluorescence-quenched gold nanoprobes exhibited dose-dependent fluorescence-recovery

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signals immediately upon exposure.[97]

3.3. Inflammation, cell migration and homing Inflammation drives the progression of multiple diseases that affect the brain, such as atherosclerosis, GBM and stroke. Accordingly, developing novel targeting strategies tailored to different aspects of inflammation will advance nanocarriers for the site-specific delivery of drugs.[98] Cerebral ischemia progresses in several stages. After ischemic episodes, inflammation contributes significantly to the poor outcome.[99] Pro-inflammatory cytokines are extensively upregulated within the ischemic cortex and in response, endothelial adhesion molecules are increased, mediating

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the recruitment and activation of leukocytes.[100] The selectins are transmembrane proteins that interact with glycosylated ligands such as Lewis X (SX), mediating leukocyte recruitment in the complex response following cerebral ischemia. Farr et al. tested the stroke homing quality of the SX functionalized nanoparticles SX@MNPs in their earlier work. Although the nanoparticles were anticipated to specifically target the stroke lesion via SX’s interaction with E and P selectins, the

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results failed to prove the site-specificity: magnetic resonance imaging (MRI) and subsequent total

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dark voxel counting did not reveal a significant difference between SX@MNP and HO@MNP,

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while site-specificity seems to be higher in the HO@MNP treated group, which might be attributed

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to the fact that selectin expression is upregulated throughout the brain.[101] In contrast, Fas ligand,

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which is known to trigger ischemia- initiated brain damage through exaggerated inflammation and microglia recruitment, was found to be a potential biomarker selectively upregulated in ischemic

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tissue.[102] Based on this discovery, further work was carried out by the authors to develop a

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Fas-ligand-decorated nanoparticle for cerebral ischemia targeting. This was successfully achieved,

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obvious lesion accumulation in mice was observed 24 hours (h) after intravenous injection of the PEGylated-lipid nanoparticles, showing the great potential of targeted nanoparticles to treat cerebral ischemia.[103]

Active invasion and metastasis is a major phenotype of cancers. Although the details are still unknown, the roles of cell-cell/matrix interactions are well established in the process of tumor invasion and metastasis. As a general rule, adhesion molecules related to cytostasis are downregulated, while the expression of the molecules facilitating cell migrations is often upregulated in carcinomas.[104] Neural cell adhesion molecule (NCAM) is a cell adhesion molecule that participates in a wide

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variety of cellular functions including tumor metastasis.[105] This protein is universally expressed in GBM tissues regardless of their stages, qualifying it as a molecular signature of the GBM.[106] Site-specific nanoparticles were developed against NCAM, using NCAM-targeting peptide (NTP, C3 peptide) as a targeting moiety.[107] Similarly, CD44, a cell-surface glycoprotein overexpressed on tumor cells, is involved in cell-cell interactions, cell adhesion and migration.[108] The main ligand

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of CD44 is HA, but other extracellular matrix components can also interact with it. In a study

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conducted by Hayward et al., HA-coated liposome nanoparticles can induce the cellular uptake of

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drugs loaded in the nanoparticle by interacting with CD44, thus prolonging the survival of

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GBM-bearing mice.[109] In addition, the specific expression of fibronectin extra domain B (EDB)

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on both angiogenic endothelial cells and glioma cells was also exploited in the construction of GBM-targeted APT-nanoparticles which were modified with the EDB-targeted APTEDB peptide.

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[110]

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3.4. Proliferation and stem cells

Uncontrolled proliferation is a feature of malignant tumors including GBM. As has recently become known, the aberrant activation of growth factor signaling is a causal factor underlying the malignant transformation of GBM.[111] While overexpressed growth factors provide sustained stimulation for oncogenesis, dysfunctional tumor suppressor genes trigger the imbalance of pro- and anti-tumor signals, activating downstream proliferation pathways, leading to malignant transformation.[112] The discovery holds great promise for clinical applications, including translation into tumor markers and therapeutic targets, and importantly, as target molecules for drug delivery systems. Increasing interest in nanomedicine has prompted the use of these molecular patterns as delivery targets. To

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date, a variety of those molecules have been exploited to guide the active delivery of nanoparticles, including nucleolin, VEGFR, EGFR, somatostatin receptor 2, disialogangliosidase2 and neuropeptide Y Y1 receptor.[113–121] The presence of stem-like cells in the central nervous system is a possible explanation for the pathogenesis of malignant brain tumors.[122–124] Similarly to normal stem cells, glioma stem-like

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cells (GSCs) are characterized by the self-renewal and differentiation properties that allow the

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seeding and regrowth of tumors.[125,126] They also share similarities with normal stem cells in

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terms of phenotypic markers, including CD133, CD44 and CD15.[127] Their unique attributes as

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resistant to standard chemotherapy and radiotherapy present a challenge in treating drug-resistant

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tumors.[128] Fortunately, antibodies and small peptides, such as CBP4 for CD133, have been proved

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3.5. Matrix remodeling

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efficient in specifically targeting to the glioma stem cells.[13]

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The extracellular matrix is a three-dimensional protein network that supports the physiological activities of cells and contributes to the structure of organs and tissues.[129] Matrix remodeling serves an important role in neuronal functions, as well as cancer progression and various brain disorders associated with a damaged BBB.[130,131] MMPs are a family of zinc-dependent peptidases essential in degrading extracellular matrix proteins and regulating molecules involved in signal transduction.[132] A disrupted balance between MMPs and their inhibitors, TIMPs, often results in the pathogenesis or deterioration of brain diseases.[133] Due to the unique properties of MMPs as extracellular enzymes, nanomedicines have been functionalized with various navigational methods to specifically deliver agents to pathological sites.

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As previously reported, small peptides such as chlorotoxin (CTX, ClTx) and CTT peptide have been tested and validated as targeting ligands which enable an enhanced accumulation of nanomedicines in the pathological site characterized by an increased level of MMPs.[134–143] Other than targeting ligands, MMP-sensitive nanoparticles have also been established, with MMP-cleavable substrates serving as core components to release drugs specifically at the lesion.[143]

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Mohanty et al. fabricated an MMP-14-sensitive nanoprodrug by coupling ICT, an

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MMP-14-cleavable azademethylcolchicine-peptide conjugate, to cross-linked iron oxide

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nanoparticles.[144] Similarly, gelatin-based nanoparticles loading siRNA were also developed for

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hope for an improved outcome.[145]

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intranasal administration, in an effort to reduce the infarct volume of cerebral ischemia, providing

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3.6. Abnormal metabolism

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The metabolism of living systems is a highly dynamic process which changes over time in line with

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physical and mental changes.[146]

Carriers and transporters mediating the transportation of nutrients often demonstrate varying activity to meet energy demands according to the metabolic condition, which applies to both molecules present on the BBB and those on the parenchymal cells.[147–150] Using substrates of those molecules as the targeting groups, nanoparticles can be delivered through the BBB and internalized by parenchymal cells in a pathological site-specific manner. Widely used substrates include transferrin for Tf receptors, apoE/apoB/angiopep-2 for lipoprotein receptors and folic acid for folate receptors (FR).[10] Moreover, transporters such as d-Glucose transporter protein (GLUT), LAT1 and

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P-gp have also been exploited to aid brain drug delivery towards glioma cells and with an epileptic focus, respectively.[151–153] Notably, the substrates mentioned above often serve a dual-targeting function in drug delivery, with the overexpression of their receptors by both the BBB and the pathological cells, as exemplified by

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Tf,[147] angiopep-2,[154,155] apoE3,[156] etc.

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4. Dual and multi-targeted nanoparticles for site-specific drug delivery into the brain

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For decades, targeted nanomedicines have been extensively studied, incorporating a wide range of

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molecular recognition patterns. These particles provide elegant approaches to combat neurological diseases.[2] However, while the targeting moieties allow interaction with pathological cell

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populations to some extent, they are far from exclusive to the pathological site. Single

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molecule-based targeting still faces the possibility of off-target effects, which hinders their further

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application in highly complex clinical situations. Moreover, due to the presence of the BBB, single targeting strategies cannot guarantee both the infiltration and retention of nanoparticles into the pathological brain tissue.[21] By integrating both the BBB-targeting group and the pathological site-specific moiety, dual and multi-targeted nanoparticles have shown the great potential to tackle this problem. Dual and multi- targeted nanomedicines are combination nanoplatforms in whic h, in addition to the scaffold, two or more targeting moieties, including ligands and/or bioresponsive components, are incorporated as an integral part of the nanoplatform to deliver the drugs to the intended site. Since there are two or more types of targeting moieties, these nanomedicines are designed to recognize and

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respond to more than one molecular signature of the pathological site, minimizing off-target delivery. Ideally, enhanced site-specificity is supposed to reinforce the theranostic effects of the payloads and offer more benefits over the conventional forms of single-targeted nanomedicines. According to the need, targeting moieties can be engineered to act either simultaneously or sequentially in vivo. The latter often includes a BBB-targeting moiety to ensure accessibility into the brain. These

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meticulously designed nanoparticles may exhibit pathology-specific control over drug delivery and

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release, which leads to improved therapeutic efficacy. An overview of dual and multi-targeted

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nanoparticles for site-specific brain drug delivery is presented in Table 2.

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4.1. Parallel targeting strategy

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In parallel targeting strategies, the nanoplatforms are conjugated with two different targeting moieties, each to bind respectively with one type of receptor on the diseased cell membranes.[157]

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These approaches address a single issue during drug transportation, namely the penetration of target

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cell membranes, with dual targeting groups that interact simultaneously with different pathological

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signatures (Fig. 2). Notably, the molecular signatures can either be co-expressed by one group of target cells, or upregulated in different types of target cells. To date, the major application of parallel targeting strategies has been the delivery of anti-tumor medicines, in an effort to fulfill the following expectations: i) improving the targeting specific ity by co-targeting two tumor-specific molecules;[158,159] ii) increasing the tolerance to the antigenic alterations that occur during treatment; iii) overcoming the intratumoral heterogeneity in the expression patterns of target receptors and the resultant drug resistance;[22,160] and iv) enhancing the multivalent affinity of the nanoparticle and reinforcing drug efficacy limited by receptor saturation.[161] These parallel targeted nanoparticles also offer the possibility of targeting different

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cell types involved in the disease progression, which holds promise for brain diseases other than cancer Peptides are associated with several advantages as targeting ligands, including their small size, superior biocompatibility, high purity and relative inexpensiveness.[162,163] To overcome off-target drug accumulation, two peptides specific for the highly expressed epidermal growth factor and

f

transferrin receptors (EGFR and TfR, respectively) in brain tumors were selected by Dixit et al. to

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establish a parallel targeted nanosystem. With the linkage of covalent amide bonds, the EGF peptides

pr

and Tf peptides were coupled to gold nanoparticles (AuNPs) for photodynamic therapy of GBM (Fig.

e-

3A). As indicated by fluorescence imaging, better uptake of the dual-targeted AuNPs was achieved in

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vitro and in vivo than that of either the EGFpep-AuNPs or Tfpep –AuNPs.[164] One major drawback of targeting with peptides is the relatively low affinity. Taking advantage of the

al

multivalency effect, peptide homodimers and heterodimers have emerged with improved binding

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affinity to target molecules.[165] Moreover, based on the fact that oncogenesis often leads to the

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upregulation of a number of receptors concomitantly, heterodimeric peptides have unique potential in tumor targeting to interact simultaneously with different receptors, enhancing the cellular uptake via stronger binding.[166] For example, Kang et al. constructed a heterodimer peptide-functionalized nanoparticle based on a comprehensive view that the glioma cells and tumor microenvironments function as a pathological integration.[167] Composed of FHK and tLyp-1 coupled via a cysteine, the heterodimeric Ft peptide interacts specifically with tenascin C, an aberrant extracellular matrix component, in addition to NRP-1, an overexpressed tumor membrane receptor (Fig. 3B). Integrating the extracellular matrix and glioma cell targeting components with a tumor penetration motif, namely the Cend motif (R/KXXR/K) from tLyp-1, this system showed a significant synergistic effect in

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delivering paclitaxel to GBM, as indicated by its significant anti-tumor potential in vitro and in vivo. Immunofluorescence staining confirmed the more intense and uniform distribution of Ft-NP within the tumors compared to that of single-targeted nanoparticles (Fig. 3C). Bispecific Abs (bsAb) containing two antigen-binding sites also have a place in glioma therapy. They can be applied to recognize two different epitopes on the same or different target cells.[168] Due to

f

an antigenic alteration, VEGF inhibitors alone to treat cancer often result in drug resistance with

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increased angiopoietin-2 (Ang-2), partly through macrophage reprogramming into Tumor-associated

pr

macrophages (TAMs) and angiogenesis. To address this situation, a bsAb A2V targeting both Ang-2

e-

and VEGF was developed and demonstrated effective inhibition of tumor growth through vascular

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and/or immunomodulatory effects in two GBM models (Fig. 3D).[169] In another study conducted by Choi et. al., BsAbs targeting the immune effectors (e.g., T cells) provided an alternative way to

al

achieve enhanced glioma treatment.[170] However, antibodies including bsAbs are confronted with

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the restriction set by the BBB.[168,171] Details regarding how the mentioned bsAbs passed through

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the BBB are still unknown. How to target two molecules present on the glioma cells and simultaneously enable the nanoparticles to penetrate the BBB remains a question for the future application of bsAbs as parallel targeting groups of nanoparticles. So far, invasive methods to open the BBB (e.g., osmotic disruption) and non-invasive intranasal administration seem to be possible solutions to facilitate the application of bsAbs in parallel targeting strategies, although these approaches have not yet been validated.

4.2. Cascade targeting strategy Advances in brain-targeted drug delivery have shown great promise in the treatment of neurological

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diseases, but have also raised new questions of the subsequent flow of nanomedicines in the tissues. Recent attempts made in pathological site-specific drug delivery tend to functionalize the nanoparticles to accomplish a series of missions. Typically, a brain-targeted group serves as the first stage ligand to guide the penetration of the BBB, while in the second stage, a targeting moiety specifically for the pathological signature is adopted to allow lesion-specific accumulation of the

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drugs. To further program the nanoparticle for subcellular compartments, functional groups

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mediating endo/lysosomal escape and targeting to the subcellular organelle may be incorporated, as

pr

well as bioresponsive components allowing effective drug release. Accordingly, the targeting

e-

components function in a sequential manner along with the in vivo transport of the

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nanomedicine.[164,167]

Compared with traditional single targeting strategies mainly considering the BBB as the target,

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cascade targeting strategies may come with several benefits depending on their design, including

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deeper tissue penetration, prolonged drug retention, controlled release, cellular internalization and

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further subcellular localization.[172,173] Importantly, distinguishing between pathological microenvironments and normal microenvironments, and between diseased cells and normal cells, which is enabled by the conjugation of the second stage targeting moieties, provides a way to improve the targeting accuracy. Furthermore, for certain therapeutics that serve to interact with cellular contents, transportation into the intracellular space or subcellular compartment allows the proper function or maximization of drug efficacy.[25]

4.2.1. Cascade targeting strategy mediated by dual ligands Dual-targeted nanoparticles aimed at drug delivery into the brain can benefit from the integrated

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function of BBB penetration and target cell interaction, where one targeting moiety interacts with a receptor at the BBB and promotes transcytosis into the brain tissue, while the other specifically recognizes pathological cells and triggers internalization. Since an increasing number of targeting moieties are identified for both the BBB and brain lesions, there are an enormous number of possible combinations, which are still under active exploration.[174]

f

Cascade targeting strategies have been applied to AD treatments successfully, targeting the Aβ

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peptide within brain tissues. TGNYKALHPHNG (TGN) and QSHYRHISPAQV (QSH) are targeting

pr

peptides specific for penetrating the BBB and binding Aβ1-42 , respectively. Zhang et al. established a

e-

biodegradable PEG-polylactic acid (PLA) nanoparticle for a synergistic combination of TGN and

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QSH. The optimal density of both peptides on the surface was predetermined, with cell uptake and in vivo imaging for the TGN and thioflavin T (ThT) binding assay and surface plasmon resonance (SPR)

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experiments for QSH. In vivo results confirmed that the sequentially targeting nanoparticle improved

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the brain concentration and lesion distribution.[175] Based on this finding, H102 peptide, a β-sheet

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breaker with the potential to reduce the level of Aβ, was integrated into the nanoparticle (Fig. 4A). In contrast to the largely restricted application of free H102 resulting from its rapidly cleared nature, encapsulation into the dual-targeted nanoparticle enabled H102 peptide to produce neuroprotective effects, at least partly through site-specific delivery to the lesion.[176] Stroke homing peptide (SHp) can home to ischemic brain tissue and co-localize with apoptotic neuronal cells after intravenous administration in the middle cerebral artery occlusion models.[177] In a conducted by Zhao’s study et al., the researchers combined the stroke homing potential of SHp and the BBB targeting capacity of T7 peptide by conjugating both of the moieties to PEGylated liposomes.[178] This strategy has successfully enabled the in vivo application of a novel

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neuroprotectant (ZL006) in ischemic stroke treatment. In Gao’s study, angiopep-2 and EGFP-EGF1 were added to the surface of PEG-PCL nanoparticles (Fig. 4B).[179] While angiopep-2 enabled BBB targeting, EGFP-EGF1 was a FVII-derived fusion protein with TF-binding capacity. It precisely navigated the nanoparticle to neuroglial cells without initiating coagulation. In comparison with single-targeting systems, angiopep-2 and EGFP-EGF1

f

decorated nanoparticles (AENP) showed an increased brain accumulation and improved cellular

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distribution over the single-targeted ones, as AENP best co-localized with neuroglial cells in the

D

CDX and cyclic arginine-glycine-aspartic acid

e-

glioma targeted drug delivery have incorporated

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fluorescent in situ hybridization of brain slides. Similarly, nanoparticles developed by Wei et al. for

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peptide (c(RGDyK)) for the BBB and the blood–brain tumor barrier (BBTB) respectively.[180] Protein corona is an important attribute that influences the targeting capacity of nanomedicines.[181–

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183] It has long been noticed that apolipoproteins adsorbed by the polysorbate 80 (PS 80) coating of

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nanomedicines could mediate transcytosis of drug into the brain tissue via interaction with low

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density lipoprotein receptor (LDLR).[3,34,40,184] In a recent study conducted by Zhang et al., PS 80-containing nanoparticle was adopted to target not only the tumor neovasculature but also the tumor‐ associated macrophages (TAMs) overexpressing LDLR. Besides, the nanoparticle was conjugated to iRGD peptide to further improve the penetration of the BBB and enable the binding with tumor cells.[185] As suggested by the results, targeting macrophages within tumor microenvironment has great potential in circumventing triple negative breast cancer. In addition to the TAMs and other stromal cells, extracellular matrix components represent another potential target within the tumor microenvironment for the site-specific ligands. Heparan sulfate proteoglycan (HSPG) is an extracellular matrix proteoglycan upregulated in the tumor

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microenvironment. It is thought to regulate the signal transduction between the extracellular matrix and cancer cells by interacting with adjacent signal molecules such as growth factors and cytokines.[186] Therefore, in a nanoplatform designed by Hu et al., neuropilin-1 (NRP-1) binding peptide was combined with an extracellular matrix-binding peptide targeting to the HSPG, in which the PEG–PLA nanoparticles loading PTX was coupled with the fused peptides of ATWLLPPR and

f

CGKRK. Through NRP-1-dependent internalization, ATWLLPPR peptide mediates blood vessel

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extravasation and tumor penetration. CGKRK peptide, the substrate of HSPG, increases the

pr

nanoparticle retention in the pathological site. Consequently, the dual-decorated nanoparticle

e-

mediated increased vessel extravasation and enhanced tumor accumulation and penetration, which

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was demonstrated in both in vivo and in vitro glioma models (Fig. 4C).[187]

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4.2.2. Cascade targeting strategy combining ligands and bioresponsive materials

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Nanoparticles integrating both the property of bioresposiveness and targeting potential can

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significantly enlarge the scope of targeting, by allowing a variety of internal environmental factors, including the redox potential, pH and temperature, to be landmarks of the lesion, which cannot be achieved with nanoparticles merely decorated with targeting ligands (Fig. 5).[188] Epileptic seizures are characterized by a sudden, uncontrolled discharge in the brain.[189] Taking advantage of the electrophysiological mechanism of seizures, a hydrogel nanoparticle was designed to release the antiepileptic drug (phenytoin sodium, PHT) in response to the electric field (Fig. 6A).[190] With sodium 4-vinylbenzene sulfonate-based polyelectrolyte in the hydrogel nanoparticle, it swelled rapidly when exposed to an electric field due to the ionization of the sulfonate groups. Increased particle size triggered the release of PHT. Moreover, the nanoparticle was also decorated

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with angiopep-2, so as to ensure the penetration into the brain. As demonstrated by in vitro and in vivo experiments, hydrogel nanoparticles accumulated in the brain after administration. Upon exposure to the electric field, PHT was released within the brain tissue and inhibited the discharge, alleviating seizure behavior (Fig. 6A). Hua et al. reported the successful integration of ligands and biosensitive materials in glioma

f

targeted-delivery.[191] Resistance to radiation is a tough challenge in glioma treatment, with the

oo

underlying mechanism being the intratumoral hypoxia. Although radiosensitizers have been widely

pr

explored in recent years, their applications are fairly limited, owing to the off-target effect resulting

e-

from low specificity to pathological tissues. To solve this problem, Hua’s group constructed a

Pr

hypoxia-sensitive targeted delivery system that conjugates angiopep-2 to a hydrophobic P-(MIs)n core. Nitroimidazoles, one of the ingredients, serve as both hypoxia-sensitive controllers and

al

oxygen-mimetic radiosensitizers.[192] With the conversion of the nitro groups of the core into

rn

hydrophilic groups, the dual-targeted nanoparticle can release the cargo specifically within the tumor

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in response to hypoxia. The same process acted to induce DNA strand breakage and cell death in an oxygen-mimic manner, causing radiation-sensitizing effects in gliomas (Fig. 6B). External signals such as radiation and ultrasound convey energy through tissues. Upon reception of the signals, specially engineered nanoplatforms can convert the energy into a subsequent physicochemical reaction and activate the theranostic agents.[193] Among these nanoplatforms, those responsive to magnetic fields are thought to be more “active”, migrating under the navigation of a configurable magnetic force.[194,195] Interestingly, in research conducted by Cui et al., magnetic nanoparticles in conjugation with T7 peptides have shown great potential to achieve site-specific drug delivery to the brain lesion.[196] The magnetic PLGA-PEG-T7 nanoparticles

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(MNP/T7-PLGA nanoparticles) were prepared for the glioma delivery of curcumin (CUR) and paclitaxel (PTX). Due to the fact that magnetic guidance alone can hardly benefit transcellular penetration and the cellular uptake of nanoparticles, which may be a result of magnet-induced nanoparticle aggregation, it is necessary to enhance drug accumulation in the brain by ligand- mediated transcytosis. As indicated by in vivo investigations, the bioresponsive nature of

f

nanoparticles and the T7-mediated targeting strategy have a significant synergic effect when

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MNP/T7-PLGA nanoparticles are administered to glioma-bearing mice followed by exposure to a

pr

magnetic field (Fig. 6C).

e-

Notably, a current research conducted by Xu et al. presented us with a novel strategy to develop

Pr

targeted nanoparticles.[197] Inspired by the recruitment of platelets during thrombus formation, the authors adopted the platelet membrane to develop a bioengineered “nanoplatelet” which can be

al

recruited to the ischemic site. Consisted of a ZL006e-loaded core and platelet membrane shell

rn

functionalized with thrombin-cleavable Tat-peptide-coupled rtPA, the nanoplatelets can sequentially

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home in on the thrombus, release the rtPA and expose Tat peptide in response to activated thrombin. Under the navigation of the Tat peptide, ZL006e penetrate across the BBB into the ischemic tissue and take effect. The “nanoplatelets” succeeded in delivering the thrombolytic rtPA and the neuroprotective ZL006e respectively to their active sites, i.e. the blocked vessel and the damaged tissue in the penumbra, providing a novel paradigm for targeted drug delivery into the brain. The same research team has also introduced a nanocarrier composed of a ROS-responsive core and a SHp inserted red blood cell (RBC) membrane shell for stroke treatment.[198] Likewise, RBC-coated nanocrystal functionalized with c(RGDyK) has also shown great promise in facilitating the chemotherapy of glioma.[199] Interestingly, this nanosystem appears to have a pH-sensitive property

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and releases the drug quickly in PBS at pH 6.5 but not at pH 7.4. Given the acidic microenvironment of glioma, this may further ensure the site-specific delivery of drugs. In addition to above-mentioned nanosystems, pH-sensitive bovine serum albumin (BSA) nanoparticle[200], high-intensity focused ultrasound-responsive PLGA nanoparticle[201], GSH-sensitive prodrug[88], MMP-responsive nanoparticle[202], etc. have also been engineered with

oo

pr

sites within the brain and improve therapeutic efficacy.

f

both bioresponsive components and targeting ligands to enhance their accumulation at pathological

e-

4.3. Multi-targeting strategy

Pr

In addition to the dual-targeted nanoparticles mentioned above, multi-targeted nanoparticles have also attracted extensive attention. Consisting of a more complex group of targeting components,

al

these systems can be highly programmed and tailored to the needs of the clinical context (Fig. 7).

rn

The toxin trichosanthin (TCS) is a ribosome-inactivating protein with anti-tumor activity. However,

Jo u

due to rapid renal clearance, poor BBB penetration and cell permeation, its clinical application has been restricted to a few gynecological diseases.[203] Chen et al. have successfully established a multi-targeted delivery system loading TCS for the treatment of glioma. This rocket-like nanosystem consisted of four sequential components: Lf to overcome the BBB, MMP-cleavable peptide as a separator, low molecular weight protamine (LMWP) to penetrate the tumor and TCS as the anti-tumor therapeutic (Fig. 8A). After crossing the BBB with the aid of Lf, the nanoparticle was subjected to MMP-2 cleavage. The TCS/cell-penetrating peptide (CPP) portion of the nanoparticle was released and penetrated the tumor cells with LMWP as a CPP, so that TCS could enter the cytoplasm and inactivate the ribosomes of glioma cells. Compared with unmodified TCS, the

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nanohybrid protein exhibited rather better accumulation in the tumor, a moderate improvement in treatment efficacy and an alleviation of systemic toxicity.[204] Similarly, An et al. designed a multifunctional nanoparticle to deliver doxorubicin (DOX) into glioma cells. LAT1 is a branched-chain amino acid transporter highly expressed by the brain capillary endothelial cells and glioma cells, which supports the rapid growth of tumors. 3CDIT, a

f

substrate of LAT1, can serve as a targeting moiety to facilitate drug delivery across the BBB and into

oo

the tumor cells. The authors intercalated DOX into the ATP-responsive DNA scaffold and condensed

pr

the complexes with 3CDIT-decorated pOEI to form the 3CDIT-targeting pOEI/ DOX/ATP aptamer

e-

nanoparticles. When pumped into the glioma cells, the nanoparticles disintegrated in response to a

Pr

high concentration of GSH through the reduction of disulfide bonds within pOEI. Then, elevated ATP levels in cancer cells rapidly triggered the further release of DOX into cytoplasm from the

al

DOX/ATP aptamer complexes (Fig. 8B). As demonstrated by in vitro and in vivo experiments, the

rn

targeted delivery system enabled better drug accumulation in gliomas than free drugs, improving the

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anti-tumor effect without causing increased systemic toxicity.[205] Cells are composed of subcellular compartments and spaces within compartments are separated from the cytosol by lipid membranes. Many therapeutic agents need to be transported into a certain compartment to exert maximal effects, depending on their mechanisms of action, which poses a challenge for the further development of drug delivery systems.[206,207] After nanoparticles become internalized, most of their cargo is entrapped within the endosomes and lysosomes. Escaping from the degradation in the endo/lysosomes is a primary step before orienting to most subcellular compartments other than the lysosomes.[208,209] Components mediating endosomal destruction via various mechanisms are needed to avoid drug degradation by the lysosomal enzymes and release the

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drugs into the cytoplasm. Widely used components identified for this process include polyethylenimine (PEI), glutamic acid-alanine- leucine-alanine (GALA) motifs and membrane-perturbing peptides, etc.[210,211] Utilizing targeting components specific for an organelle is a key step in designing organelle-targeted nanomedicines, such as peptide D[KLAKLAK]2

(KLA) for drug delivery into the mitochondria and nuclear localization signal (NLS)

f

peptide motif for nuclear targeting.[212,213] The interaction between nanoparticles and subcellular

oo

structures can be detected by fluorescence microscopy with the aid of organelle trackers and

pr

transmission electron microscopy exclusively for metallic nanoparticles.[214] Stimulus-sensitive

e-

components are also needed in some instances to ensure a proper release of the drugs.

Pr

Gene therapies based on nucleic acids and oligonucleotides are the most highlighted novel treatment for a number of diseases lacking effective therapy. However, nucleic acids are examples of agents

al

that cannot simply be administered in their free form to cause therapeutic effects, due to their

rn

sensitivity to enzymatic degradation and the low permeability of the BBB. Successful nucleic

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acid-based therapies for the brain disorders require functionalized delivery systems mediating the in vivo processes of cell uptake, endosomal/lysosomal escape and drug release in the correct intracellular compartment.[215]

Although diverse drug delivery systems have been developed, traditional vectors such as viruses are not universally applicable for the delivery of all nucleic acids.[216,217] With this consideration, non-viral delivery systems, including multifunctional nanoparticles, seem to be promising alternatives to nucleic acid-based therapeutics. For instance, Qiao et al. have successfully adjusted the activity of lymphocytes in gliomas by downregulating the expression of TGF-β with siTGF-β with a multi-targeted nanoparticle ALBTA (Ang-LiB(T+AN@siTGF-β)).[218] Angiopep-2 was

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selected to guide the system across the BBB and facilitate the internalization of tumor cells. Endosomal/lysosomal escape and cytoplasmic translocation of the nanosystem was mediated by the zwitterionic lipid DSPE-PCB. Finally, the ROS-responsive BAP polymers were adopted for the release of the TMZ and siTGF-β within tumor cells. The nanosystem was traceable by MRI with SPIONs encapsulated by the BAP polymers (Fig. 8C). Subsequent in vivo data proved the efficacy of

f

this drug delivery system: After administration, the treatment group exhibited significant ly improved

oo

ratios of both CD4+ Teff/Treg and CD8+ CTL/Treg, with the percentages of CD8+ CTL and helper T

pr

cells elevated, and the percentages of Treg reduced. An improvement in the survival time was also

Pr

as effective carriers for enhanced gene therapy.

e-

observed in the treated mice. These findings demonstrate that nanoparticle-based systems could act

rn

al

5. Conclusion and perspectives

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The studies described in this review emphasize the potential use of dual and multi-targeted nanoparticles in site-specific brain drug delivery. The past years have witnessed the development of the concept of site-specific delivery from the organ level to the organelle level. With a new wave of technologies, various targeting functionalities have been combined to develop dual and multi-targeted nanoparticles for site-specific brain drug delivery, which exhibit great potential to enhance therapeutic efficacy by delivering drugs specifically to the pathological cell populations or further, into a certain organelle and release the drug in response to a trigger stimulus. These dual and multiple-targeting strategies allow more smart control of drug delivery systems and promising therapeutic treatment of brain diseases. In this review, we mainly illustrate the targeting strategies

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applied in intravenous nanotherapy. Other routes of drug administration, such as intranasal administration, have shown great potential in drug delivery to the brain. They are faced with various barriers (e.g. the nasal epithelial barriers in intranasal administration, the round window membrane in intratympanic delivery) and should be further studied before translation into clinical practice. In general, progress in dual and multi-targeting strategies have inspired us to explore more

f

possibilities of site-specific brain drug delivery. Developing these sophisticated nanoparticles

oo

requires a proper combination of diverse targeting components. Dual and multi- ligand-decorated

pr

nanoparticles can help to deliver drugs to specific organs, tissues and cells. However, this strategy

e-

confronts a dilemma: due to the steric hindrance, both an inadequate level and an excessive level of

Pr

ligand density can impair targeting.[47] Thus, a common difficulty in establishing dual ligand-decorated nanoparticles is optimizing the density and 3-dimensional arrangement of the

al

ligands, which is essential for the active site on the ligand to bind the target molecules. [219] In

rn

contrast, nanomedicines incorporating ligands and the bioresponsive groups may escape the setback

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of steric hindrance, offering an alternative way of dual- targeting strategy. Unlike ligands which should be conjugated to the surface in a proper orientation, bioresponsive groups can be arranged in a more flexible manner. Moreover, targeted nanoparticles equipped with bioresponsive components can avoid premature drug leakage within the circulation and are more versatile in terms of the responsive modes.[220] Despite the recent advances achieved in the design and development of dual and multi-targeted nanoparticles, the applications of these highly complicated strategies are still at the proof-of-concept stage. In animal models, the amount of drug that can arrive in the brain via dual and multi-targeted nanoparticles remains unclear in most cases, and accumulation rates under 10% were reported in

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some studies.[36,156] The clinical translations that have been achieved so far are rather limited, including mostly simple nanoparticles lacking active targeting or stimulus-responsive drug release components. Understandably, dual and multi- targeted nanosystems are more difficult to be adapted to mass production in the pharmaceutical industry due to the complex preparation process. [220,221] Besides, there is an ongoing concern about the safety profiles of nanomedicines. [220] More data are

f

needed to provide better insight into how well these nanomedicines work in vivo. As a result, there is

oo

still a wide scope of challenges to address before the clinical translation of these dual and

e-

pr

multi-targeted nanotherapeutics.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (no. 81901212 to

Jo u

rn

2018YFC1312200).

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YFZ, no. 81820108010 to BH), National Key Research and Development Program of China (no.

*Color should be used for Fig. 1 in print.

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Journal Pre-proof

Table 1. Pathological processes involved in brain diseases and the corresponding molecular signatures Molecular signatures

Apoptosis and tissue injury

HSP72, caspase, IL13α2, Fn14

Oxidative stress

ROS, H2 O2

Inflammation, cell migration

P/E selectin, FAS, MMP-2, NCAM, CD44, fibronectin extra domain B

f

Pathological process

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and homing

NPY Y1 receptors, nucleolin, VEGFR, GD2, SSTR, CXCR4, CD133

Matrix remodeling

MMPs

Abnormal metabolism

Hyperthermia, low pH value, LRP1, LDLR, LAT1, TfR, FR, GLUT

Pr

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pr

Proliferation and stem cell

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Abbreviations:

rn

heat shock protein (HSP); interleukin (IL); reactive oxygen species (ROS); neural cell adhesion

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molecule (NCAM); matrix metalloproteinase (MMP); neuropeptide Y (NPY); vascular endothelial growth factor receptor(VEGFR); cxc chemokine receptor (CXCR); somatostatin receptor (SSTR); transferrin receptor (TfR); low density lipoprotein receptor-related protein 1 (LRP1); low density lipoprotein receptors (LDLRs); glucose transporters (GLUT)

Table 2. An overview of dual and multi-targeted nanoparticles for site-specific brain drug delivery.

Journal Pre-proof Targeting

Molecular

Animal Target sites

strategies

Zeta

Carriers

Payload

Size (nm)

Potential

signatures

Ref. model

(mV) Parallel targeting with dual ligands

f o

pathological cell gold Tf

peptide/EGF

glioblastoma

TfR/EGFR

1/pathological

nanoparticle

cell 2

s

o r p

Pc4

peptide

pathological cell angiopoietin-2

angiopoietin-2/

glioblastoma Ab/VEGF Ab

VEGF

n r u cell 2

Cascade targeting strategy Congo amyloid AD

al

1/pathological

o J

red/boronate ester

41

-

mice

[164]

-

-

SCID mice

[169]

e

r P

bispecific

-

Abs

microenvironme nt/microenviron

APPswe/PS1 USPIONs

rutin

27

-5

dE9

plaques/ROS bonds

ment

transgenic

[222]

Journal Pre-proof mice

ICR

f o

PEG-PLA TGN peptide/QSH AD

BBB/microenvir -/Aβ

nanoparticle

peptide

l a n

r u o

-/Aβ peptide

J

100

subjected

-20

-6/DiR

s

AD

p e

onment

TGN peptide/QSH

ro

coumarin

BBB/microenvir

intracerebrov [175]

injection

ICR PEG-PLA

s

of

Aβ1-42 mice

subjected H102

to

intracerebrov 120

onment

to

entricular

r P

nanoparticle

mice

-28

peptide

[176] entricular injection

of

Aβ1-42 AD

angiopep-2/D1

LRP1/Aβ

BBB/microenvir

GNRs

-

20

-10

Caenorhabdit [223]

Journal Pre-proof peptide

onment

is elegans

BBB/microenvir Lf/disulfide amnesia

polymersom bacoside LfR/pH /GSH

onment/microen

linkages

Swiss albino 150

es

s

hydrogel epilepsy

96.8 (10

nanoparticle field

s

glioblastoma

BBB/microenvir

al

TfR/pH bonds

T7

onment

peptides/ TfR/magnetic

glioblastoma MNPs

o J

field

n r u

BBB/pathologic

PHT

e

onment

Tf/acyl hydrazone

r P

PAMAM

DOX

Sprague-Daw -

-1

mg mL )

[190] ley rats

71

-

-

[225]

130

-15.9

mice

[196]

60

11

mice

[226]

magnetic paclitaxe PLGA l/curcum

al cell

f o

o r p

BBB/microenvir

angiopep-2/PSS

[224] mice

vironment

LRP1/electric

-26

nanoparticle in s

TfR/magnetic glioblastoma

BBB/pathologic

PEG-PLL

Tf/MNPs

siPLK1 field

al cell

nanoparticle

Journal Pre-proof s

BBB/pathologic glioblastoma

angiopep-2

LRP1

BALB/c nude UCNPs

Gd

47.9

al cell BBB/microenvir glioblastoma

iNGR/pOEI

o r p

siRNA

e

PEG-PCL BBB/pathologic glioblastoma

angiopep-2

LRP1

al

al cell

glioblastoma

apoE3

LDLR

o J

n r u

r P

13(10μg/μl)

[228] mice

paclitaxe

nanoparticle s

BALB/c nude

μl)

nanospheres

[227] mice

90(10μg/

RNAi

NRP-1/GSH onment

f o

30.8

70

−3.28

mice

[155]

l

lipid

BBB/pathologic

porphyri nanoparticle

al cell

BALB/c nude 30

-

[156]

n

mice

Temozol

C57BL/6

s lipid BBB/pathologic

glioblastoma

Lf

LfR

nanoparticle al cell

68~81 omide

s

−10

[229] mice

Journal Pre-proof metronid tertiary

microenvironme azole/do

glioblastoma

amines/metronida

pH/hypoxia

nt/microenviron

liposomes

BALB/c nude [230] 167.96

-

xorubici zole

mice

ment n doxorubi PLGA

glioblastoma

LRP1/HIFU

BBB/-

r P

nanoparticle

nanoparticles

l a n

r u o

s

o r p

cin/perfl

e

angiopep-2/PLGA

f o

uoroocty

41

BALB/c nude -20.7

[201] mice

l

bromide

BBB/pathologic

cRGD/electrostati glioblastoma c interaction

J

αv β3 and αv β5 al integrins/pH

BSA

DOX

241

-12.6

-

[200]

97.08

-

mice

[88]

cell/subcellular compartment

Pep-1/ glioblastoma

disulfide

pathological IL-13Rα2/GSH

bond

Paclitaxe prodrug

cell/subcellular

l

Journal Pre-proof compartment BBB/pathologic angiopep-2/hydraz glioblastoma

al

Kunming

LRP1/pH

AuNPs

one

DOX

35

cell/subcellular

BBB/pathologic

glioblastoma

CD13/pH

MSN

ne

cell/subcellular

l a n

o r p

e

r P

al

DOX

160.1

[231] mice

f o

compartment

NGR/polydopami

-20

BALB/c nude −22

[232] mice

compartment

EGF glioblastoma

disulfide

J

BBB/microenvir

bond/ EGFR/GSH/pH

hydrazone bond

glioblastoma

r u o

peptide/

BALB/c nude AuNPs

-

[23] mice

BBB/microenvir

lipid

ed

onment/patholo

nanoparticle

gical cell

s

lipopeptide

80

onment

angiopep/PEGylat cleavable LRP1/MMP

DOX

siRNA

192

−7.0

-

[233]

Journal Pre-proof PEG– BBB/pathologic glioblastoma

tLyp-1

NRP1

BALB/c nude PLA nanopa PTX

111.30

−24.3

al cell

[234] mice

rticles PEG–PLA

f o

BBB/pathologic glioblastoma

peptide-22

LDLR

nanoparticle

PTX

o r p

al cell s

angiopep-2/nitro glioblastoma

BBB/microenvir LRP1/ hypoxia

groups

FHK

l a n

onment

o J

peptides/ NRP1/tenascin

glioblastoma tLyp-1

C

ur

e

r P

DOX/

metronidazo

124.7

prodrug

BALB/c nude −29.2

[235] mice

90

-25

ICR mice

[191]

les PEG-PLA

BBB/microenvir

BALB/c nude nanoparticle

PTX

132.5

−30.3

onment

[167] mice

s CPEGGM-

des-octanoyl glioblastoma

BBB/pathologic GHSR/FR

ghrelin/folate

BALB/c nude PDSGM

al cell

DOX

88.6

-

[236] mice

polymersom

Journal Pre-proof es/copolym ers angiopep-2/c(RG

LRP1/αv β3 and BBB/pathologic

Gd3+-DT

PAMAM

glioblastoma

10 DyK)

αv β5 integrins

al cell

dendrimers

αvβ3 and αvβ5 RGD/IL-13 glioblastoma

integrins/

s

[237] mice

120

−10

mice

[238]

l a n

r P

8

-

mice

[239]

superparam

BBB/pathologic

agnetic iron

al cell

oxide

r u o

J

10

-6

αv β3 and αv β5

receptor

o r p

e

al cell

integrins/folate

f o

coumarin nanoparticle

IL13Rα2

c(RGDyK)/folate

PA

PEG-PCL BBB/pathologic

peptide

glioblastoma

BALB/c nude

-

nanoprobes

BBB/ PEG–PLA

K237 peptide/CK glioblastoma

pathological cell VEGFR-2/SHH

peptide

BALB/c nude nanoparticle

(VM channels)/

117.36

-26.72

[240] mice

s pathological cell

PTX

Journal Pre-proof (tumor cells) DTX/VE BBB/pathologic glioblastoma

angiopep-2/tLyP-1

LRP1/NRP

BALB/c nude liposomes

GF

110~150

25.1

al cell

mice siRNA AONs

anti-TfR

PMLA

mAb/anti- EGFR

TfR/EGFR al cell

mAb

mediated

glioblastoma mannose

l a n

r u o

absorption ethylenediamine/

r P

nanobioconj

J

transcytosis/GL

ugates

f o

o r p

against

e

BBB/pathologic glioblastoma

[241]

CK2α

15.1±0.7

BALB/c nude -3.7

[242] mice

and EGFR

BBB/pathologic

38.2(c-HAS); HSA

DOX

BALB/c nude

90.5

al cell

[243] -26.2(m-HAS)

mice

UT ATWLPPR glioblastoma

BBB/pathologic

PEG–PLA

NRP1 /HSPG peptide/CGKRK

BALB/c nude PTX

al

nanoparticle

123

−11.4

[187] mice

Journal Pre-proof peptide

cell/microenviro

s

nment disulfira albumin T12

m/copper

glioblastoma

nanoparticle peptide/mannose

nnose receptors

IL-13Ra2/fibrin –fibronectin

al

rn

al cell

u o

complexes

glioblastoma

MAN/Tf

J

GLUT1/TfR

o r p

regorafe

Balb/c

nude

-18.7

[244] mice

e

r P

nib

PLGA-PEG

BBB/pathologic Pep-1/CREKA

complex/ 132.2

al cell s

glioblastoma

f o

TfR/SPARC/ma BBB/pathologic

nanoparticle

BALB/c nude PTX

101.1

-25.6

[245] mice

s

BBB/pathologic

Spraque– liposome

DNR

122.80

− 7.46

al cell

[246] Dawley rats

αvβ3 and BBB/pathologic glioblastoma

cRGD/AS1411

αvβ5 integrins/

AuNC al cell

nucleolin

DOX

3

-15.13

nude mice

[247]

Journal Pre-proof BBB/pathologic glioblastoma

NGR/T7

CD13/TfR

RBCNPs

VCR

123.67

DTX

170.6

-

ICR mice

[248]

al cell PEG-PCL BBB/pathologic glioblastoma

TGN/AS1411

nanoparticle

-/nucleolin al cell

s/copolymer s

nicotine acetylcholine receptors glioblastoma

D

r u o

(nAChRs)/αv β3

integrins

[22] mice

o r p

e

r P

BBB/BBTB/pat

CDX/c(RGDyK)

and

l a n

f o

BALB/c nude -8.79

BALB/c nude liposomes

DOX

93.9

-

hological cell

[180] mice

J

αv β5

αv β3 and αv β5 BBB/microenvir glioblastoma

glioblastoma

c(RGDyK)/-

Tf/anti-Nestin Ab

integrins/pH

onment

TfR/nestin

BBB/pathologic

RBCNPs

DTX

70

-23

BALB/c mice

[199]

SPIONs

TMZ

84.37

-11.6

BALB/c nude [249]

Journal Pre-proof polysorbate-80/

lipoprotein

anti-Nestin Ab

receptor/nestin

al cell

101.56

brain

-20.1

mice

DOX LDLR/αvβ3 an

metastases of polysorbate-80

terpolymer BBB/pathologic

and ‐ lipid

d triple negative /cRGD

f o

126

al cell

mitomyc

αvβ5 integrins

in C

e

r P

platelet membrane the

pathological

l a n

/thrombin stroke cleavable

ed

bin

nanoplatelet

ur

amino

acid sequence

AMD3100/NH2 -L

o J

CXCR4/MMP-

GRMGLPGK-C-S 2

nment

rtPA

[185]

rat

bioengineer

thrombus/throm cell/microenviro

with -

female

NRG mice

o r p

nanoparticle

breast cancer

old 43.6

-

model middle

cerebral

[197]

artery occlusion

BBB/microenvir

polymeric

onment

micellar

glyburid

stroke

C57BL/6 226.3

H

CXCR4/Throm

BBB/microenvir

nanoparticle

AMD3100/NH2 -n

bin

onment

s

e

5.85

[202] mice

Journal Pre-proof orleucine-TPRSF L-C-SH BBB/pathologic stroke

T7 peptide/SHp

TfR/-

liposomes

ZL006

96.24

al cell

stroke

SHp/boronic ester

-/-

site/microenviro nment

r u o

Multi-targeted nanoparticles to intracellular compartment breast cancer FA/hydrazone bon and

brain ds/dNP2 peptides

l a n

FR/pH/-

J

membrane-c

o r p

e

r P

oated

ICR mice

[178]

f o

RBC pathological

3.237

NR2B9C

194.6

Sprague– 12.3

[198] Dawley

rats

nanoparticle s

BBB/microenvir liposomes

PTX

100

-6

siTGF-β/

120(N/P

10(N/P

BALB/c mice

[250]

mice

[251]

onment/BBB metastasis angiopep-2/DSPE glioblastoma

BBB/pathologic LRP1/-/ROS

-PCB/BAP

SPIONs al

TMZ

ratio=10)

ratio=10)

Journal Pre-proof cell/endosomal escape /subcellular compartment

f o

BBB/pathologic al

e

DOX/ATP 3CDIT/pOEI/ATP

LAT1/GSH/AT

r P

cell/subcellular

glioblastoma

aptamer aptamer

P

compartment/su bcellular

al

o r p

DOX

-

BALB/c nude -

[205] mice

complexes

n r u

αv

o J

compartment BBB/pathologic al

iron

oxide

iRGD/CGKRK/[K glioblastoma

[KLAKL integrin/NRP-1/ cell/subcellular

nanoparticle

LAKLAK]2

80~100 AK]2

-/-

compartment/su bcellular

s

BALB/c nude -

[252] mice

Journal Pre-proof compartment BBB/pathologic Tf/AS1411/disulfi

TfR/nucleolin/

al

[Ru(bpy)

glioblastoma

MRNs de bonds

GSH

2+

cell/subcellular

2 (tip)]

compartment BBB/microenvir LF/MSP/ LMWP

LRP1/MMP/-

onment/patholo

r P

protein gical cell

l a n

TCS

38.6

-

[253] mice

f o

o r p

e

nanohybrid glioblastoma

BALB/c nude 138

BALB/c nude -

[204] mice

BBB/pathologic

Parkinson’s

B6/Mazindol/boro

o J

TfR/DAT/ROS disease

nate ester

ur al

cell/subcellular

αS-overexpre SPIONs

EGCG

80

-0.20~-0.70

[254] ssing mice

compartment

Abbreviations: transferrin receptor (TfR); epidermal growth factor receptor (EGFR); antibody (Ab); vascular endothelial growth factor(VEGF); reactive oxygen species (ROS); ultrasmall superparamagnetic iron oxide nanoparticle(USPION); amyloid beta (Aβ); 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR); low density lipoprotein receptor-related protein 1 (LRP1); gold nanorods (GNR); lactoferrin (Lf); magnetic nanoparticles (MNPs); neuropilin-1 (NRP-1); glutathione (GSH); high-intensity focused ultrasound (HIFU); bovine serum albumin (BSA); gold nanoparticles (AuNP); mesoporous silica nanoparticle (MSNs); low density lipoprotein receptors (LDLRs);

Journal Pre-proof paclitaxel (PTX); docetaxel (DTX); doxorubicin (DOX); gadopentetate (Gd-DTPA); Sonic hedgehog (Shh); antisense oligonucleotides (AONs); naïve albumin (HSA); daunorubicin (DNR); gold nanoclusters (AuNC); red blood cell membrane-coated nanoparticles (RBCNPs); vinca alkaloid vincristine (VCR); temozolomide (TMZ); recombinant tissue plasminogen activator (rtPA); folate (FA); folate receptor (FR); mesoporous ruthenium nanoparticles (MRN); phenytoin sodium (PHT); toxin trichosanthin (TCS); matrix metalloproteinases (MMPs); p-aminophenyl-α-D-manno-pyranoside (MAN); low-molecular weight protamine (LMWP); ghrelin/growth hormone secretagogue receptor (GHSR); dopamine transporter (DAT); epigallocatechin gallate (EGCG); large amino acid transporter 1 (LAT1); polyelectrolyte poly(sodium 4‐ vinylbenzene sulfonate) (PSS); heparan sulfate proteoglycan (HSPG); blood–brain tumor barrier (BBTB)

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Fig. 1. Schematic illustration of multi-targeting strategy for site-specific brain drug delivery. To

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optimize control over the biological process of nanomedicines, a proper design of the dual or multi-targeted nanoparticles is essential, with several key points along the way to the target (usually

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the active site of the drug) being the focus of attention: (1) overcome the BBB; (2) specifically target

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to the lesion and trigger internalization; (3) enable the endo/lysosomal escape and navigate to the

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target organelle; (4) release the drugs in a controlled manner.

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Fig. 2. Schematic illustration of parallel targeting strategy. In parallel targeting strategies, the

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nanoplatforms are conjugated with two different targeting moieties, each to bind respectively with

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one type of receptors on the diseased cell membranes. These approaches address a single issue during drug transportation, namely the penetration of target cell membranes, with dual targeting

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groups interacting simultaneously with different pathological signatures.

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Fig. 3. Parallel targeting strategies applied in the site-specific brain drug delivery. (A) Design of

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a dual-targeted AuNP loaded with Pc. a) cap exchange (20% SH-PEG-COOH/80% SH-mPEG); b)

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EDC/Sulfo-NHS (Formation of covalent amide bond); c) Pc4 encapsulation (Pc4 PDT drug). [164] (B) Schematic design of the Ft peptide-modified poly (ethyleneglycol)–poly (lactic acid)

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nanoparticulate system (Ft-NP-PTX). Composed of FHK and tLyp-1 coupled via a cysteine, the

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heterodimeric Ft peptide interacts specifically with tenascin C, an aberrant extracellular matrix

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component, in addition to NRP-1, an overexpressed tumor membrane receptor. (C) In vivo distribution of mice intravenously treated with a dosage of 1 mg/kg DiR-labeled NP, tLyp-1-NP, FHK-NP and Ft-NP. [167] (D) i) Gene expression in TAMs isolated from Gl261 tumors suggests a A2V treatment- induced increase in M1 function-related genes and decreased expression of M2 TAM-associated genes. ii) A2V decreases tumor vessel density in Gl261 tumors. iii) In the Gl261 model treatment with A2V shifts TAMS toward the M1 state. [169] The M1/M2 ratio showed a trend toward the M1 polarization state in A2V-treated animals compared with IgG-treated animals. Reprinted with permission from ref [164,167,169].

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Fig. 4. Cascade targeting strategy mediated by dual ligands. (A) Schematic illustration of the

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H102 peptide-loaded TGN and QSH peptide dual-decorated nanoparticles in the treatment of

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Alzheimer's disease. [175,176] (B) Schematic illustration of the angiopep-2 and EGFP-EGF1

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decorated NP (AENP). [179] (C) i) Preparation of AC peptide conjugated PEG−PLA nanoparticles. The ATWLLPPR peptide and CGKRK peptide were coupled to overcome the tumor microenvironment barrier and increase the tumor parenchyma penetration. ii) After administration, AC-decorated nanoparticles are capable of targeting tumor blood vessel endothelial cells through utilizing the specific affinity between ATWLPPL peptide and NRP-1, and targeting tumor cells by taking advantage of the high selectivity of CGKRK and HSPG with enhanced penetration. iii) In vivo glioma distribution of NP, ATWLLPPR-NP, CGKRK-NP, and AC-NP after treatment for 3 h. iv) Survival curve of U87MG bearing-mice following the treatment of saline, Taxol, NP-PTX, ATWLPPR-NP-PTX, CGKRKNP-PTX, and AC-NP-PTX (PTX dose 5 mg/kg). [187] Reprinted with

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permission from ref [175,176,179,187]

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Fig. 5. Schematic illustration of cascade targeting strategy combining ligands and biosensitive

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materials. Integrating the biosensitive quality and the targeting property can further enlarge the scope of targets: It allows a variety of internal environmental factors including redox potential, pH

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and temperature to be landmarks of the lesion, which cannot be achieved with nanoparticles

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decorated with merely targeting ligands.

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Fig. 6. Cascade targeting strategy combining ligands and bioresponsive materials. (A) i)

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PHT-loaded electro-responsive hydrogel nanoparticles modified with angiopep-2

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(ANG-PHT-ERHNPs) for antiepileptic therapy. ii) Representative EEGs recorded from the right amygdala and the corresponding energy spectra of the solvent, PHT solution and PHT preparation at the dose of 50 mg kg-1 . The black arrows denote the kindling stimulation artifacts. iii) The transport percentage of FITC, ERHNPs, and ANGERHNPs across an in vitro BBB model at various times. iv) PHT concentrations in the hippocampus after the administration of PHT, PHT-ERHNPs, and ANG-PHT-ERHNPs at the dose of 50 mg kg-1 for various times. [190] (B)i) Schematic illustrating ALP-(MIs)n applications: hypoxic ce ll radiosensitizer, hypoxia-responsive release of DOX into the cytoplasm, and then transports it to the nucleus to kill tumor cells. ii) Mechanism of ALP-(MIs)n RT sensitization and DOX release under hypoxic condition and formation of ALP-(MIs)n/DOX. Six

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electrons are transferred in the complete reduction of nitro (R-NO2) to amine (R-NH2) under hypoxic conditions via a single-electron reduction catalyzed by a series of intracellular nitroreductases. [191] (C) i) Schematic illustration of BBB-penetrating and tumor-targeting delivery via the T7-mediated and magnetic- guided, dual-targeting MNP/T7-PLGA NPs. ii) In vivo distribution of the nanoparticle systems by IVIS animal imaging system after injection via tail vein at

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4 h. [196] Reprinted with permission from ref [190,191,196]

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Fig. 7. Schematic illustration of multi-targeting strategy. Multi-targeted delivery system consist

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of several components to address a series of tasks: overcome the BBB, induce cellular internalization,

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release.

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mediate the escape from endo/lysosomes and target to the subcellular compartment and trigger drug

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Fig. 8. Multi-targeted nanoparticles for cellular and intracellular targets. (A) Illustration of the

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rocket-like, multi-stage booster delivery strategy of the BBB-penetrating nanohybrid TCS toxin for

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antiglioma treatment. [204] (B) Preparation, LAT1-mediated transport, and GSH & ATP

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dual-responsive DOX release of 3CDIT-targeting pOEI/DOX/ATP aptamer NPs. [205] (C) i) Chemical structural formula of each component and preparation of the targeting nanoparticles

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(Ang-LiB(T+AN@siTGF-β), ALBTA). (1) BAP polymers could self-assemble into nanoparticles by

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encapsulating SPIONs in the hydrophobic region. (2) The positively charged AN could load siTGF-β

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by electrostatic attractions. (3) Then zwitterionic lipid-based envelopes were coated to the nanoparticles. (4) The targeting molecules angiopep-2 were conjugated to nanoparticles by the reaction between maleimide groups of DSPE-PCB-mal and sulfhydryl groups (-SH) of angiopep-2. ii) Schematic diagram of cellular uptake and subcellular drug delivery of ALBTA. (1) The cellular uptake of targeting NPs via receptor-mediator endocytosis, (2) the acidifcation and perturbation with the membranes of endosomes/lysosomes, the endosomes/lysosomes escape and the release of (3) TMZ molecules and (3′) nanoparticles into cytosol, (4) TMZ enter into nuclei. After being oxidized by ROS (4′), nanoparticles release (5-1′) siTGF-β and (5-2′) SPIONs into cytosol. SPIONs could serve as contrast agents for MRI. (6′, 7′) SiTGF-β could down-regulate the secretion of TGF-β and

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(8′). [251] Reprinted with permission from ref [204,205,251]

Journal Pre-proof Graphical Abstract Highlights The pathological cells and organelles are a more specific target for nanomedicines Nanomedicines for brain disorders should cross the BBB and target to the damaged site Dual and multi-targeting strategies can address both the processes

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The targeting moieties can act in a parallel or sequential manner in vivo