Accepted Manuscript

Recent progress of drug nanoformulations targeting to brain

Abdur Rauf Khan, Xiaoye Yang, Manfei Fu, Guangxi Zhai

PII: S0168-3659(18)30575-3
DOI: doi:10.1016/j.jconrel.2018.10.004
Reference: COREL 9487
To appear in: Journal of Controlled Release
Received date: 12 September 2018
Revised date: 3 October 2018
Accepted date: 4 October 2018

Please cite this article as: Abdur Rauf Khan, Xiaoye Yang, Manfei Fu, Guangxi Zhai ,
Recent progress of drug nanoformulations targeting to brain. Corel (2018), doi:10.1016/
j.jconrel.2018.10.004

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 ACCEPTED MANUSCRIPT

RECENT PROGRESS OF DRUG NANOFORMULATIONS

TARGETING TO BRAIN
Abdur Rauf Khan, Xiaoye Yang, Manfei Fu, Guangxi Zhai*

Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan 250012, China

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* Corresponding author:

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Guangxi Zhai, Ph D

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Professor

Department of Pharmaceutics
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School of Pharmaceutical Sciences, ShandongUniversity
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44 Wenhua Xilu, Jinan 250012, China

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Tel.: (86) 531-88382015.

E-mail: professorgxzhai@126.com
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Abstract

Most of the potential therapeutic agents capable to modulate the pathophysiology or treat the
neurological disorders and brain tumors are useless in the current modern and advanced era of
neuroscience due to the impeding action of biological barriers. Among various therapeutic
strategies applied for translocation of drug delivery across the blood-brain barrier (BBB),
nanoformulations set an excellent platform for brain targeting by overcoming the biological and

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chemical barriers and protecting drug from efflux to promote the optimum therapeutic drug

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concentration in brain parenchyma tissues. Nanocarriers are the most widely studied delivery

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vehicles for BBB translocation with the efficiency of selectively targeting or exploiting inherent
biological molecules, receptors, carriers or mechanisms of the brain. Nearly all of the available

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drug delivery nanocarriers explored in recent years for brain therapeutics and theranostics are
based on lipid or polymeric materials. Polymeric nanoparticles (NPs) and lipid based
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nanocarriers including liposomes, solid lipid NPs (SLNs) and micelles, etc. are under the direct
focus of neuroscientists due to the promising attributes and vast applications in neurological
disorders. Surface modification of nanovehicles with proper targeting moiety or coating with
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surfactants promotes the interaction with endothelial cells and passage of nanocarriers to the
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brain. This review comprehensively depicts challenges to the brain targeted drug delivery,
mechanisms of drug transportation across the BBB, and potential contributions of endogenous
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cells as NPs delivery cells and novel targeting ligands decorated nanoformulations in imaging,
treating and controlling neurological disorders.
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Chemical compounds studied in this review article:

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Ethylene glycol (PubChem CID: 174); 1-methyl-2-pyrrolidinone (PubChem CID: 13387);

Polyamidoamine dendrimers (PubChem CID: 161547); Polyisohexylcyanoacrylate (PubChem
CID: 108097); Poly lactic-co-glycolic acid (PubChem CID: 23111554).

Keywords: Blood-brain barrier, Receptor mediated transport, Stem cells, Exosomes,

Nanoformulations, Dendrimers, Solid lipid nanoparticles, Nanogel.
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1. INTRODUCTION

Neurological disorders are life threatening abnormalities of brain and nervous system severely
affecting the quality of human life. Neurological disorders are prevailing in America with more
than 100 million American people experiencing central nervous system (CNS) diseases such as
Alzheimer’s disease and Parkinson’s disease. Annual cost of neurological disorders in US
accounts to be $800 billion and in 2050, this figure will exponentially increase with

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elderly population becoming double [1]. High level research and polices at government

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level are highly demanded to overcome an individual's loss of health-related quality of life as

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well as the personal and socio-economic burden. Pharmaceutical industry seems unarmed to
impart the potential therapeutic remedy for CNS ailments. Neuroscientists considered
impervious and tightly compacted BBB endothelial cells accountable for slow growth and

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progression of CNS global market. In addition to the BBB, early diagnosis of neurological
disorders is challenging in the development of neuropharmaceuticals. Neurological disorders
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such as Alzheimer’s disease progress slowly for many years, and have variable and silent
symptoms among the patients, even in the same patient [2]. Damage has already been done upon
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the appearance of symptoms. To understand the underlying development mechanism of disease

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is another challenge for successful therapy of neurodisorders and avoidance of unwanted off-
target effects.
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Neurological disorders such as Alzheimer’s disease, Parkinson’s disease, epilepsy,

schizophrenia, traumatic brain injury, brain stroke, ischemia, lysosomal storage disease, anxiety,
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depression and multiple sclerosis are major CNs disorders. Alzheimer′s disease involves
deposition of Aβ plaque in brain microvasculature and accumulation in the lymphatic cleaning
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system [3]. Immunotherapy of Alzheimer′s disease based on active and passive targeting of Aβ
antigen has been conducted in mice and clinical trials in human [4]. Astrocytic gliosis is
primarily contributing towards pathology of Alzheimer’s disease [5]. Recently, researchers
reported that astrocytes together with microglial cells penetrated the plaque and Aβ protein
[6]. Perivascular microglia/macrophages have the ability to clear Aβ. Strategy based on
modulation of Toll-like receptors and scavenger receptors found on the microglia could be
helpful in microglia clearance of Aβ. Phosphodiesterase (PDE) III is highly expressed in the
cerebral amyloid angiopathy (CAA) and Alzheimer’s patients. A study on CAA model mice was
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conducted using cilostazol. Cilostazol, a selective PDE III inhibitor, promoted eradication of Aβ
[7]. Ependymal cells produce CSF and are situated inside of brain ventricles [8]. In vivo
experiment based on intraventricular injection of galAd.RSV exhibited successfully transfer of
beta-galactosidase to the ependymal cells. Exosomes derived from neuroblastoma could be
therapeutically beneficial in the Alzheimer’s disease therapy [9]. Poly (D,L-lactide-co-glycolide)
(PLGA) NPs have wide applications in Alzheimers therapy. Peptide iAβ 5 is highly efficient in
amyloid β aggregate inhibition in Alzheimer’s disease. Encapsulation of iAβ 5 in PLGA NPs

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guided through OX26 mAb and anti-Aβ (DE2B4) resulted in an elevation of porcine brain

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capillary endothelial cells uptake [10]. Similarly, hybrid PLGA NPs coated with polyethylene

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glycol (PEG) and tween 80 promoted rosmarinic acid BBB translocation and retarding the Aβ
neuron degeneration through decreased apoptic SK-N-MC cells viability [11]. Monoclonal

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antibodies (mAbs) interacting with plasma Aβ peptide 1-42 decorated cyanoacrylate NPs
effectively decreased the soluble Aβ oligomers level in the brain and enhanced its blood level
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[12]. Theranostic potential of multifunctional liposomes for Alzheimer’s disease was also
studied. Liposomes were decorated with curcumin ligand targeting amyloid and other two BBB
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targeted ligands: one binding with transferrin receptor (TfR) and the other one binding with low
density lipoprotein receptor (LDLR) on BBB [13].
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Parkinson’s disease is associated with the degeneration of dopaminergic neurons, glial cells
activation, infiltration of inflammatory cells and inflammation based progression of disease.
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Mesenchymal stem cells (MSCs) interact with inflammatory cells and play an
immunomodulatory role through the rectification of compromised inflammatory response in
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Parkinson’s. Embryonic stem (ES) cells have potential to generate dopaminergic neuron slots in
Parkinson’s disease [14]. Early studies showed L-dopa generating neurons differentiated from
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ES cells transplanted into the brain of rat and mice [15]. However, recent study provided
evidence of successful differentiation of ES cells into dopaminergic neurons with the
reinstatement of motor functional activity in rat model [16]. Lodgment of misfolded α-Syn
agglomerates in the neurons, nerve fibers or microglia portrays contributive position in the
pathogenesis of Parkinson’s disease, dementia with Lewy Bodies (DLB) and multiple system
atrophy [17]. Snca gene is alpha-synuclein (α-Syn) encoding gene and it was investigated that
mutation of this gene led to altered expression of aberrant protein and increased risk of
Parkinson’s disease. Therapeutic strategies based on α-Syn targeting are to restrict production,
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aggregation and extracellular uptake of α-Syn and elevate intracellular and extracellular
degradation of α-Syn. Liposomes conjugated with rabies virus glycoprotein peptide (RVG29)
were explored to amplify the therapeutic efficacy of dopamine derivative. Liposomes efficiently
translocated the BBB and were taken up by the endothelial cells. Liposomal delivery expedited
the therapeutic effect of neuropharmaceutical in Parkinson’s disease model of mouse [18]. Gold
NPs (AuNPs) were explored as a nonviral gene vector to carry siRNA targeting and down
regulating the genes responsible for α-Syn expression. Composite transported pDNA into cells

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through receptor mediated endocytosis and inhibited p12 cells apoptosis [19].

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Traumatic brain injury (TBI) is a non-degenerative condition experienced by the mechanical

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force applied externally to the head with clinical manifestation of altered level of consciousness,
amnesia, with or without neurologic deficits [20]. Pharmacological interventions of TBI failed to

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produce impressive results in the clinical trials or couldn’t reach the stage of clinical trials. Many
processes are involved in the pathophysiology of secondary injury and available treatment
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options just work by inhibiting single participating event [21]. Pre-clinical and phase I clinical
trials based on intravenous bone marrow (BM) mononuclear cells for severe TBI adult therapy
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manifested declined neuroinflammatory cascades and restoration of CNS structures [22]. Probe
conjugated NPs could be exploited for their diagnostic and therapeutic applications to recognize
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the traumatic brain lesion and cargo the therapeutic agents to the necrotic tissues. In one study,
PEGylated PLGA NPs conjugated with 800CW imaging agent penetrated and accumulated in the
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necrotic area. Imaging agent promoted the NPs recognition of dead cells and visualized the NPs
accumulation in TBI lesion [23]. Dendrimers could be effective drug delivery vehicles for brain
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injury and neuroinflammation therapy such as TBI.

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Besides neurological disorders, glioblastoma multiforme (GBM) is another major CNS disease
with high morbidity and mortality rate. It is a malignant type of brain tumor and difficult to treat
due to poor prognosis and rapid growth. Despite of enormous research on GBM, the survival rate
of glioma patients is disappointedly very low [24]. Available treatment options based on
radiotherapy and chemotherapy have certain limitations of reoccurrence, resistance and
impermeation of tumor and CNS damage. In this current scenario, highly efficacious, BBB
permeable and potent chemotherapeutic agents delivered via innovative technology-based
nanoparticulate systems are urgently demended with no peripheral toxicity and safety profile.
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In recent decades, various invasive and non invasive strategies based on overcoming the
impeding aciton of impermeable BBB and targeting the required diseased sites of brain have
been elucidated [25]. The researchers, in the last few years, put all their efforts and energy to
design carrier systems that effectively overcome the obstacles and transfer drug molecules to the
brain parenchyma [26]. The clinical translation of formulations based on the applications of
nanotechnology opened a new gate for selective and targeted delivery of drugs.
Nanoformulations, especially, polymeric NPs made challenging therapy of world leading CNS

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diseases successful [27]. Nanoscopic formulations of biodegradable and biocompatible synthetic

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or natural polymers can load hydrophilic or lipophilic neuropharmaceutical agents in solid or

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solution form and protect them from biological and chemical hazards [28]. Nanoformulations,
for neurotherapeutic drug transportation, consisting of polymeric or lipid materials offer unique

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benefits of diverse size distribution and morphological characterization, surface modulation by
targeting moieties and surfactants, various pathways of administration and stimuli sensitive drug
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release behavior [29]. In this review, novel therapeutic approaches, under current development
and investigations for brain disease targeting, based on nanoformulations and endogenous cells
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such as astrocytes, macrophages and exosomes to improve the efficacy and safety profile of
neurotherapeutics are summarized in detail.
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2. Challenges to brain targeted drug delivery

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CNS poses many challenges for effective drug delivery to brain [30]. A drug has to cross many
barriers to achieve desired therapeutic level in CNS. Brain is typically characterized with four
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main barriers; BBB, blood-cerebrospinal fluid barrier, blood–brain tumor barrier and efflux
protein, which are explained below.
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2.1 The blood-brain barrier (BBB)

The BBB has a regulatory role in the proper functioning of brain and keeps peripheral circulation
separate from CNS. BBB offers protection from entry of hazardous materials to the brain. It is
composed of tightly packed endothelial cells presented along the capillaries in brain as shown in
Fig. 1. BBB performs filtering function and selectively permeable to water, nutrients and
hydrophobic molecules through passive transport [31]. Its protective mechanism involves P-gp
mediated efflux of lipid soluble toxins and bacteria. Additionally, it keeps fluid level of brain at
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required volume and prevents the frequent transport of fluid and salt to extracellular matrix.
Astrocytes are crucial for proper functioning of the BBB and provide support to the endothelial
cells of the BBB. Tight junctions are basic structural components essential for functional activity
of endothelial cells. Normal capillaries of the blood circulation are deprived of these tight
junctions of endothelial cells but the tight tight junctions of BBB endothelial cells protect the
brain [32]. Astrocytes and endothelial cells are joined together by the tight junctions. These
junctions consist of transmembrane proteins (occludin, claudins, junctional adhesion molecule

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(JAM), ESAM, and others). Endothelial cells bind to subunits of tight junction via complex of

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protein known as zo-1 and associated protein [33]. Some parts of brain including

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circumventricular organs do not have BBB [34]. In some disease conditions, BBB loses its
functions to some extent and allows the entry of bacteria and toxins to brain. Meningococcal

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bacteria open slightly the tight junction of endothelial cells and cross the BBB. The majority of
the drugs are unable to cross this barrier and lose their therapeutic effects, leaving neurological
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patients untreatable.
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Fig.1: Pictorial depiction of the BBB [27].

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2.2 The blood–brain tumor barrier (BBTB)

Normal functions of the BBB are altered during different disease conditions of brain such as
brain tumor, multiple sclerosis, stroke and dementia. Glioma is the primary brain tumor
originating from glial tissues and characterized by the loose “tight junctions” of endothelial cells
[2]. Low grade glioma has normal functioning of the BBB [35], whereas high grade glioma has
altered the permeability of brain vasculature [36]. High grade glioma comprises of enhanced

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angiogenesis and vascular endothelial growth factor (VEGF) to fulfill the demand of accelerated

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metabolism. This leads to formation of blood vessels and BBTB with altered functions [37].

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There might be many reasons of the altered permeability of endothelial cells. Astrocytes in the
glioma brain are not well differentiated and unable to secrete required chemicals for proper
functioning of the BBTB, which leads to leaky blood vessels [38]. Expression of claudins has

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also been studied to evaluate its potential role in the leaky endothelial junctions. It has been
reported that downregulation or loss of claudins and occludins enhanced the permeability of the
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BBTB. In previous research, downregulation of claudin-3 and claudin-5 expressions [39] and
loss of claudin-1 led to leakage of tight junctions [40]. It is highly needed to explore the
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mechanism through which claudins damage the BBTB. Claudin-1 controls the function and
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expression of cell-cell adhesion molecules through different signaling pathways [41]. The role of
claudin-5 to make endothelial junction leaky has been revealed in brain metastatic tumor [42].
Occludins are transmembrane proteins present in microvessels and their potential involvement in
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regulation of BBTB permeability is highly expected. Loss of their expression in astrocytes and
metastasis of adenocarcinoma has been found. Astrocytoma and other brain tumors secrete
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VEGF [43], cytokines and hepatocyte growth factor that downregulate the expression of
occludins [44, 45]. Hence, the BBTB is a major hindrance to the effective therapy of brain tumor
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leading to subtherapeutic level of chemotherapeutic agents in brain tumor tissues.

2.3 Efflux transporters

Multidrug resistance (MDR) is the resistance of tumors to the structurally unrelated drugs or
cytotoxins [46]. Presence of the BBB and different efflux transporters are responsible for poor
penetration of drugs to the brain and resistance of tumor. P-glycoprotein (P-gp) is the transport
protein dependent on ATP for its functioning of drug transportation. P-gp, belongs to ATP-
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binding cassette (ABC) gene family, decides about the fate of drug disposition into the CNS
[47]. P-gp is present on the different epithelial cells in the body [48]. Luminal membrane of the
BBB endothelial cells has phosphorylated protein that helps BBB to play its prominent role of
barrier to drug delivery. Multidrug-resistance-1(MDR1; ABCB1) gene encodes P-gp [49]. P-gp
is a substrate for variety of structurally similar drugs and hence, called multidrug transporter. It
keeps the brain safe from fat soluble substances and toxic material. Most of the lipophilic drugs
are expected to easily permeate the BBB and approach the brain. A large number of lipophilic

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drugs have shown high affinity to P-gp mediated efflux and low BBB permeability. P-gp results

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in the efflux of large number of fat soluble drugs from the brain and responsibles for the poor

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uptake of hydrophobic drugs to the brain. The active efflux mechanism of P-gp prevents the
uptake and cytotoxicity of anticancer agents to the brain tumor. In addition to P-gp, MDR

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proteins (MRPs) belong to ABCC family responsible for transport of anionic substances,
whereas P-gp mediates efflux of cationic substances from the brain [50]. However, some drugs
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are transported by the both proteins. Some of the MRPs are present in the luminal membrane of
the endothelial cells which is an ideal place for proper functioning of efflux transporter. Breast
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cancer related proteins (BCRP) are found to be present in the apical membrane of the BBB
endothelial cells and expectedly responsible for the inhibition of brain uptake of some potential
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drugs [51]. Members of organic anion transporting-polypeptide family (OATP-family) and the
organic anion transporter-family (OAT-family) are also located in the brain and may involve in
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the efflux of drugs. These transporters work along the concentration gradient. OATP and OAT
transporters are ATP independent reversible transporters and depend on the concentration of
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drug [52].
3. Transport mechanisms
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Various mechanisms involved in the transport of drugs across the BBB are shown in Fig. 2 and
summarized in Table 1. Passive diffusion and active transport are the major transport systems for
drug delivery to brain. Diffusion is the movement of molecules across the membrane along the
concentration gradient, between the cells (paracellular) or across the cells (transcellular). Small
size fat soluble molecules are transported through transcellular transport, which is easier than the
paracellular transport of water soluble compounds. Active transport may involve receptor
mediated endocytosis, adsorptive mediated endocytosis and carrier mediated transport [53].
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3.1 Receptor mediated endocytosis

Many types of receptors are found in the endothelial cells of the BBB and participate in the
uptake of drugs to the brain. Peptide and protein based drugs and other high molecular weight
substances are carried to brain through receptor mediated endocytosis [54]. Transferrin (Tf)
receptor, insulin receptor, low-density lipoprotein receptor–related protein, nicotinic
acetylcholine receptor, insulin like growth factor receptor, diphtheria toxin receptor, scavenger

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receptor call B type, leptin receptor and the neonatal Fc receptor are the key receptors binding

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with the ligands thus promotingthe endocytosis [55-71]. The binding of ligands with the

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receptors to trigger receptor mediated transcytosis has become the most widely used strategy to
cross the BBB and target the brain. It is assumed that nanocarriers release drug in the endothelial
cells and the drug is transferred through the transcytosis or diffusion to brain. Tf receptors (TfR)

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are the glycoprotein present on the endothelial cells of the BBB and majorly explored receptors
for brain targeting [69]. Tf could be conjugated on the surface of NPs to exploit the TfR for
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endothelial cells targeting [72, 73]. However, the applications of Tf as a targeting moiety to cross
the BBB are limited. Endogenous Tf competes with exogenous Tf for binding to TfR.
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Endogenous Tf has higher concentration in plasma than nanocarriers conjugated exogenous Tf

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and displaces the exogenous Tf from TfR, leading to poor targeting efficiency of delivery
systems [55]. Antibodies such as OX-26 recognize the TfR and have brain targeting properties. It
has been studied that OX-26 binds to TfR other than Tf binding sites and hence, endogenous Tf
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couldn’t displace it from TfR [48]. Mechanism through which Tf and OX-26 cause transcytosis
is unclear. It has been reported that Tf and OX-26 can cross the BBB but Tf mediated
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endocytosis prevails. One study supported the accumulation of OX-26 in the endothelial cells
[74]. Moss and Morgan hypothesized that accumulation of OX-26 by the endothelial cells might
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be the triggering factor of transcytosis. [75]. Low plasma level of iron leads to the over
expression of TfR and ultimately, OX-26 mediated endocytosis [76].

Low density lipoprotein receptor (LDLR) is another family of protein receptors located on the
BBB. Members of this family, LDLR, LRP1, LRP2, apolipoprotein E2 (ApoE2) and very-low-
density lipoprotein receptors have some resemblance in structure [77]. Lactoferrin (Lf), ApoB,
ApoE, lipoprotein lipase and plasminogen activator bind with these receptors and their potential
to target the brain through LDLR has been studied, extensively. Many drugs have been
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investigated for their brain targeting efficiency though LRP1 and LRP2 receptors. Tf, hepatic
lipase, factor VIIa, factor VIIIa, Aβ1−40, amyloid-β precursor protein (APP), apoE, HIV-1 Tat
protein, rhinovirus, matrix metalloproteinase 9, collagenase-3, heparin cofactor II, α1-antitrypsin,
and heat shock protein 96 are ligands for LRP1 receptor. ApoJ, amyloid beta (Aβ) bound to apoJ
and apoE, aprotinin, and very-low-density lipoprotein (VLDL) are specifically substrates for the
LRP2 receptor [78, 79]. Lf is another glycoprotein of Tf family binds to the Lf receptors on the
endothelial cells of brain capillaries [80]. It is an efficient ligand for nanocarriers’ delivery to the

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brain. Lf has less competition with endogenous Lf than Tf and hence, preferred over Tf as brain

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targeting moiety [81]. Besides iron transportation, Lf has some functional activity against virus

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and carcinogens. Lf modulates the factors which have potential contribution in the inflammation
and tumor growth. It potentiates cell signaling pathway through modulation of cytokines,

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chemokines, growth factors and interleukins [82]. Lf is cationic in nature and has capability to
bind with anionic receptors. This cationic structure helps Lf to bind with variety of receptors in
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the brain and other organs. So, LRP1 and LRP2 expressed in the brain have binding affinity with
Lf [83]. LRP facilitates the Lf mediated transcytosis. Neurons, astrocytes and other brain cells
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have endogenous Lf that plays an important role in the progression of neurological disorders and
brain lesions. During neurodegenerative diseases, LRP mediated transcytosis is evident for
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transport of Lf from blood circulation through the BBB endothelial cells. Exogenous Lf could be
an efficient ligand to bind with LRP and facilitate the delivery of drug vehicles to the brain while
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treating neurological disorders and brain tumor.

Melanotransferrin (p97) is another protein similar to Tf, most abundantly found on the surface of
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melanoma cells than normal melanocytes and binds to iron in the body. Two types of p97 exist,
one is membrane-bound and the other is secreted. P97 is highly expressed in malignant
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melanoma cells than normal melanocytes. It is a substrate of LRP1 receptors [84]. Beliveau’s
group coupled doxorubicin (DOX) with melanotransferrin and conducted animal experiments to
study the effect of melanotransferrin on the transport of doxorubicin to the brain [85]. They were
successful to deliver melanotransferrin conjugated DOX to the brain [85]. BioMarin
Pharmaceuticals Inc. is developing melanotransferrin based drug delivery technology to deliver
enzyme replacement therapy to the brain [86]. Melanotransferrin is a marker of brain tumor and
also brain lesion related to the Alzheimer ′s disease [87]. LRP1 receptors of melanotransferrin
have association with Alzheimer′s disease and supposed to be involved in the metabolism and
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processing of APP and neuronal uptake of Aβ through α2-macroglobulin (α2M) carrier protein
[78]. In addition to LRP1, LRP2 receptors have been reported to facilitate the brain uptake of
apoJ and apoE conjuagated Aβ [78]. The serum level of p97 is markedly increased in
Alzheimer’s disease. The association of Alzheimer’s disease with LRP1/LRP2 made it
convenient for the scientists to target the brain through LRP1/LRP2 mediated delivery.
Melanotransferrin activates the plasminogen and its high amount is required to attain brain
targeted delivery, leading to excessive iron binding of the body.

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Fig.2: Representation of transport mechanisms across the BBB

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Insulin receptors are widely explored receptors for the targeted delivery of drug to the brain
through receptor mediated endocytosis. Insulin and insulin like growth factors are found in the
BBB and many other parts of the CNS. Endogenous insulin cross the BBB and enter the brain
through receptor mediated endocytosis. Based on this strategy, insulin has been exploited as a
targeting moiety for brain directed delivery. Serious problem of hypoglycemia and disorder of
insulin limited the application of insulin as brain targeting moiety. Moreover, high dose of
insulin is required for this purpose, which is lethal [54]. Monoclonal antibodies (mAbs) bind to

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exofacial epitope of insulin receptors and have been researched for brain directed delivery

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through insulin receptor mediated endocytosis [88]. Peptidomimetic mAb binds to insulin

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receptor and was studied for delivery of genes and drugs to the brain of rhesus monkey. These
antibodies delivered 10-fold more drugs and genes compared to mAbs directed against Tf

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receptors of rodent’s brain [89]. Murine 83-14-SA mAb is another ligand for insulin receptors
and promoted the uptake of I-bio-A1-40 to the rhesus monkey brain when complexed with I-bio-
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A1-40 and administered intravenously. Immunogenicity problems related with 83-14 mAb
limited its clinical applications in human [90]. Genetically modified 83-14-SA is a big hope for
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the successful transport of drugs and genetic material to the brain. Insulin like growth factor
receptor (IGFR) delivers drugs to the brain based on the same mechanism of insulin receptors
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and consists of IGFR1 and GFR2.

Nicotinic acetylcholine receptors (nAchR) are the ligand gated positively charged channels
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present throughout the CNS [91]. These channels play a vital role in the normal functioning and
pathological conditions of the brain. Cationic receptors are expressed on the endothelial cells of
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brain capillary and inhibited by the peptide based neurotoxins and viral protein. These receptors
have been extensively exploited to deliver drug delivery systems (DDSs) modified with cell
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penetrating peptides for brain targeting [92].LCDX (FKESWREARGTRIERG) is a candoxin

derived peptide based ligand for nAchR [93]. It has less binding affinity to nAchR than candoxin
but it is more important for brain targeting. A study was conducted to evaluate the mechanism of
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nAChR-mediated transcytosis of CDX. Fluorescence had shown intracellular distribution of
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CDX in punctuate fashion and lysosomes played a potential role in the transcytosis of LCDX
peptide [93]. LCDX is proteolytically unstable in the endothelial cells of BBB and inefficient to
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target the brain through nicotinic receptors. CDX was synthesized with stability against the
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proteolytic enzymes activity and had profound transcytosis efficiency. CDX had binding
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affinity to α7 nAChR of the neurons through electrostatic, cation– π, and hydrophobic

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interactions. Conformatory changes occurred in CDX facilitated it to bind with the binding
pocket of α7 nAChR. A comparative study was conducted to confirm the binding mechanisms of
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CDX and CDX to the nAChR. The two ligands were bound to the receptors in different
manners. DGlu10 of DCDX penetrated deep to the packet and connected to the NH group of
Ser147 residue through hydrogen bonding. Besides, Van der Waals forces played prominent role
in the binding of DGlu10 to Pro193, Tyr194, Ser147, and Trp148 residues in subunit A. whereas;

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Glu7 in CDX was found outside the packet and bound to Arg185 in subunit A through

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hydrogen binding. The side chain in CDX was found deep into the packet and made

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hydrophobic connection with Trp54, Leu36, and Leu37 residues in subunit B. Whereas, Trp5 in
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CDX was unable to make hydrophobic interaction with the packet and just was connected with

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Tyr167 in subunit B. There was hydrogen binding between DArg5 of CDX and Gln116 in
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subunit B while, hydrogen bonding was prominent between Arg12 in CDX and Glu188 in
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subunit A. Van der Waals interaction of DIle4 residue in DCDX with Met159 in subunit B was
found while no such interaction was present in LCDX. Hence, D
CDX made strong interaction
with nAChR than LCDX and is potent antagonist of of α7 nAChR. Moreover, D
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CDX is more
efficient in α7 nAChR mediated transcytosis than LCDX [91].DCDX could be exploited in brain
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targeted delivery of potent antitumor drugs and had shown improved uptake and efficacy of
nanocarriers to the brain tumor.
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Virus is transported to brain through short peptide sequence derived from virus protein and could
be a better choice as brain targeting moiety. Rabies virus glycoprotein (RVG) peptide has
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binding affinity with nAChR located in the BBB [94] and its potential to improve the brain
uptake of targeted delivery systems has been widely explored. This RVG peptide is derived from
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RVG which is located in the lipid layer outside the virion. RVG binds with α1 subunit of muscle
class of nAChR and enters the CNS [95]. Other subtypes of nAChR facilitate the entry of RVG
to the brain through synaptic pathway. α7 subunit has also been reported to interact with RVG
and expected to be involved in the transport of RVG to the brain. RVG is cationic in nature
which enables it to bind with negatively charged cell membrane and enhance the uptake through
electrostatic interaction [96]. In addition to receptor mediated transcytosis, positively charged
RVG peptide makes complex with negatively charged nucleic acid and improves the brain
uptake of nucleic acid through electrostatic interaction. The positive charge on the surface of
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RVG peptide is highly needed for its interaction with nucleic acid. Attachment of nona-arginine
to the C-terminal of the RVG peptide via glycine or histidine linker assigns positive charge to the
RVG peptide [97, 98]. Moreover, RVG peptide could be directly conjugated to the nanocarriers
through maleimide-thiol-coupling reaction [99]. RVG peptide-coupled DDSs have been
extensively studied to target the brain tumor and neurodegenerative conditions. nAChRs are also
highly expressed in the nasal cavity, especially in the trigeminal nerves ending and could be
targeted for direct nose-to-brain delivery of drug. SLNs loaded with RVG-9R-BACE1 siRNA

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complex have been studied for this route of delivery and successfully delivered BACE1 siRNA

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to the brain for Alzheimer’s disease therapy [100].

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Diphtheria toxin receptors (DTRs) are located in the BBB, glial cells and neurons. DTRs
participate in the internalization of receptor mediated drug delivery across the BBB [55]. The

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expression of these receptors is up-regulated in brain disease conditions such as brain stroke,
multiple sclerosi, Alzheimer’s disease and gliomas, resulting in enhanced efficiency of
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nanocarriers designed for neurological disorders [101]. These receptors have no endogenous
targeting moiety, thus no existence of endogenous competitor to the exogenous ligands. DTR
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mediated endocytosis avoids the lysosomal enzymes degeneration, which enables DTRs as
useful receptors for treatment of neurological disorders and brain tumor targeted therapy.
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Diphtheria toxin is a toxic substance and its clinical applications are limited. CRM197 is a safe
carrier protein and mutant of diphtheria toxin, which have been investigated for its brain
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targeting efficiency. Gaillard’s group discovered CRM197 for the first time and described it as
efficient ligand for the brain delivery of conjugated proteins through DTR mediated transcytosis
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[102]. The existence of Abs against the diphtheria toxin formed or already existed in the blood
circulation of the vaccinated patient is the major concerning issue; leading to lower targeting
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efficiency of CRM197 conjugated DDSs across the BBB. The level of the already existing
antibodies is reduced after continuous CRM197 treatment for 30 days [103]. An Ab reaction
mostly occurred to subcutaneous and intramuscular injection and is less likely reported with
intravenous (i.v.) injection.
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Table 1: Summary of transport mechanisms studied for brain targeting

Receptor/ Ligand Investigational Targeted Disease Ref

Transport Carrier/ Drug/ Agent
Mechanism Target
TfR Tf, OX26 Azidothymidine, Brain tumor 104
methotrexate,
loperamide,
LDLR Melanotransferrin, DOX, Rotigotine, Alzheimer’s disease, 77,

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ApoB, ApoE, Lf, apoJ and apoE Parkinson’s disease, 78

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HIV-1 Tat protein, conjuagated Aβ enzyme replacement
APP, heparin therapy and brain tumor

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cofactor II, α1-
antitrypsin and heat
shock protein 96

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Insulin Murine 83-14-SA Genes, I-bio-A1- Alzheimer’s disease and 143
receptors mAb, IGFR1 and 40 and Secreted diabetes-induced
GFR2 amyloid precursor encephalopathy
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Receptor
protein alpha
Mediated L D
Acetylcholine CDX , CDX and BACE1 siRNA Brain tumor and 126
Endocytosis
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receptors RVG Alzheimer’s disease

DTR CRM197 Irinotecan Brain stroke, multiple
Alzheimer’s
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clerosis,
disease and gliomas 144
TRP Capsaicin, TRPV1 Neurodegenerat-ive 110
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N-arachidonoyl- antagonists conditions

serotonin
Integrins RGD, cRGD, SMIA Tysabri and Parkinson's disease, 145
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natalizumab, etc Alzheimer's disease,

multiple sclerosis and
gliomas
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Folate - farletuzumab and Gliomas 112

receptors vintafolide

Adsorptive The negative Albumin, anti- DOX Brain tumor 123

Mediated charge surface ras oncogene
Transport of the antibodies, anti-
endothelial betaA4 protein
cells antibodies and CPPs
GLUTs D-glucose, and 2- Ethanol, nicotine, Alzheimer’ disease, 129,
deoxy-D-glucose, chlorambucil and ischemic stroke and 130
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mannose, and ibuprofen inflammatory

dehydroascorbic acid conditions
Carrier Mediated AA Phenylalanine, Ketoprofen, Brain tumor and pain 139
Transport transporters tyrosine, leucine, L- gabapentin
dopamine, and
thyroid hormone
MCTs Lactic acid, Statins Alzheimer’s disease, 105
AZD3965 and epilepsy and gliomas

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7ACC2

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The transient receptor potential (TRP) family consists of 28 mammalian TRPs and 6 subfamilies

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on the basis of structural similarity. Vanilloid family is the most widely characterized subfamily
and named on the basis of ligand-containing vanillyl moieties. Vanilloid-receptor 1 or capsaicin

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receptor (TRPV1) is a well known receptor of TRP family [106]. TRP1 is the ligand gated
positively charged ion channel sensitive to heat, pH, electric current and endovanniloids.
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Endovanniloids are fat soluble endogenous substances including anandamide (AEA), Nacyl-
dopamine or the lipoxygenases products 12-and 15-(S)-HPETE (hydroperoxyeicosatetraenoic
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acid) [106]. Some compounds have shown synergistic response to TRPV1 and cannabinoid
receptors in the CNS, simultaneously [107]. TRPV1 receptors have been extensively recognized
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in the CNS and their functional activity and wide dissemination in the brain made them special
for targeting the neurological disorders. The expression of TRPV1 is regulated in different parts
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of the CNS based on age, physiological and pathological conditions. TRPV1 channels are under
the initial stage of study. These receptors are highly controversial and further work is extremely
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demanded to elucidate the pharmacological response of these receptors [106]. Published studies
used capsaicin, agonist of TRPV1, to investigate the response of TRP1V in the brain [107]. In
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future, potential role of TRPV1 in the brain will be exploited with the help of new generation of
molecules such as I-RTX or N-arachidonoyl-serotonin. It is expected that drug delivery
strategies focused on TRPV1 will be designed and helpful in targeting the neurodegenerative
diseases.

Endothelial cells of the BBB and astrocytes regulate the basement membrane via extracellular
matrix (ECM) protein. Brain capillaries have ECM receptors such as integrin and dystroglycan to
link cellular compartment with ECM. Integrins are the receptors consist of  and 
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transmembrane glycoprotein and interact with ECM via the peptide sequence of ECM [108].
Integrins are found on the neuronal cells, leukocytes, tumor cells, cardiac cells, skeletal muscle
cells and vascular cells. In recent years, Integrin-based drug delivery approach has been attracted
in brain therapy of neurological disorders. A novel therapeutic drug delivery strategy is to
explore the ligands for integrin receptors. Arginine–glycine–aspartic acid (RGD), a tripeptide,
has binding affinity with integrin receptors and has been extensively studied for targeting
integrin receptors. Cyclic RGD has a high binding affinity to the integrin receptors than RGD

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and Qin et al. investigated the capability of cRGD as a ligand to bind with integrin expressed on

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the surface of leukocytes [109]. During neuroinflammatory conditions of brain such as

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Parkinson's, Alzheimer's disease and multiple sclerosis, leukocytes cross the BBB and migrate to
the brain lesion. Thus, cRGD coupled leukocytes could be a better choice to target the lesion in

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brain. Natalizumab, mAb against α4-integrin, was reported to reduce the seizure associated with
multiple sclerosis. RGD-binding integrins were reported to be present in the glioma,
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significantly, higher than normal tissues [110]. Integrin antagonists such as small molecule
integrin antagonists (SMIA) are under clinical trial for evaluation of their safety and efficacy in
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the synergistic therapy of tumors with antitumor agents. SMIA have shown positive outcomes in
in vitro and in vivo experimental studies [111].
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Folate receptors are glycolipid receptors over-expressed in different types of cancer tissues and
involved in the internalization of folate and folic acid derivatives. Folate conjugated therapeutic
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moieties or folate ligand guided nanocarriers are useful strategies in the development of brain
tumor targeted therapy. However, there are controversies regarding the potential impact of folate
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on BBB crossing efficiency of nanovehicles before reaching the tumor sites and FR mediated
endocytosis of folate conjugated carriers. While, two functional ligands conjugated nanovehicles:
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one ligand for overcoming the BBB and other ligand with folate receptor binding affinity and
trigger signaling pathway could be beneficial for glioma therapy. Various folate receptor
targeting chemotherapeutic agents are under clinical trial, such as farletuzumab and vintafolide;
however, none of them has shown positive outcomes in clinical settings [112]. Although, Folate
receptor mediated drug delivery enhances the efficacy and reduces the toxicity of glioma therapy
but the mechanism of folate trafficking is required to be elucidated.
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3.2 Adsorptive mediated endocytosis

Most hydrophilic molecules including peptide- and protein- based drugs are unable to cross the
lipophilic cytoplasmic membrane of BBB endothelial cells, which limited their applications in
neuroscience. Further, tight junctions of the capillary endothelial cells impede the migration of
hydrophilic substances into the microenvironment of brain [113]. Improvement of lipophilic
characteristics of the hydrophilic neurotherapeutical agents is insufficient for BBB

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transmigration. Electrostatic interaction of positively charged molecules with anionic endothelial

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cells cytoplasmic membrane overcomes the impeding action of the BBB and triggers the site

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specific transport of drug molecules to the brain. The adsorptive mediated endocytosis (AMT) of
cationic protein-and-basic oligopeptide relys on the above-mentioned electrostatic interaction
[114]. The negative charge on the endothelial cells luminal surface is associated with

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glycoconjugates and heparan sulfate proteoglycans (HSPGs) [115]. Syndecans and glypicans are
the major HSPGs found at the surface of endothelial cells. HSPGs are vital for the internalization
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of protein-, peptide-and nucleic acid-based drugs and genes by the endothelial cells. Chondroitin,
glycosaminoglycans and perlecan situated at the basement membrane confer anionic barrier.
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Basement membrane and abluminal surface of the endothelial cells contribute anionic charge to
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the electrostatic interaction of cationic peptides and proteins [116]. Macromolecular drugs, and
nanovectors coupled with cationic ligand easily penetrate through brain parenchyma tissues.
Proteins could be internalized through AMT without the involvement of carriers. Cationization
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of protein by amidation of its carboxylic acid groups with natural or synthetic diamines and
polyamines achieves AMT of protein [116]. However, it was reported that cationization modified
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the biological activity and potency of proteins. Amidation of proteins at the site away from
ligand binding sites conserves the biological activity of protein. Cationization technique has been
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applied to various proteins such as albumin [117], anti-ras oncogene Abs [118], anti-betaA4
protein Abs [118], anti-amyloid beta peptide Abs [119] and nerve growth factor [120] for
diagnostic and therapeutic purposes. Adsorption of cationic protein to the endothelial cells of the
blood capillary has been reported and higher doses of cationic proteins are required to achieve
therapeutic concentration in the targeted site, leading to endothelial cells destruction [116]. The
toxicity and immunogenicity of cationic protein limit their therapeutic applications in the brain
specific and other organs targeted delivery. Strategic use of the cell penetrating peptides (CPPs)
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for endothelial transmigration of hydrophilic neuropharmaceuticals exploits non-receptor

mediated endocytosis. CPPs are cationic peptides interfacing with anionic cell membrane of
microvascular endothelial cells and cargo transport of drugs and genes to the specific sites in the
brain [121]. Transcription-activating factor (Tat), penetratin and the Syn-B vectors are peptides
originated from naturally occurring proteins. Various other CPPs are synthesized from
engineering of other short peptides. Initial studies ruled out the involvement of endocytosis in the
internalization of CCPs and this process was temperature-dependent. Recent studies

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demonstrated AMT played a vital role in the translocation of CPPs through electrostatic

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interaction with negatively charged cell surface. A study was conducted for the evaluation of

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TAT protein internalization mechanism. Internalization of TAT-conjugated protein was mediated
through HSPGs electrostatic interaction. Furthermore, it was suggested that glypicans, sugar

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residue of cell membrane, might interact with TAT protein. Involvement of HSPGs at the
luminal cell surface in the internalization of TAT peptide and TAT conjugated protein
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strengthens the crucial role of adsorptive endocytic mechanism in the uptake of TAT [122]. In
recent decade, CPPs has been widely used for the successful delivery of potential therapeutic
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agents to the brain. For example, Syn-B enhanced the DOX brain uptake, significantly, by
inhibition of P-gp mediated efflux compared to unmodified DOX [123]. The toxicity-,
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immunogenicity- and stability- related problems of CPPs restricted their applications in the field
of neuroscience; however, limited studies regarding these issues have been performed and
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further work will be highly appreciated.

3.3 Carrier mediated transport

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Nutrients required for normal physiological functions of the brain are carried through
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transporters located in the microvasculature of BBB. These transporters are carriers for
transportation of drugs with specific recognition and binding capability to these carriers. Solute
carriers (SLCs) are ATP independent carrier protein and the major transporters of hexose,
glutathione monocarboxylate, vitamin and nucleoside across the endothelial cells of brain [124].

For normal functions of neurotransmission, oxidative defense and biogenesis, carbohydrates,

especially D-glucose are rich source of fuel for brain. Glucose transporters (GLUTs) and
sodium/glucose co-transporters (SGLTs) are main SLCs for carbohydrates transport [124].
GLUTs are sodium independent facilitated carriers whereas, SGLTs are sodium dependent
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transporters. GLUT1s are highly expressed on the luminal and abluminal surface of mammalian
endothelial cells [125]. In pathological conditions, the level of GLUT1 is altered, resulting in the
modification of neuronal cells regulatory process and neurovascular functions. Decreased level
of GLUT1 in the brain capillaries of Alzheimer’s patients reduces the brain uptake of glucose
and slows down the cognition. GLUT3 expressed on neurons carries glucose from extracellular
space into neurons with higher binding affinity and capacity than GLUT1. They meet the
frequent demand of neuronal energy [126]. SGLT1s are found on the endothelial cells and

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neurons [127], whereas, SGLT2s present on the endothelial cells [128]. SGLTs mediate transport

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of glucose against the concentration gradient to meet higher glucose demand in stress conditions

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such as ischemia or brain stroke and prevent neuronal damage. Ethanol and nicotine suppress the
level of GLUTs in the BBB and reduce the brain uptake of glucose. Intake of alcohol or nicotine

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has been reported to elevate the neuronal injury in ischemic stroke conditions [129]. Down or up
regulation of GLUT1 expression in the endothelium of BBB could be beneficial in the strategic
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therapy of various neurological disorders. Elevated level of glucose is marker of brain stroke and
GLUT1s at BBB are potential target to prevent further ischemic damage to brain. GLUTs could
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be better carriers for delivery of neurotherapeutics effective in neurological disorders and

neurodegenerative conditions of brain. Many drugs were conjugated with glucose to exploit
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GLUT1 and improve BBB penetration ability of drugs. For example, chlorambucil, a
chemotherapeutic agent, conjugated with D-glucose prevented erythrocytes uptake of C-glucose
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through GLUT1 [130]. Similarly, ibuprofen was conjugated with D-glucose and the prodrug
exhibited three-fold higher concentration of drug in rat brain than control group [131]. Ligand-
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conjugated nanocarriers have been investigated for improvement of drug level in the brain via
GLUTs. Multivalent glucoside conjugated liposomes [132], mannose derivatised liposomes
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[133], dehydroascorbic modified micelles [134], glucose coated gold NPs [135] and 2-deoxy-D-
glucose functionalized NPs [136] have been explored to enhance the brain distribution of drugs
via GLUTs.

Amino acids (AA) are essential for various brain functions such as synthesis of glutathione,
neurotransmitter recycling and regulation of precursor availability for AA metabolism. Four
families of AA transporters have been recognized to mediate AA transport across the BBB [137].
These SLCs are classified, on the basis of charge on the substrate they carry, into three
categories; cationic, anionic and neutral. On the functionality basis, AA transporters are sodium
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ion dependent and independent transporters. Large neutral AA transporters are plentiful
transporters present on the luminal and abluminal surfaces of brain endothelium. Phenylalanine,
tyrosine, and leucine belonging to large neutral amino acid class are carried through system L of
sodium ion independent transporters [138]. System L has binding affinity to large number of
substrates and broader applications in drug delivery of macromolecules. LAT1 genes of system L
are highly expressed in the microvasculature of BBB. LAT1 had demonstrated transport of non
AA substances such as L-dopamine, gabapentin and thyroid hormones. Recently, Puris et al.

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performed in vivo pharmacokinetic study of LAT1 utilizing a prodrug of ketoprofen.

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Phenylalanine conjugated ketoprofen presented enhanced brain delivery of drug through LAT1

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mediated transport with reduced peripheral exposure of drug [139]. AA and nutrients are
essential for tumor progression. The expression of LAT1 in tumors has strengthened the belief of

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scientists on the clinical values of LAT1 in brain tumor targeted delivery [140]. LAT1 inhibitors
could be prospective chemotherapeutic agents and big hope for critical cancer patients [141].
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Cationic AA transporters, CAT1 and CAT3, are located at BBB endothelium belongs to system
y+ of sodium independent transporters and carry L-arginine, L-histidine and L-lysine across
BBB. Cationic AA are carried through system y+ and broad scope AA transporter (systems b0, +,
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B0, +, and y+L) [142].

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In ischemia and brain stroke, brain has insufficient level of glucose to meet its demand of energy
and lactate is utilized to prevent the neuronal damage. Astrocytes in the brain supply lactate and
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carried to the neurons for fuel supply. Lactate is transported across the BBB by the uptake and
efflux monocarboxylate transporters (MCTs). MCTs proteins are affiliated with
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monocarboxylate porter (MCP) family. MCTs family consists of ten members (MCTs 1-10) but
MCTs1-4 participate in the monocarboxylate transport [146]. MCT1 has instant association with
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lactate and abundantly found in the normal and cancer cells whereas; MCT4 has the lowest
binding affinity. These transporters adjust the dispersal of lactic acid and ketone bodies in the
brain. The appearance of MCTs is modified in different neurodegenerative conditions of brain.
Decreased expressions of MCT2 and lactate amount were reported in the rat model of
Alzheimer’s disease after Aβ25–35 fragment injection. Similarly, reduced expression of MCT1
in the blood vessels and increased expression in astrocytes of hippocampus in rat epilepsy
models were observed [147]. To evaluate the expression of MCTs in the brain ischemia, focal
ischemia was induced in rat and it was found that microglial cells linked MCT1 and MCT2 were
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highly expressed [148]. MCTs have been found in different types of cancers and MCT1 is
responsible for lactate distribution to cancerous cells. MCT4 is expressed on bladder and prostate
cancer and glioma. Cancer cells are opportunistic and adapt to hard situations through MCTs
contribution. Targeting of MCTs could be advantageous in the therapy of neurological disorders
and brain tumor. MCT1 inhibitors could be helpful to prevent tumor cells migration and
acidification of cancer cells, ultimately leading to death. MCT4 targeted therapy could be
exploited for inhibition of tumor cells migration and eradication of brain tumor. Traditional MCT

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inhibitors lack the specificity and novel AZD3965, a second-generation MCT1/2 inhibitor, is

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under the phase of clinical trials [149]. 7ACC2, a selective inhibitor of MCT1/MCT4, has been

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recently explored [150, 151] and further studies will confirm its potential implications in cancer
therapy.

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4. Novel neurotherapeutic approaches for brain targeting
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Various conventional and newly developed invasive and non invasive drug delivery strategies for
brain targeting have been explained in detail in our previous published review article [25]. Novel
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therapeutic approaches under current development and investigation for brain disease targeting
are discussed here with focus to exploit endogenous cells and mechanisms for brain therapy.
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4.1 Astrocytes targeting therapeutic strategy

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Astrocytes are crucial for signal processing and normal physiological functions of the brain.
They are vital for the regulation of brain metabolite homeostasis. Astrocytes work in
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collaboration with neurotransmitters through communication network between astrocytes and

synaptic neurotransmitters [152]. Astrocytes are essential in the normal cognition and astrocytes
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dysfunction contributes to the origination and progression of neurodegenerative conditions of

brain. Astrocytes support and protect the brain during pathological conditions and brain lesion
through astrogliosis. Astrogliosis is characterized by the modification of cellular components and
functional activity of astrocytes in pathological injuries to brain [153]. In case of severe injury to
brain such as necrosis, tumor and infection, astrocytes keep in line with various cells and form
glial scar to save normal cells from inflammatory cells infiltration [154]. Astrocytes proliferation
under stress condition excites glutamate-induced glutathione production and maintains the
metabolic and ionic homeostasis of brain. Astrogliosis is the stamp of authenticity of
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neurological disorders; radial and fibrillary gliosis are associated with hippocampal sclerosis
[155]. Astrocytes elevate extracellular neuronal uptake of K + through Kir 4.1 K+ channels in
neuronal excitation and depolarization, eventually contributing to the pathophysiology of
epilepsy [156]. A higher expression of glutamate receptors [157], voltage gated Na+ [158] and
Ca++ ion channels [159] in the astrocytes of epileptic brain tissue than normal brain tissue
demonstrated the contribution of these channels and receptors in the pathogenesis of scelrotic
hippocampus. Targeting abnormal astrocytes in epilepsy could be a revolutionary strategy for

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controlling and modifying the disease. Similarly, astrocytic gliosis is primarily contributing

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towards pathology of Alzheimer’s disease [160]. Astrocytes hypertrophy [161] and increased

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expression of glial fibrillary acidic protein (GFAP) [162] have been found near to amyloid
plaque. Astrocytes, once activated, penetrate and metabolize the plaque. Recently, researchers

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reported that astrocytes together with microglial cells penetrated the plaque and Aβ protein
[163]. Aβ trigger the proliferation of astrocytes and Aβ42 deposit in the astrocytes is the
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proportionally indicator of disease progression. Aβ42 deposited astrocytes are destroyed forming
GFAP+ amyloid plaque. Expression of GLT-1 and GLAST, glutamate transporters, is highly
reduced in Alzheimer’s patient [164]. Hence, glutamate is not removed from astrocytes and its
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increased level results in the occurrence of seizures. Enhanced astrocytic GABA production is
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also a contributing factor. Mitew et al. demonstrated the increased level of GABA along with
GAD67 and GAT3 in the astrocytes of Alzheimer’s patients [165]. GABA transporters can
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reverse their transport direction in presence of excessive intracellular GABA. GAT3 could be
exploited as a new drug target. Astrocytoma is a subclass of glioma, characterized by the
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structural and functional modification of astrocytes. GFAP has been recognized in the glioma
tissues and highlights the involvement of astrocytes in tumor progression and astrocytes origin
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[166]. Increased peritumoural glutamate concentration associated with reduced GLT-1

expression has been reported, which also caused the seizure in glioma patients [167]. Enhanced
expression of system X and SLC7A11 were declared responsible for glutamate release and tumor
progression and invasion to normal tissues. Altered Na + and K + channels expression in astrocytes
have been found to produce spike-like events and depolarize membrane potential, respectively
[168].

Astrocyte targeted drug delivery for neurological disorder treatment is expected in the near
future. One study based on targeting ephrine-A5 present on the astrocytes in infarct surrounding
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area reported that ephrine-A5 targeting potentiated the recovery process in stroke [169].
Glutamate expression contributes to depression and releases D-serine from astrocytes. Treatment
based on D-serine exhibited antidepressant effects in rodents [170]. Similarly, Autism spectrum
disorders could be treated by controlling tumor necrosis factor alpha (TNFα) signaling in
astrocytes [171]. As discussed above, glutamate is important for normal neurological functions
and its abnormalities lead to amyotrophic lateral sclerosis (ALS), stroke and brain tumor. β-
lactam antibiotics increased the expression and activity of GLT-1 in the brain and helpful in

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ALS therapy [172]. Further, astrocytes transplantation-based replacement therapy has shown

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fruitful results in ALS treatment.

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4.2 Therapeutically targeting of Lymphatic Drainage System

Lymphatic system has innate ability to regulate the level of fluid and salt in tissues, immune

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response, metabolic pathway and homeostasis of the body. Lymphatic system comprises of
vessels that carry fluid, white blood cells, immune cells and nutrients through tissues, organs and
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lymph node. This cleaning system travels alongside of the blood vessels and takes away dead
cells, waste material and lymph [173]. Brain, which has a highly complicated structure, lacks the
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well-organized lymphatic cleaning system and has its own identical glymphatic drainage system
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that works under the control of glial cells. Recently, Daniel et al. scanned dye injected human
and monkey brains via magnetic resonance imaging (MRI). They observed lymphatic vessels
containing unique cells and protein markers alongside of blood vessels. They suggested
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lymphatic system worked in collaboration with blood circulatory systems in human and
mammalian brains [174]. The brain lymphatic cleaning system consists of many pathways such
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as perivascular route, glymphatic system, olfactory/cervical cleaning passage and cerebrospinal

fluid (CSF) draining meningeal lymphatic vessels [175]. Brain lymphatic system performs
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various physiological functions of brain including maintenance of water and solute balance
[176], serving as a communication network between CNS and immune system via afferent and
efferent pathways [177] and controlling CSF and interstitial fluid (ISF) pressure. Aging, genetic
factors, sleep, cardiac activity, respiration and body position are the regulatory elements of
lymphatic performance. Dysfunction of lymphatic cleaning system has a critical contribution in
the pathogenesis of neurodegenerative and inflammatory conditions and neurovascular diseases
of brain [178, 179]. Various kinds of brain stroke are associated with altered lymphatic drainage
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system of brain [180]. Pathogenesis of stroke involves the triggering of microglial cells,
ICF/CSF pressure, glymphatic system controlled fluid homeostasis and immune system
activation by meningeal vessels. Blockage of cerebral artery leads to acute stroke that requires
immediate break down of the clot to prevent further damage and restore normal functioning of
brain tissues [181]. Thromobolytic therapy based on i.v. administration of recombinant
tissue plasminogen activator (t-PA) dissolved the clot in cerebral artery of ischemic stroke
patient through fibrinogen degradation and plasminogen conversion. Clot removal may reinstate

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the glymphatic system removal of metabolic waste and toxic materials from the brain tissues and

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further ameliorate patient condition. Clot formation in the glymphatic system and reduced

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removal of metabolic waste from the brain parenchyma tissues of subarachnoid hemorrhagic
(SA) patient have been reported [182]. However, i.v. administered plasminogen activator

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restored the glymphatic clearance of metabolites. I.v. administered t-PA therapy restored
glymphatic system perfusion in the ischemic stroke and SA patients [183]. Alzheimer′s disease
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involves deposition of Aβ in brain microvasculature and accumulation in the lymphatic cleaning
system [184]. Immunotherapy of Alzheimer′s disease based on active and passive targeting of
Aβ antigen has been conducted in mice and clinical trials in human [185]. Bapineuzumab, mAb,
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was investigated in clinical trials for passive immunization of Alzheimer′s patients. Similarly,
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clinical trials on aducanumab have shown reduction of amyloid plaque [186]. Among other
mechanisms, Ab restored the lymphatic drainage system and caused efflux of brainAβ to the
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blood [187]. Phosphodiesterase (PDE) III is highly expressed in the cerebral amyloid angiopathy
(CAA) and Alzheimer’s patients. PDE inhibitors facilitate the drainage of Aβ through restoration
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of lymphtic cleaning system. A study on CAA model mice was conducted using cilostazol.
Cilostazol, a selective PDE III inhibitor, promoted eradication of Aβ [188]. In another study on
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mild cognitive impairment patients, cilostazol reduced cognitive decline through tuning of
vascular elasticity [189]. Perivascular microglia/macrophages have the ability to clear Aβ. Toll-
like receptors and scavenger receptors found on the microglia help microglia to eliminate Aβ.
Strategy based on modulation of these receptors expression could be helpful in microglia
clearance of Aβ. Ependymal cells produce CSF and are situated inside of brain ventricles [190].
Ependymal cells could be targeted for the delivery of gene, protein or peptides. In vivo
experiment based on intraventricular injection of galAd.RSV exhibited successfully transfer of
beta-galactosidase to the ependymal cells [191]. Ependymal route has been exploited in gene
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therapy. Achariyar et al. investigated fluid transporting system of brain for CSF generated apoE
delivery using lentivirus vector and demonstrated new route for delivery of genes to the brain
parenchyma. IGF-1 enters the brain via ependymal route [192]. IGF-1 based delivery of genes is
expected to treat neurodegenerative conditions of brain in near future. Intranasal route bypasses
the BBB and delivers neurotropic factor while exploiting ISF-CSF exchange mechanism.
Intranasal delivery enhanced the distribution of granulocyte colony-stimulating factor (G-CSF)
into the brain parenchyma and CSF in cerebral ischemic rat model [193].

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4.3 Alpha-synuclein based therapy

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Alpha-synuclein (α-Syn) is a mysterious protein comprised of 142 amino acids and abundantly
found in hippocampus, dentate gyrus, olfactory bulb, thalamus, cerebellum, amygdale,
presynaptic terminals and other brain regions [194]. α-Syn consists of N-terminal repeat region,

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C-terminal region and central region. N-terminal is positively charged region with repeatedly
seven residues and fatty acid binding protein (FABP). FABP binds with lipophilic micelles and
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forms helical structure [195]. C-terminal is negatively charged unfolded in structure, preventing
the aggregation of αSyn and its interaction with N-terminal or NAC region and retains the
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unfolded structure of a-Syn [196]. Despite of plenty of research articles published on the role of
α-Syn in the pathophysiology of neurological disorders, the functional activities of α-Syn are still
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not well known. α-Syn has physiological importance in the maintenance of neural plasticity,
release of pulse transmitting synaptic chemicals, dopamine synthesis, and development and
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functions of neurons. Lodgment of misfolded α-Syn agglomerates in the neurons, nerve fibers or
microglia portrays contributive position in the pathogenesis of Parkinson’s disease, dementia
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with Lewy Bodies (DLB) and multiple system atrophy [197]. Snca gene is α-Syn encoding gene
and the mutation of this gene altered the risk related to Parkinson’s disease and α-Syn level in
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Parkinson’s disease [198]. Families with Snca gene variants have been found and it was
investigated that mutation of this gene led to altered expression of aberrant protein and increased
risk of Parkinson’s disease. However, there are still controversies regarding the involvement of
aberrant α-Syn protein accumulation in the pathogenesis of Parkinson’s disease. Aggregation of
α-Syn has been found in cells stress condition and might also accumulate in the case of
antioxidant defense, protein metabolism or increased energy requirement of cells and not cause
Parkinson’s disease [199]. It was suggested that aggregation-prone released in the extracellular
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space might be taken up by the adjacent neurons and propagate disease in the nearby area of
brain [200]. Therapeutic approach to restrict the release of α-Syn species and prevent uptake by
the nearby brain area could be useful in Parkinson’s disease therapy. It is also not clear which
part of the brain is first exposed to Lewy crisis and lewy may not disseminate all parts of the
brain, simultaneously [201]. Correlation of α-Syn with neural cell death and abnormal
neurological manifestations is doubtful. Therapeutic strategies based on α-Syn targeting are to
restrict production, aggregation and extracellular uptake of α-Syn and elevate intracellular and

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extracellular degradation of α-Syn. Reduction of α-Syn synthesis by inhibiting the RNA

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translation of protein might recover the neuronal activity. Lentiviral vector delivered short

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hairpin (sh), α-Syn RNA, in the rat striatum reduced the level of α-Syn after two weeks of
injection without any sign of toxicity [202]. Di Monte et al. infused siRNA in squirrel monkey

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and demonstrated 40-50% reduced level of α-Syn compared to control group [203]. In another
study, 35% reduced expression of α-Syn was reported in rats received shRNA's [204]. Therapies
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involving significant reduction of α-Syn expression exhibited neural and peripheral toxicity.
Currently, Mittal et al. targeted transcription gene for α-Syn protein through beta-2-adreno
receptor. They declared clenbuterol, beta-2AR agonist, reduced 35% level of α-Syn protein in
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blastoma cell line [205]. Normal functional activity of α-Syn protein could be restored by
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inhibiting its aggregation and misfolding. Heat shock proteins (HSP) are effective in removing
aggregates of protein as the characteristic feature of neurological disorders [206]. To prevent the
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oligomerization of α-Syn monomers is another strategic way of aggregate inhibition. Intrabodies

are mAbs have specific binding affinity with NAC, vulnerable aggregating region, and played
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neuroprotective effects [207]. Similarly, viral vector delivered VH14 PEST and NbSyn87 were
injected intranigrally in rats, clearly removed aggregated α-Syn and restored neuronal functions
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[208]. Clinical investigational studies involving NPT200-11 and NPT088 are in going phase.
NPT200-11 inhibited oligomerization of α-Syn to form aggregate [209, 210]. Autophagy has
been utilized to eradicate the aggregate and promote the degradation of α-Syn protein.
Autophagy inhibitors such as trehalose augmented the clearance of protein aggregates through
lysosomal biogenesis but neuronal toxicity limited its applications [211, 212]. C-abl is an
oncogen and neurology studies explained alleviated level of c-abl in Parkinson’s brain
parenchyma promoted the aggregation of α-Syn through phosphorylation [213]. C-abl inhibitors
such as viral vector delivered nilotinib reduced α-Syn level in mice and secured neurons from
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toxicity [211]. Further trials are required to evaluate the safety and efficacy of C-abl inhibitors.
Active and passive immunotherapies protect brain tissues from inflammatory reaction in
neurodegenerative conditions. Phase1 clinical trial involving active immunotherapy vaccine,
AFFITOPE® PD03A, was conducted and an immune response in a dose dependent manner was
noted with mild toxicity [215]. Passive immunotherapy of PRX002, mAb, reduced serum level
of α-Syn up to 96% in phase 1 clinical trial without toxicity and is under phase 2 trials [216].
Uptake inhibition of extracellular α-Syn is helpful in managing Parkinson’s disease. α-Syn is

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released from glial cells into the extracellular space and taken up by the nearby neuronal cells to

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propagate disease. It was suggested that α-Syn fibril bound with HSPGs content of cell

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membrane and picked up by endocytosis [217]. HSPGs inhibitors could be potentially
efficacious in preventing endocytosis of α-Syn and further progression of neurodegenerative

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disease. Clinical trials regarding α-Syn based therapy are faced with many challenges such as
recombinant oligomers aggregate into Lewy bodies. Numbers of monomer required for oligomer
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formation is not known and it is also unclear whether oligomers aggregate is necessary for
toxicity. Various species of α-Syn exist and abundantly found species in disease should be
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identified to evaluate its toxicity and further target that specific protein [199].

Table-2: Clinical trials based on various therapeutic approaches for neurological disorders
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therapy.
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Trial No/ Name Therapeutic Phase of trial Strategy for Outcomes Ref.
Agent disease targeting
NCT01885494 AFFITOP® randomized, Active Induced Abs 215
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and PD01A and placebo- immunotherapy preferentially binded

NCT02267434 PD03A controlled Phase of Parkinson’s to α-Syn fibrils and
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1 trial disease immune response in

86% of Parkinson’s
patients. Well
tolerated and safe
NCT02157714 PRX002 Phase 1 Passive Decreased α-Syn 216
immunotherapy serum level in up to
of Parkinson’s 96% Parkinson’s
disease patients. Well
tolerated and safe.
Trials are still
ongoing.
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NCT00568776 scyllo-inositolphase 2 Amyloid Not significantly 218

randomized trial inhibition efficacious.
AN1792 Amyloid-beta phase I Amyloid plaques Diminished Aβ 219
peptide Ab42 randomized, clearance in plaque but no
placebo- Alzheimer’s improvement in
controlled trial disease. cognition
NCT00594568 Semagacestat Double-blind, γ-secretase No remarkable 220
placebo- inhibition in cognitive
Alzheimer’s

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controlled improvement was
disease. reported. Trials were

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terminated due to
serious toxicity

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related issues
NCT00905372 Solanezumab Phase 2 and phase Clearance of Aβ Outcomes were 221

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and 3 plaque negative in cognition
NCT00904683 amelioration.
UMIN000000465 Leuprorelin 1. randomized, inhibiting the 1. suppressed the 222,
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. placebo- accumulation of deterioration of 223
controlled pathogenic neuromuscular
phase 2 trial androgen impairment
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2. randomized, receptors in 2. No effect on

double-blind, Spinal and bulbar Swallowing function
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placebo- muscular atrophy

controlled trial
NCT01510028 ARSA gene Phase I/II gene Stem cell therapy Trial exhibited high 224
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therapy trial based on ARSA enzyme expression

gene throughout
hematopoietic
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lineages and CSF.

Inhibited
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progression of
disease
NCT01515462 Gene encoding phase I/II clinical Reinfusion of Restoration of 225
functional WASP trial genetically WASP expression
corrected led to
hematopoietic immunological and
stem/progenitor hematological
cell improvement
NCT00221104 Statin randomized trials 3-hydroxy-3- Reduced lipid level 226
methylglutaryl- and associated
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CoA reductase stroke risks.

inhibition
MOVES-PD GZ/SAR402671 Phase 2 trial GABA1-mutation Inhibited the
production of
glycosphingolipids 227
which is substrate
for GCase
NCT02941822 Ambroxol Phase 2 trial To evaluate Trials are ongoing 228

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and safety, tolerability
NCT02914366 and efficacy in

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Parkinson’s
patients

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NCT02525432 BM mononuclear Preclinical and Adult severe TBI Declined 249
cells Phase 1 trial neuroinflammatory
cascades and

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restored CNS
structures
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4.4 Stem cells mediated brain therapy as a promising therapeutic option for
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neurological disorders
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Neurons and glial cells are of utmost importance for normal physiological functions of brain.
Loss or dysfunctional activity of neuronal or glial cells is causative agent for neurovascular or
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neurodegenerative disorders. In addition, hyperactive glial cells provoke inflammatory response

and accelerate inflammation through peripheral immune cells invasion [229]. Abnormal glial
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cells mediated immune response leads the way to neurodegenerative conditions. Mechanism
involves in the glial cells induced inflammation is based on activation of toll like receptors
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(TLRs) expressed on glial cells. TLRs activated glial cells release the chemokines and facilitate
the infiltration of immune cells and inflammatory response. For example, Aβ plaque is
aggregated in Alzheimer’s disease and eliminated through TLRs activated glial cells [230].
However, it has been reported that prolonged activation of TLR4 resulted in abnormal
accumulation of Aβ plaque and progression of Alzheimer’s disease. Pharmacological
interventions of neurological disorders provide symptomatic relief and have compromised
efficacy with serious side effects. These factors pondered the scientists to search for alternative
options based on cell therapy. Stem cells have gained attention of neurologist and psychiatrists as
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an alternative auspicious choice for neuroinflammatory therapy. Stem cells are continuously
regenerating and only therapeutic cells could be applied in regenerative conditions. These cells
are efficacious in the hematological disorders and their clinical applications are limited in the
field of neurology, cardiovascular and gastroenterology, etc. Studies involving stem cells failed
to regenerate dysfunctional or damaged neuronal or glial cells. Fetal tissue transplantation (FTT),
embryonic stem (ES) cells and reprogrammed somatic cells have been investigated for cell
therapy. Preclinical trials based on FTT have shown efficacious results but placebo controlled,

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double blind trials have shown discouraging results in Parkinson’s disease cell therapy [231].

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FTT has limitations of diverse fetal donor’s requirement, loss of neurons after grafting, and the

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impact of abortifacient on the fetal tissue may compromise the soundness of transplant material
and ethic related issues [232, 233]. ES are self renewable with ability to generate any type of

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human cells and have potential to generate dopaminergic neuron slots in Parkinson’s disease
[234]. Early studies showed L-dopa generating neurons differentiated from ES cells transplanted
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into the brain of rat and mice [235]. However, recent study provided evidence of successful
differentiation of ES cells into dopaminergic neurons with the reinstatement of motor functional
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activity in rat model [236]. Animal studies had shown encouraging performance but ES
differentiated neurons replicated poor in vivo results and further work is required to explore the
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in vivo restoration of dopamine release [237]. Reprogrammed somatic cells are non-fetal or non-
embryonic stem cells source for derivation of dopaminergic neuron transplant. Induced
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pluripotent stem cells or dopaminergic cells derived from fibroblast were exploited with little
success [238]. However, immunomodulatory features of mesenchymal stem cell (MSC) enabled
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them to play promising therapeutic contribution to the psychiatric therapy. Interplay between
MSC and immune cells renders immunomodulation based therapeutic efficacy of MSC [239]. In
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neurological disorders, inflammatory response is activated by the destruction of glial or neuronal

cells and eventually, inflammatory cells, especially macrophages are infiltered. In brain,
microglial cells are called habitant macrophages, capable of inducing immunologic response
thorough release of interleukins and nitric oxide. Upon transfusion, MSC were guided through
TNF and activated gene/protein 6 at the injury site, leading to glial cell inactivation [240].
Neurodegenerative conditions are associated with inflammation and the inactivation of immune
response is the key to prevent the degeneration of neurons. Parkinson’s disease is associated with
the degeneration of dopaminergic neurons, glial cells activation, infiltration of inflammatory
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cells and inflammation based progression of disease. In1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA) or lipopolysaccharide (LPS)
induced animal models of Parkinson’s disease, MSC exhibited microglia inactivation and
inhibition of cytokines [241, 242]. Similarly, intranigrally transfused adipose derived MSC
enhanced the peripheral level of IL 10 and IL4 in rat Parkinson’s disease model induced by 6-
OHDA. MSCs interact with inflammatory cells and play an immunomodulatory role through the
rectification of compromised inflammatory response in Parkinson’s. In Alzheimer’s disease, β-

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amyloid aberrantly activates the glia cells and astrocytes, followed by the infiltration of

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inflammatory cells and increasing the level of peripheral cytokines. It has been reported that in

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animal model, MSC switched microglia from detrimental phenotype to neuroprotective with
enhanced level of anti-inflammatory cytokines and diminished level of pro-inflammatory

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cytokines [243, 244]. Traumatic brain injury (TBI) is a non degenerative condition experienced
by the mechanical force applied externally to the head with clinical manifestation of altered level
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of consciousness, amnesia, with or without neurologic deficits [245]. Pharmacological
interventions of TBI failed to produce impressive results in the clinical trials or couldn’t reach
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the stage of clinical trials. Many processes are involved in the pathophysiology of secondary
injury and available treatment options just work by inhibiting single participating event [246].
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Up to now, Food and Drug Administration (FDA) has not accorded approval to any therapeutic
agent for TBI treatment [247]. Tissue restructuring, neuroprotective, cell death prevention and
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inflammation modulating abilities of SCs made them suitable choices for TBI treatment [248].
MSC had shown auspicious potential in clinical trials than other SCs for TBI therapy. Pre-
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clinical and phase I clinical trials based on i.v. bone marrow (BM) mononuclear cells for severe
TBI adult therapy manifested declined neuroinflammatory cascades and restoration of CNS
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structures. These trials are blinded, randomized, placebo-controlled and at an ongoing stage. BM
MNCs would be infused after 48 hrs of injury induction and infusion associated toxicity should
be monitored. In another study, BM derived SB623 cells were transplanted in rat TBI contusion
model and improved motor functions, significantly, without any noticeable toxicity, histological
or clinical abnormalities and side effects [249].
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4.5 Exosomes as carriers for neurotherapeutics

Exosomal vesicles (EVs) are lipid bilayer membrane surrounded vesicles containing adhesive
and transporter proteins with endocytic commencement or released from cytoplasmic membrane
[250]. EVs serve as a mediator in signal based communication between the cells and categorized
on the size basis into exosomes (30-150nm), microvesicles (50nm-1µm) and apoptotic bodies
(50nm-5µm) [251]. Normal physiological functions and pathological conditions of brain are

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based on the EV-mediated cross-talking between neuron, glial cells and astrocytes [252]. Crucial

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role of exosomes in transferring genetic and biochemical information between the cells endows

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them to deliver macromolecular drugs, lipids, protein and nucleic acids into the brain. Exosomes
are superior to other nanocarriers in term of non-immunogenicity, BBB crossing potential, non
toxicity, stability and tissue targeting capabilities. Exosomes have long circulation half life, and

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avoid immune system detection, phagocytosis and lysosomal degradation. They are small size
and have ability to load protein and wide range of therapeutic molecules. Endogenous markers
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expressed on their surface guide them to the specific tissue and their safety profile render them
most versatile and suitable vehicles for cargo transport of therapeutic moieties to the brain [253].
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Recently published research articles focused on biological impact of exosomes derived from
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tumor cells and stem cells on the neurological disorders and brain tumor. Human neural stem
cells (HNSC) derived exosomes exhibited phenotype similar to HNSC after head-only
irradiation. Baulch et al. demonstrated exosomes prevented neuroinflammation and restored
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neural functions in specific brain part [254]. Multipotent MSC generated exosomes when
administered i.v. to the rats with TBI, enhanced production of endothelial cells and neurons in
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dentate gyrus region and reduced inflammation, significantly [255]. Dendritic cells (DC)
enhance the release of EV and DC-EVs loaded with micro RNA provided pro-myleniating effect
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and avoided oxidative stress condition [256]. Exosomes derived from neuroblastoma could be
therapeutically beneficial in the Alzheimer’s disease therapy. Alzheimer’s disease pathogenesis
involves altered release of EV and accumulation of Aβ exaggerates Alzheimer’s disease. EVs
derived from neuroblastoma were transfused to Alzheimer’s disease mouse model promoted Aβ
transportation to microglia and clearance of Aβ [257]. Methods to load drug into exosomes
involve sonication, incubation and electroporation. Exosomes are excellent vehicles impeding
BBB and cargo transport of macromolecular drugs and small molecules to the targeted site in
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brain. Protein and genetic contents could be loaded into exosomes and efficiently transferred
across BBB [253]. Exosomes are secreted by the B cells, T cells and macrophages of immune
system. Haney et al. reported that macrophages loaded with catalase or catalase encoded plasmid
DNA released exosomes incorporating catalase in mouse model of Parkinson’s disease and
promoted delivery of catalase into the targeted neurological cells [258]. They further conducted
another study and loaded catalase in naive exosomes. In in vitro and in vivo model, exosomes
decreased oxidative stress and enhanced neuronal activity [259]. Brain endothelial exosomes

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could be utilized to deliver small RNA for brain tumor therapy. Yang et al. investigated

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exosomal delivery of SiRNA in in vitro and in vivo zebra fish model of U87 glioblastoma.

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Exosomal delivered SiRNA reduced VEGF gene level in astrocytoma cells of tumor and
inhibited xenograft brain tumor aggregation [260]. Further, RVG engineered exosomes

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encapsulating BACE-1 siRNA for targeting β-secretase in AD, with 60% reduced mRNA and
BAC1 protein knockdown [261]. Naive exosomes could be exploited for neurological and
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neurodegenerative disorders therapy. Macrophages derived exosomes have been studied for
protein based therapy of neuroinflammatory conditions. Similarly, Yuan et al. explored
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macrophages exosomes as carriers for the delivery of the brain derived neurotrophic factor
(BDNF) protein intravenously. They reported that i.v. administered Mφ exosomes crossed the
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BBB and transported protein to the brain. Mφ exosomes interacted with endothelial ICAM-1
through LFA-1 protein inherited from parent cells and mediated migration across BBB. LFA-1
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and ICAM- 1 further mediated cellular uptake of exosomes [262]. Despite of the therapeutic
applications of exosomes in gene therapy, delivery of protein and chemotherapy, exosomes are
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facing problems in the clinical settings. Mammalian cells release very less quantity of exosomes
and their purification involves troublesome process. Drug loading and targeting abilities of
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exosomes without structural and functional loss of exosomes are other factors.

4.6 Sonodynamic therapy of glioma

Glioma is one of the life threatening brain disease and currently available therapeutic options are
incapable to restrict the tumor growth and improve patient outcomes. Synergistic therapy of
tumor with ultrasonic wave and sonosensitizer attained positive outcomes in suppressing tumor
progression [263, 264]. Ultrasonic waves open the tight junctions of endothelial cells, penetrate
to the deep tissues, trigger sonosensitizer nearby tumor tissue and kill the cancer cells without
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harm to normal tissues [265]. Hematoporphyrin monomethyl ether (HMME), porfimer sodium
(Photofrin), di-sulfo-di-phthalimidomethyl phthalolcyaninezinc (ZnPcS2P2), Photolon, 5-
aminolevulinic acid (5-ALA), and rose bengal (RB) are sonosensitizers used in sonodynamic
therapy (SDT) [265]. The combined effect of ultrasonic waves with sonosensitizer is to generate
reactive oxygen species (ROS) and decrease mitochondrial membrane potential which has
central place in the apoptosis and cancer cell death [266]. Malignant glioma involves stem like-
glioma cells with self renewable potential and responsible for reoccurrence of disease after

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therapy. Sonosensitizer showed apoptotic activity against malignant glioma cells and effective in

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recurrent malignant glioma [267]. It has been demonstrated that chemotherapeutic or anti-

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inflammatory agents experiencing synergistic effect with ultrasonic waves could act as
sonosensitizer [268]. Sono-photodynamic therapy (SPDT) is a novel approach with characteristic

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attributes of PDT and SDT. Various studies reported SPDT killed the tumor cells, inhibiting
primary and metastatic tumors with reduced dose of sonosensitizer [269, 270]. Further,
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encapsulation of sonosensitizer into or conjugating onto the surface of NPs boosts up their
antitumor efficacy and efficiency. SDT is remarkable therapeutic option for glioma patients but
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innovative sonosensitizers are demanded with low phototoxicity, and improved sonosensitivity
and intra-tumor accumulation. Currently available ultrasonic devices are only suitable for animal
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skin penetration of ultrasonic waves; but human skin requires advanced devices with potential to
provide deeply skin penetrating waves and activate sonosensitizer in the targeted site.
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5. Recent progress of drug nanoformulations targeting to brain

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Most of the available drug delivery nanocarriers explored in recent years for brain therapeutics
and theranostics are based on lipid or polymeric materials. Polymeric nanoformulations include
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polymeric NPs, carbon based nanovehicles and polymeric micelles. Lipid based
nanoformulations are liposomes, solid lipid NPs, niosomes and transferosomes. Nanogel and
nanoemulsion have also gained considerable attention of researchers for trans-endothelial
delivery of neuropharmaceuticals. Various nanoformulations targeting to brain are sketched in
Fig. 3.
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Fig.3: Sketch of various drug nanoformulations targeting to brain

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5.1 Polymeric nanoformulations

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Polymeric NPs are nanoscopic formulations of biodegradable and biocompatible synthetic or

natural polymers loading hydrophilic or lipophilic neuropharmaceutical agents in solid or
solution form or chemically linked to the surface and protecting drugs from biological and
chemical hazards [271]. Polymeric NPs for neurotherapeutic drug transportation offer unique
benefits of diverse size distribution and morphological characterization, surface modulation by
targeting moieties and surfactants, various pathways of administration and stimuli sensitive drug
release behavior. BBB transmigrated polymeric NPs consisted of natural polymers are chitosan,
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gelatin and sodium alginate NPs; whereas, NPs fabricated from synthetic polymers are poly
(D,L-lactide-co-glycolide) (PLGA), poly (D,L-lactide) (PLA), polybutyl cyanoacrylates (PBCA),
polycaprolactone (PCL) and poly (ethylenimine) (PEI) NPs [272].

5.1.1 PLGA NPs

FDA accorded approval to one of the safest, biodegradable and biocompatible polymers, PLGA,

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as a drug delivery vehicle. Synthesis of PLGA polymer is based on the co-polymerization of two

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hydroxyacids, PLA and PGA [27]. NPs constructed of PLGA polymer are conferred targeted
transport, safer and low toxicity profile, compatibility with the biological systems and surface

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modulation. PLGA NPs have been proven as splendid nanovectors for BBB transmigrated and
brain tissues targeted delivery of neurotherapeutics. Surface modulation of these nanoagents with

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surfactants or receptor targeting proteins/Abs promotes their entry across brain capillary
endothelial cells by receptor mediated mechanism [273]. To study the impact of surfactants on
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the biodistribution of PLGA NPs in brain, polysorbate 80 (PS80) functionalized PLGA NPs
encapsulating carboplatin for malignant glioma therapy were constructed and in vivo
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biodistribution studies in rats were conducted. Surfactant modified NPs manifested 3.27 fold
enhanced concentration of drug in brain than free drug solution [274]. Similarly, poloxamer 188
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coated NPs for intracranial glioblastoma treatment and their mechanism of transportation across
BBB were explored. Cellular studies showed clathrin-mediated endocytosis was the major
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mechanism involved in the internalization of NPs, depending on the surface characteristics of

NPs. After entry to U87 glioma cells, NPs accessed and released DOX in lysosomes and the drug
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was transported to nuclei for its cytotoxicity. NPs transportation across BBB was mediated
through binding of Apo lipoprotein anchored NPs with LRP1 or scavenger receptors. These
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results were in consistence with the potential of poloxamer 188 binding to Apo proteins and
facilitate receptor mediated endocytosis of nanocarriers [275]. Schuster et al. investigated
lysosomal hydrolase arylsulfatase A (ASA) bound and surfactants coated PLA, PLGA and
human serum albumin (HSA) NPs for lysosomal storage disorder (LSD). Various methods
employed for ASA binding to NPs and preventing the detaching of ASA from NPs surface in
blood circulation were compared. It was observed NPs were inefficient to achieve desired brain
concentration of ASA in mice and animal models. The surfactant coating was not up to the mark
and unable to prevent the rapid clearance of PLGA NPs by the macrophages of peripheral
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organs; whereas, HSANPs were not rapidly cleared by the macrophages but still not reached the
brain efficiently compared to free ASA. ASA glycoprotein was declared responsible for the
diminished transmigration of HSANPs across endothelial cells of BBB. PLGA NPs recognition
and clearance by the reticuloendothelial system shortened the biological half life and limited
their active and passive targeting capabilities of cerebral disorders [276].

PEGylation, a covalent or non-covalent conjugation of polyethylene glycol to the surface of

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nanostructures, protects NPs from opsonization and phagocytic clearance, modulates hydration

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capacity and stability, extends plasma half life and improves efficiency. Epigallocatechin-3-
gallate is a potent therapeutic moiety for temporal lobe epilepsy but stability concerning issue

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hampers its efficacy. Construction of PLGA NPs and surface modulation with PEG improved the
stability of drug, reduced the convulsion and provided neuroprotection in kainic acid mouse

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model of epilepsy. Further, PEGylated NPs attained sustained release characteristics, reduced the
drug degradation and immunogenicity and optimized the bioavailability [277]. Particle size of
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nanovehicles influences their tissue targeting efficiency while treating neurological disorders. To
probe the impact of particle size of PLGA nanoformulations on their TBI targeting potential and
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study the diffusion of NPs to the injured area of brain in TBI mouse model, PEGylated PLGA
NPs were formulated. Further, to target the dead cells and visualize the NPs, PEG coating was
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conjugated with 800CW imaging agent. In vivo biodistribution analysis showed particles with
smaller size penetrated and accumulated in the necrotic area to a greater extent than lager NPs.
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Imaging agent promoted the NPs recognition of dead cells and visualized the NPs accumulation
in TBI lesion [278]. These results suggested that 800CW conjugated NPs could be exploited for
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their diagnostic and therapeutic applications to recognize the traumatic brain lesion and cargo the
therapeutic agents to the necrotic tissues. Decreased non-specific adhesivity and receptor
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targeting (DART) therapy of brain glioma based on ligand guided PLGA-PEG NPs binding
affinity to Fn14 receptors rather than ECM and their efficiency to optimize the retention time in
tumor is a robust approach. PLGA NPs were densely coated with PEG to prevent ECM
interaction and conjugated with ITEM4 to bind with Fn14 receptors. SPR Biacore assays
analyzed the strong binding of PEG-PLGA-ITEM to the Fn14 Biacore chip than NPs without
ITEM modification. Flow cytometry determined the expression of Fn14 receptors and affiliation
of ligand guided NPs with KR158 cells. Confocal microscopy imaging and 3-D projection of
cells further confirmed the higher cell uptake of PEG-PLGA-ITEM4 than unguided NPs and
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deep penetration of cells, respectively. In vivo biodistribution studies in mice implanted with
KR158-Luc glioma and i.v. injected NPs revealed distribution of NPs in brain tumor tissues and
higher co-localization of ITEM4 conjugated NPs than unconjugated NPs, represented Fn14-
targeted delivery of NPs to the tumor cells [279].

Various CPPs have been reported to bind with nAChRs and promote BBB translocation of
nanoconstructs for neurotherapeutic drug transportation. In recent years, CPP coupled PLGA

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nanovehicles were studied for treatment of neurodegenerative brain conditions and brain tumors

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therapy. Biological NPs such as red blood cell membrane-coated nanoparticles (RBCNPs) are
novel nanostructures with the properties of plasma membrane, showing excellent anti-tumor

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efficiency, low toxic effects, prolong plasma half life and less immune response in previous
published research experiments [280]. Chai et al. modified RBC membrane coated PLGA NPs

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encapsulating DOX with CDX peptide. In vitro cellular experiments exhibited higher
endothelial cells uptake of DCDX modified RBCNPs than unmodified RBCNPs. Pre-incubation
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of BBB monolayer with CDX and significant competitive reduction of CDX RBCNPs
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transcytosis across monolayer confirmed nAChRs mediated transcytosis. CDX-RBCNPs
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reduced apoptosis and angiogenesis, significantly, in U87 glioma compared to RBCNP,

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confirming the CDX promoted the delivery of nanocarriers to the glioma. RBC coating
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D
protected nanocarriers from cardiotoxicity as free CDX exhibited cardiotoxicity; whereas,
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CDX-RBCNPs had no effect on cardiac function. Similarly, RVG peptide bounded to nAChRs
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on the brain endothelial cells and translocated drug delivery to brain [280]. Fu et al. revealed that
RVG cell uptake is inhibited by GABA [281]. To predict whether RVG is substrate for GABA
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receptors or nAChRs, mechanism of RVG peptide conjugated PLGA NPs for BBB
transmigration was explored by neural cells uptake studies. DOX loaded RVG modified
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nanocarriers showed improved cytotoxicity of DOX in neural cells. SH-SY5Y neuroblastoma

cells were pretreated with inhibitors of GABA (bicuculline, saclofen) and nAChRs
(hexamethonium, mecamylamine). Cells pretreated with hexamethonium exhibited 96% cell
viability whereas, GABA inhibitor-pretreated groups exhibited decrease in cell viability,
confirming the cell death was associated with nAChRs mediated endocytosis of RVG DOX-NPs.
These results suggested the binding of RVG with nAChRs instead of the GABA receptors [282].
It is usually known that NPs transported across the BBB enhance the biological effect of payload
but it is unclear whether BBB transportation is consistent with the efficacy of drug. For this
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purpose, pegylated PLGA NPs via surface linker (DSP-PEG) and PLA-hyperbranched
polyglycerol (PLA-HPG) NPs were designed and surface modified with three transport ligands:
RVG, TGN and Adenosine (Ad). In vivo uptake experiments in mice revealed RVG modified
PLGA NPs did not show any significant improvement in brain uptake than unmodified NPs.
Whereas, TGN modified NPS demonstrated an increased accumulation in brain compared to
unconjugated NPs. Brain uptake efficiency of PLA-HPG NPs was superior to PLGA-DSPE-PEG
NPs. Ad conjugated PLA NPs were additionally efficient in improving the brain uptake which

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was correlated with the contents of Ad. To study the efficacy, camptothecin encapsulated PLA

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NPs were injected i.v. to U87 tumor bearing mice and NPs exhibited no survival benefits at

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maximum tolerated dose which was inconsistent with BBB transmigration efficiency of NPs.
However, the cytotoxic effect of these NPs was appeared in glioma cells. Repeated

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administration of PEG NPs exhibited immune response but HPG coating prevented detection of
NPs by immune system [283].
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Targeting the TfRs located on the BBB endothelial cells or glioma cells is another robust
approach for neurological disorders and brain tumor therapy. Tf surface modified PLGA NPs
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have been successfully implied in brain delivery. Dual targeting strategy based on T7 peptide
binding affinity with TfR and magnetic NPs for active targeting of the glioma cells was implied
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(as explained in Fig.4). T7 conjugated PLGA NPs encapsulating magnetic NPs and two
combined drugs (paclitaxel and curcumin) showed, significantly, higher cell uptake and brain
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accumulation compared to unmodified NPs. The dual drugs synergistically inhibited tumor
progression via mitochondrial destruction and apoptosis induction [284]. Similarly, in another
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study, surfactant modified PLGA NPs delivered methotrexate-Tf (MTX-Tf) conjugate to tumor
cells through TfR receptor mediated endocytosis. Surfactants, pluronic F-68 and/or PS80,
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promoted BBB penetration and inhibited P-gp efflux of drug loaded PLGA NPs. The highest
fluorescent intensity of fluorescein isothiocyanate (FTIC) labeled NPs in glioma cell uptake
experiment confirmed the surfactant enhanced permeation and TfR targeting potential of MTX-
Tf conjugate. Sustained release behavior of nanoformulations, selective targeting of tumor cells
and peripheral metabolism protection, augmented the cytotoxicity of drug conjugate [285].
Macromolecule such as peptide and protein based drugs are therapeutically potent in cerebral
disorders therapy but the challenging barrier of brain microvascular endothelial cells hinder their
brain targeting efficiency. Peptide iAβ5 is highly efficient in amyloid β aggregate inhibition in
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Alzheimer’s disease but BBB restriction led to the loss of its therapeutic beneficial results.
Encapsulation of iAβ5 in PLGA NPs guided through OX26 mAb and anti-Aβ (DE2B4) resulted
in an elevation of porcine brain capillary endothelial cells uptake. OX26 supplemented NPs
transportation through TfR internalization whereas; DE2B4 helped to recognize amyloid β.
Substrates for Lf receptors located on capillary endothelial cells of brain could be ligands for
trans-BBB drug delivery [286]. Lf coated PEG-PLGA NPs loading Shikonin (SHK), a
chemotherapeutic agent, were designed to improve the bioavailability of drug and overcome the

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BBB penetration-related issues. Lf augmented transport of NPs to brain thorough LfR mediated

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passage. Lf NPs had shown more cytotoxicity of SHK in C6 cells than SHk alone and it was

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observed that targeting capabilities of Lf promoted C6 cells uptake of NPs. Nanovehicles further
improved the pharmacokinetic profile of SHK [287].

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Hybrid NPs of PLGA with other biocompatible and biodegradable polymers have been widely
studied for trans-endothelial cells and brain tissues targeting delivery. Polyacrylamide-chitosan-
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PLGA NPs (PAAM-CH-PLGA NPs) encapsulating rosmarinic acid and modified with ApoE on
surface and cross-reacting material 197 (CRM197) for Alzheimer’s therapy. Surface modulation
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with tween 80 and PEG prevented nanocomposite uptake by the phagocytes, promoting RA BBB
translocation and retarding the Aβ neuron degeneration through decreased apoptic SK-N-MC
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cells viability [288].

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Fig.4: (A) dual drugs encapsulated MNP/T7-PLGA NPs; (B) transport ratios of different NPs
across the BBB; (C) synchrotron radiation X-ray highlighting iron distribution in brain; (D) in
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vivo biodistribution of i.v. injected NPs; (E) confocal microscopic imaging of brain tissues.
Reproduced from [284]
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5.1.2 Dendrimers
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Dendrimers are smart, nanoscale in size, three dimensional, safe, efficacious and peculiar
engineered architecture polymeric nanocarriers with cationic/neutral surface which facilitates
membranous and biological barrier interaction. Characteristic attributes of dendrimers to target
the neuroinflammatory cells and neurons, penetrate endothelial cells efficiently, and cargo
neurotherapeutics, antitumor agents and nucleic acid materials, made them promising
nanovehicles for brain targeting [289, 290]. In recent decades, intratumoral brain targeting based
on dendrimers drug conjugates of chemotherapeutic agents for glioma therapy has gained fame
and attention of researchers [291, 292]. Polyamidoamine (PAMAM) dendrimers have been
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widely characterized for BBB transmigration-based neurological disorders therapy [293]. The 4th
generation of PAMAM dendrimers (G4 dendrimers) could effectively enter the brain, target
microglia and neurons, and the neutral surface prevents plasma protein binding. However, their
smaller size made them vulnerable to the clearance by glomerular filtration and shortened the
half life. Dendrimers with larger size such as G6 are protected from renal clearance and hence,
prolonged the half life and enhanced brain accumulation. Flow of drug molecules from blood
brain to the CSF is limited by the tightly packed endothelial cells. However, in injury and

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inflammation to brain, this barrier becomes leaky and allows the passage from blood to CSF and

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parenchyma. Zhang et al. while keeping in consideration of above hypothesis compared G4 with

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G6 dendrimers in brain injury model [294]. G6 dendrimers were superior in CSF targeting and
accumulation than G4 dendrimers which was based on neural cells and microglia targeting of

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injured brain. Larger size of G6 dendrimers protected them from renal filtration and augmented
transportation to the CSF [295]. Dendrimers could be effective drug delivery vehicles for brain
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injury and neuroinflammation therapy such as TBI, ischemic brain stroke and also in brain
tumor. Animal models of ischemic adult and neonate strokes, ischemic optic neuropathy and
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brain injury related to cardiac arrest have shown accumulation of dendrimers in injured brain
[289]. BBB disruption, microglia sensitization and injury condition are the deciding factors on
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the fate of the dendrimers entered to microglia. In another research to deliver drug molecules to
the microglia and treat neuroinflammation in brain injury condition, dendrimers being neutral
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nanovehicles were applied and their uptake by the microglia in in vivo clinical rabbit model was
evaluated. Altered expression of claudin-5, ZO protein and occludin in disrupted BBB, and
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inflammatory condition associated transcytosis promoted BBB transmigration of dendrimers. G4

dendrimers were localized only to the microglia of injured brain regions (periventricular and
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choroid plexus) frequently diluted with blood and CSF. The uptake of G4 and even G6
dendrimers was correlated with severity of injury [289]. Smart version of nanotechnology
engineered dendrimers enabled transport of gene, nucleic acid and nucleotides to the brain.
Second generation dendrimers as nonviral vectors set a novel platform for antiretroviral SiRNA
delivery to transfect the astrocytes in HIV-associated dementia (HAD). In vitro experiment
exhibited BBB transcytosis of carbosilane dendrimers and siRNA complex. Serramia et al.
conducted in vivo study and observed siRNA dendriplexes reduced the level of HIV infection in
astrocytes compared with controlled astrocytes. Dendriplexes achieved successful transportation
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and transfected siRNA to the infected astrocytes with safety profile [295]. G4 dendrimers were
explored for DNA transfection. G4 dendrimers guided by Serine–Arginine–Leucine (SRL)
peptide and loading DNA were designed and biodistribution and cellular experiments were
conducted. Nanocarriers were transported across the BBB through LRP receptor mediated
endocytosis. Peptide modified nanocarriers showed significant cell uptake than unmodified NPs
and the uptake was inhibited by Lf, confirming LRP mediated mechanism of uptake. Fluorescent
imaging analysis of mice brain revealed accumulation of SLR G4 dendrimers in brain higher

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than unmodified dendrimers. SLR nanocarriers promoted gene expression in brain with low

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toxicity [296].

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5.1.3 Cyanoacrylate nanoformulations

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Nanoarchitectured vehicles based on PBCA polymers are leading nanocarrier discovery to
improve the permeability of BBB impassable drugs. Surface alteration of PBCA NPs with
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surfactants enhances their BBB passage through surfactants-plasma Apo-E interaction with LDL
receptors [297]. PBCA NPs could be innovative drug delivery carriers to target the specific cells
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of brain such as endothelial cells through LDL receptors mediated endocytosis. I.v.
administration of PS80 coated PBCA NPs encapsulating puerarin, a stroke treating moiety,
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provided neuroprotection in rats with focal ischemic cerebral injury at much lower dose of drug
[298]. PS80 PBCA NPs have been reported to have diagnostic applications in neurological
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disorders. PBCA NPs improve BBB permeation of imaging probes, contrast agents and staining
reagents thus, beneficial in whole brain MRI, multiphoton and electron microscopy. Delivery of
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fluorophores across BBB based on PS modified PBCA NPs enabled the neuroscientists to design
Nissl stain for cell imaging, recognize Aβ plaque in Alzheimer′s model and conduct whole brain
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MRI through gadolinium-based contrast agents transport to the mice brain. Conjugation of
surfactant modified PBCA NPs with ApoE receptors binding ligands mediates trans-BBB
delivery to the brain tissue. ApoE-3 conjugation to PS80-coated PBCA-NPs highly potentiated
nanovehicles permeation across porcine brain capillary endothelial cells (BCEC) monolayer
compared to un-conjugated NPs [299]. To achieve high drug content in nanoformulations and
brain concentration of itraconazol is challenging for cryptococcal meningitis therapy. CuricA et
al. were successful to formulate PBCA NPs with 99% of drug contents. Innovative nanocapsules
with solid core of drug instead of liquid core and shell consisted of polymeric material were
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constructed. Further, covalently linked ApoE on the surface of NPs targeted LDLR on the BBB
endothelial cells. Among various surfactant-based formulations, sodium cholate containing
nanosuspensions were stable and had maximum drug loading [300]. Modification of NPs with
the components of cell membrane helps their passage across BBB. In this connection, PBCA
NPs were coated with cholesterol and PEG with the purpose to overcome the endothelial cells
impermeability and immune system detection, respectively. Coumarin-6 (C6) loaded
nanocarriers were safe and provided sustained release and higher brain concentration of a

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fluorescent probe than controlled solution. Cholesterol efficiently enhanced the cell uptake and

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BBB passage of NPs [301]. Molecular simulation has brought about revolutionary change in the

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field of pharmaceutical technology. Simulation could be helpful for selection of suitable
compositions and compatibility of drug and polymers. To explore and compare the interaction of

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tacrine with PBCA and chitosan for Alzheimer′s therapy, molecular dynamic simulation as a
promising tool was applied. Low Interaction energy and value of Flory-Huggins interaction
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parameter of PBCA/tacrine system represented strong interaction and more compatibility of
tacrine with PBCA compared with chitosan, respectively. There was direct relationship between
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chain length of PBCA and interaction with the drug molecule and this relation was not applicable
to chitosan [302].
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In addition to PBCA NPs, other cyanoacrylate based NPs were also exploited for drug delivery
across brain endothelial cells. Impact of PEG coating and monomer types of poly (alkyl
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cyanoacrylate) (PACA) on the interaction of NPs with monolayer and cell uptake by the brain
endothelial cells was evaluated using cell membrane model. PEG type was the deciding factor
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for the interaction of NPS with cell membrane and monolayer. Cell uptake was dependent on the
density of PEG whereas, monolayer association was independent of PEG density. Morphological
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characteristics and monomer types of NPs had no contributive effect on the cell association
[303]. Alzheimer’s disease is accompanied with the loss of memory and the design of suitable
DDS is highly demanded to target the Aβ, enhance its transportation to the plasma and recover
the memory functions. In this connection, mAbs interacting with plasma Aβ peptide 1-42 (Aβ1-
42) and increasing its removal from the brain and plasma were decorated on poly [hexadecyl
cyanoacrylate-comethoxy poly (ethylene glycol) cyanoacrylate] NPs and injected to the
transgenic mice. Novel object recognition (NOR) test demonstrated complete recovery of
memory in mice. NPs effectively decreased the soluble Aβ oligomers level in the brain and
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enhanced its blood level. Plasma clearance of Aβ1-42 facilitated the distribution of Aβ1-42 from
brain to blood and decreased its brain accumulation [304].

Critical attributes of

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nanoformulations

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Large scale Surface US High
No Off-
target
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production modification Small size drug efects
Biodegradable loading
Biocomaptible Long
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half life
Serum
stability
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Fig.5: Critical attributes of drug nanoformulations targeting to brain

5.1.4 Carbon based nanoformulations

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5.1.4.1 Carbon quantum dots

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Carbon quantum dots (C-dots) are carbon particles with size range of 2 to 10nm. C-dots have
versatile applications in the field of nanomedicine attributing to their unique physicochemical
and biological properties, biocompatibility, ease of preparations, low toxicity and conjugation of
wide variety of ligands. C-dots are important pillar of neurotherapeutics delivery to pass the
BBB and deliver the therapeutic molecules to the targeted site. High florescence and imaging
qualities of C-dots enabled neuroscientists to explore their therapeutic, theranostic and diagnostic
applications in neurological disorders and brain tumors. Glioma tumor imaging and targeting
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multifunctional C-dots conjugated with RGERPPR peptide were constructed and injected to the
glioma bearing mice. RGERPPR with strong tumor penetrating abilities potentiated the
penetration of C-dots to the glioma through neuropilin-1 receptors located on the cancer cells and
blood vessels. Furthermore, the peptide overcame the restriction imposed by the EPR effect and
facilitated nanocarrier entrance to the tumor tissues. Higher distribution of C-dots in the glioma
tissue than normal tissues indicated their enhanced permeation across leaky BBB in the glioma
tissue. Peptide functionalized C-dots could be a future target of scientists for glioma targeted

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delivery of chemotherapeutic agents [305]. Less quantum yield of C-dots is a big hindrance in

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their labeling and imaging potentials. Doping of C-dots with heteroatoms such as nitrogen could

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improve quantum yield and imparts electronic and chemical properties to C-dots for
nanotheranostic purposes. Various methods have been applied to construct nitrogen-doped

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carbon dots (N-CDs) including hydrothermal synthesis, liquid-phase polymerization,
electrochemical oxidation, and microwave methods. Lu et al. performed hydrothermal treatment
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of citric acid to construct N-CDs and PEI, which was used as doping agent as well as passivation
material. The synthesized N-CDs had high quantum yield, superb stability against light and were
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excellent probes for real time cell imaging. High BBB permeability of PEI enabled N-CDs as a
strong candidate for brain targeted delivery of neuropharmaceuticals [306]. Another group of
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researchers formulated N-CDs using 1-methyl 2-pyrrolidinone (NMP) as a doping agent and
source of carbon without applying additional chemical for carbon source. The resultant N-CDs
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were biocompatible, water soluble, less toxic with high quantum yield. In vitro study exhibited
penetration of N-CDs to the glioma cells and in vivo florescence imaging of glioma demonstrated
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N-CDs as an excellent contrast material [307]. This research work highlighted the importance of
common solvent in the design of N-CDs with excellent results as nanoprobe agents.
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5.1.4.2 Carbon nanotubes

Carbon nanotubes are promising nanoplatform for drug delivery across biological membranes
and thus nanocarriers of choice for the translocation of drug to the brain. CNTs are state-of-the-
art cylindrical shape nanoformulations with high drug loading and surface area, electrical
conductivity and photothermal properties. In addition to these characteristic features, hollow core
to encapsulate the drugs and surface functionalization of CNTs with targeting moieties render
them suitable nanomaterials for targeting cancerous cells and brain tumors [308]. CNTs are
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classified on the basis of layers of graphene sheets to: (1) single-walled carbon nanotubes
(SWCNTs) comprised of one layer, (2) multi-walled carbon nanotubes (MWCNTs) comprised of
many layers [309]. MWCNTs have plethora of significance in neurological disorders and brain
tumor therapy. Controversial studies have been published, explaining the cytotoxicity of CNTs to
the brain cells, microglia and neurons. The neurotoxicity was highly correlated with the dose,
type of CNTs and cell, and microenvironment [310]. In this context, a study was conducted to
compare the toxicity of MWCNTs to the tissue mimicking culture and monolayer culture of

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human brain endothelial cells, and the impact of surfactant coating or acid treatment on the

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toxicity of MWCNTs was also determined. The authors reported that the cytotoxicity was

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dependent on the cell culture type involved in testing and tissue mimicking culture was safer
than monolayer culture. Cells grown on plastic were more prone to toxicity than those grown on

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Matrigel, independent of MWCNTs surface modification. This study elucidated the importance
of environment while evaluating the toxicity of nanoformulations [311]. Many other controlling
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factors predominantly affect the toxicity of these intelligent nanoformulations such as impurities,
dimensions, surface defects, acid functionalization and plasma protein attachment to the surface
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of CNTs. Intrinsic potential of MWNTs to cross the BBB endothelial cell was implied for
therapy of neurodegenerative disorders. Angiopep-2, LRP1 binding ligand was functionalized on
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the MWCNTs. Ligand promoted the transportation of MWCNTs across primary porcine brain
endothelial cells. I.v. administered CNTs achieved higher brain concentration than unmodified
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CNTs. Wide MWNTs achieved higher brain uptake in glioma brain whereas, thin MWNT were
more accumulated in glioma brain. These findings strengthened the clinical potential of ligand
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modified MWNT in glioma targeted therapy [312]. In another experiment, in vivo biodistribution
study was performed to ensure the distribution of i.v. injected MWNTs in the brain parenchyma
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and endothelium. γ-Scintigraphy was applied for parenchyma uptake and

computed tomography imaging was conducted for distribution study. The results of imaging
analysis confirmed the presence of MWNTs in the brain parenchyma [313].

Table-3: Merits and demerits of brain targeting drug nanoformulations.

Nanostructure Merits Demerits

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Polymeric NPs Unique morphological, physiochemical Cytotoxicity of polymer and

and surface modification properties hindrance in production at industrial
scale
Carbon dots High florescence and imaging Mechanism of photoluminescence is
qualities. Small size, biocompatible unclear
and polymeric core

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Carbon Electrical conductivity, photothermal Neurotoxicity related to dose, type of

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nanotubes properties, hollow core and cylindrical CNTs and cell culture
shape

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Liposomes Long circulation half life, high BBB Plasma proteins interaction inhibits
permeability and bear multifunctional ligand binding with receptors, large

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targeting species scale manufacturing challenges, off-
target side effects and large size
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Solid lipid NPs Ease of production at large scale, and Low drug loading and entrapment
tackle solubility, permeability and efficiency, and surfactants associated
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toxicity related issues of drugs toxicity

Micelles Just self assembly to manufacture, Unsuitable for hydrophilic
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small size, high drug loading, surface molecules, self aggregation, serum
modification with BBB targeting instability leading to drug release in
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ligands, hydrophobic drug blood circulation and less drug

incorporation, and hydrophilic shell loading.
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avoids plasma protein interaction.

Nanogel Biocompatible, drug release responsive Response to external stimuli and
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to stimuli and pH , drug delivery at the tunable drug release behaviors are
targeted site, nanoscopic size, surface challenging.
modulation and prolong half life
Metallic NPs Specific morphological and surface Increase exposure of human to silver
properties. Biocompatible and easy to leading to loss of memory and
prepare cognition, neurotoxicity and
inflammation
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5.1.5 Micellar nanoformulations

Polymeric micelles (PMs) are core-shell nanoscale-sized formulations of copolymer, self

assembles in aqueous medium in such a way that the hydrophilic portion forming shell faces
towards the aqueous medium, whereas the hydrophobic part forms the core [343]. PMs as a
nanoformulation have long history approaching 1980s but their historic brain delivery touched to
2008 when Liu et al. for the first time came to know the importance of micellar nanocarriers in

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neurological disorders therapy [314]. Physiochemical properties of micelles are the driving

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factors for successful delivery of therapeutic moiety to the brain. Critical micelle concentration

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controls the serum instability and drug release from micelles in blood circulation before reaching
the brain. Particle size is an extraordinary important factor deciding the fate of nanocarriers to
achieve targeted drug delivery. There are many controversial views of scientists on the impact of

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drug loading and conjugation of ligands on the size and zeta potential of micellar nanocarriers
[315]. Surface functionalized micellar nanoformulations are in line with novel nanotherapeutic
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approaches to target the brain. In this modern era of nanotechnology, computer-aided simulation
modeling has gained considerable attention of neuroresearchers for selection and evaluation of
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binding affinity of suitable ligands to the receptors, helpful in designing glioma targeted
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nanovehicles. The receptor associated protein (RAP) has binding affinity with LRP1 present on
the glioma and BBB. A short chain RAP12 peptide was designed from RAP protein and
molecular docking study was conducted to theoretically evaluate peptide binding with receptors,
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and experimental and theoretical data were compared. In vitro and in vivo experiments confirmed
RAP peptide conjugated on PEG-PLA micelles potentiated micelles transportation across
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BBB/BTBB. Compared with the unmodified micelles and free drug, peptide modified paclitaxel
(PTX) loaded micelles significantly induced apoptosis with less angiogenesis. Overall
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multifunctional nanocarriers were efficient in targeting and treating glioma [316]. The same
group of scientists exploited another tumor homing peptide, L-VAP (SNTRVAP), binding to
GRP78 receptors expressed on glioma cells surface. Retro inverso isomer of L-VAP (RI-VAP)
and retro isomer of L-VAP (D-VAP) were constructed using solid phase peptide synthesis
method and the binding affinity with protein receptor was studied using molecular docking.
Peptides were directed on the PTX loaded micelles to investigate the effect on glioma. Cell
uptake studies elucidated significant peptide uptake by HUVEC cells and U87MG cells. RI-VAP
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and D-VAP had remarkably advantage over L-VAP in tumor accumulation. In addition, RI-VAP
and D-VAP were superior to L-VAP with respect to anti-glioma efficacy of PTX loaded micelles
[317]. Self assembled and reversibly crosslinked tandem micelles modified with Angiopep-2 and
TAT peptides, dual ligand, were loaded with docetaxel for glioma chemotherapy. The purpose of
introducing dual ligand was to bind Angiopep-2 with LRP-1, facilitate TAT peptide binding with
nAchRs and potentiate cell permeation. Nanomicelles remained in blood circulation for longer
time, achieving high brain permeability, and significantly, transporting and accumulating in

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glioma cells. Dual ligand functionalized micelles had shown strong glioma inhibitory effect than

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Angiopep-2 alone [318]. Dual ligand modification strategy based on glioma targeting and brain

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penetrating peptides could be a useful approach for glioma therapy.

5.2 Lipid based nanoformulations

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Lipid based formulations are categorized, keeping in view of their physiochemical attributes and
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method used for production, into solid lipid NPs (SLNs), liposomes, niosomes and
transferosomes.
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5.2.1 Solid lipid nanoparticles

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Polymer based nanomaterials have unique morphological, physiochemical and surface

modification properties, enabling them to earn the trust of neuroscientists as excellent carriers to
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load and release the drugs in a controlled fashion, which is potent for cerebral disorder therapy.
However, toxic chemicals generation from biodegradation and other toxicity related problems
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limited their clinical potential for brain delivery, and urged the researchers to search for reliable
alternative options [319]. SLNs have small size, biocompatibility, biodegradability, high
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entrapment efficiency and lipid in nature; all these features made them nanostructures of choice
for BBB translocation. In recent years, SLNs have gained fame over other synthetic and natural
nanostructures due to their ease of manufacturing at industrial scale. SLNs comprise of a core
based on lipid contents which are solid at physiological temperature with low melting point.
Surface decoration of SLNs with targeting moieties made them attractive choice to overcome
BBB impeding action and save therapeutic potential of potent neuropharmaceuticals. ApoE
modified SLNs were fabricated using warm microemulsion method and administered via
different routes to compare the blood brain levels achieved by different routes of delivery.
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Pulmonary delivered ApoE SLNs achieved higher brain level than i.v. and intraperitoneal
administered SLNs without causing inflammation to the lungs. The results of this study were
consistent with the study conducted by the same group of researchers involving ApoE modified
liposomes. Like liposomal pulmonary delivery, SLNs pulmonary administration delivered SLNs
from lungs to the blood circulation and then SLNs crossed brain endothelial cells to attain brain
concentration for extended period of time [320]. Pulmonary delivery could be a future
prospective for cerebral disorders and glioma targeting due to large lungs surface area, rich

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supply of blood and lymphatic vessels, and excellent permeability of alveolar capillaries, which

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could facilitate the translocation of drugs to the brain. Low drug loading capacity of SLNs is a

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big challenge while deciding their suitability for brain delivery. Nanostructured lipid carriers
(NLCs) are another class of lipid nanoformulations having liquid lipid matrix with well

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documented applications in the field of nanotechnology based drug delivery. SLNs and NLCs
encapsulating carbamazepine (CBM) were fabricated using homogenization/ultasonication
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method and compared. In comparison to previously published nanosystems, lipid NPs were of
small size, low phosphatidylinositol contents, which could provide the sustained release of drug,
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improve the encapsulation efficiency, enhance the permeability across cell monolayer and attain
stability at 4℃ for 90days. Further, lipid NPs prolonged the anticonvulsant effect of CBM
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without causing neurotoxicity [321]. Another strategic implication of NLCs in BBB permeation
is to use cationic surfactants that interact with negative charged lipid bilayer of BBB and
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overcome the impeding action of BBB. Surface of ultra small NLCs was modified with
surfactants and the safety and efficacy in glioma tumor model were evaluated. Molecular
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dynamic simulation was applied to study the effect of surfactant on the stability of lipid
membranes. Serine derived surfactants were safe, and promoted the stability and delivery of
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NLCs across cell membranes [322]. Serine derived surfactants are good choices for designing
nanosystem as an ideal carrier for brain delivery.

5.2.2 Liposomes
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Liposomes are self-aggregating lipid nanosystems incorporate and deliver lipophilic drugs,
hydrophilic drugs, protein based drugs and nucleic acid materials to the CNS. Lipophilic nature
of liposomes facilitates drug transportation across the membrane of brain endothelial cells and
endows them excellent features for brain uptake [323]. Diffusion of drug and endocytosis of
liposomes are two prominent mechanisms involved in the release of drug from liposomes.
Further, surface functionalization of liposomes with wide variety of targeting agents such as
surfactants, Tf and CPPs, etc., tackles the barrier posed by the brain endothelium and cargo

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neurotherapeutics to the targeted site in brain [324]. Tf decorated liposomes were designed to

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encapsulate and overcome the physiochemical properties related limitation of resveratol in

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glioma therapy. The synthesized liposomoal carriers encapsulated high drug contents, extended
the release of drug and had high uptake by U-87 MG cells. Compared to unmodified liposomes,

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Tf modified liposomes exhibited more cytotoxicity to glioblastoma cells, which was associated
with ROS generation in tumor cells. Besides, liposomal structures were efficient in inhibiting
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tumor growth in mouse model of glioma [325]. In addition to chemotherapy, the potential of
liposomal delivery in neurodegenerative disorders therapy was also exploited. Liposomes loaded
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with N-3,4-bis(pivaloyloxy)-dopamine (BPD) and modified with RVG29 were explored to

amplify the therapeutic efficacy of dopamine derivative. CPP conjugated liposomes efficiently
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translocated the BBB and were taken up by the endothelial cells. Biodistribution studies
demonstrated the distribution of nanostructures to the selective brain parts, and liposomal
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delivery expedited the therapeutic effect of neuropharmaceutical in Parkinson’s disease model of

mouse [326]. Theranostic potential of multifunctional liposomes for Alzheimer’s disease was
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studied. Liposomes were decorated with curcumin ligand targeting amyloid and other two BBB
targeted ligands: one binding with TfR and the other one binding with LDLR on BBB. In another
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study, the effect of curcumin ligand on the targeting efficiency of BBB targeted ligands was
evaluated in normal and transgenic mice. In vivo imaging showed no significant effect of
curcumin ligand on the performance of BBB targeting ligands [327]. Cationic liposomes offer
unique characteristics owing to their binding with negatively charged cell membrane of brain
endothelial cells; however, they are taken up by the peripheral tissues and also bind with serum
protein, ultimately requiring large amount of liposomes leading to cytotoxicity.

Table-4: Summary of ligands decorated nanocarriers with their potential outcomes in cerebral
disorders and brain tumor therapy.
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Nanocarrier Cargo Ligand Indications Outcomes Ref.

Des-octanoyl
Ligands conjugated polymersomal DOX was
Polymersomes DOX ghrelin and Glioma efficient in inhibiting glioma growth and 338
improving survival of tumor bearing mice.
folate

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Targeting ligands elevated the permeability
of the cargo across the BBB in the culture

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Niosomes Serum glucopyranose Brain uptake
model and in mice, and dual-ligand 340
albumin study
and alanine decoration of niosomes was more effective

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than single ligand labeling
Higher distribution in brain tumor tissues and
PLGA-PEG
N.A. ITEM4 Glioma cell uptake of ITEM4 conjugated NPs than 305
NPs

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unguided NPs.
D
CDX-RBCNPs reduced apoptosis and
angiogenesis, significantly, in U87 glioma
D
compared to RBCNP, confirming the DCDX
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RBCNPs N.A. CDX Glioma 280
promoted the delivery of nanocarriers to the
glioma.
OX26 mAb Elevation of porcine brain capillary
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Alzheimer’s
PLGA NPs iAβ5 Peptide and anti-Aβ endothelial cells uptake of iAβ5 encapsulated 286
disease
(DE2B4) PLGA NPs.
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TGN modified NPS and Ad conjugated PLA

NPs had more brain uptake efficiency than
PLA-HPG NPs RVG, TGN
Camptotheci RVG modified PLGA NPs. NPs exhibited no
and PLGA- and Glioma 283
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n survival benefits at maximum tolerated dose

DSPE-PEG Adenosine
which was inconsistent with BBB
transmigration efficiency of NPs.
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Magnetic Inhibited tumor progression, and

PTX and
guided PLGA T7 peptide Glioma significantly, higher cell uptake and brain 284
curcumin
NPs accumulation
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PAAM-CH- Rosmarinic ApoE and Alzheimer's Promotion of RA BBB translocation and

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PLGA NPs acid CRM197 disease retardation of Aβ neuron degeneration.
G4 PAMAM Gene Successful transfection of DNA and gene 296
DNA SRL peptide
Dendrimers delivery expression.
Achieved high drug contents in 300
Cryptococcal
PBCA NPs Itraconazole ApoE nanoformulations and brain concentration of
meningitis
itraconazol.
Brain Cholesterol efficiently enhanced the cell 301
PBCA NPs Coumarin-6 Cholesterol
disorders uptake and BBB passage of NPs.
NPs effectively decreased the soluble Aβ 304
Alzheimer's
PACA NPs N.A. mAbs oligomers level in the brain and enhanced its
disease
blood level.
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Glioma and Enhanced permeation of C-dots to the glioma 306

RGERPPR
C-dots N.A. tissue through neuropilin-1 receptors located on
peptide
imaging cancer cells
Higher brain concentration and glioma 312
CNTs N.A. Angiopep-2 Glioma
uptake than unmodified CNTs.
Significant induction of apoptosis with less 316
PEG-PLA
PTX RAP peptide Glioma angiogenesis.
micelles
RI-VAP and D-VAP were superior to L-VAP 317
RI-VAP
Micelles PTX Glioma with respect to anti-glioma efficacy of PTX

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and D-VAP
loaded micelles.

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Serine derived surfactants were safe, and 322
Serine derived
NLCs N.A. Glioma promoted the stability and delivery of NLCs
surfactants

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across cell membranes.
Receptor mediated endocytosis was evident 341
in internalization of Lf-NLC and Lf-NLC
NLC Nimodipine Lf Brain stroke
delivered nimodipine provided

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neuroprotection.
Pulmonary delivered SLNs achieved higher
BBB-
SLNs N.A. Apo-E brain level than other routes without causing 320
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permeation
inflammation to the lungs.
Aprotinin and SLNs enhanced BBB-passage and tumor
SLNs DOX Glioma 337
p97 growth inhibition efficacy of DOX.
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Tf modified liposomes exhibited significant

cytotoxicity to glioblastoma cells, which was
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associated with ROS generation in tumor

Liposomes Resveratol Tf Glioma 325
cells. Besides, liposomal structures were
efficient in inhibiting tumor growth in mouse
model of glioma.
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Liposomes Distribution of nanostructure to the selective 326

N-3,4-bis
Parkinson’s brain parts, and liposomal delivery expedited
(pivaloyloxy) RVG29
disease the therapeutic effect of neuropharmaceutical
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-dopamine
in Parkinson’s disease model of mouse.
Inhibition of p12 cell apoptosis and
AuNPs Nerve growth Parkinson’s
siRNA significantly reduced expression of α-Syn in 328
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composite factor disease

p12 cells.
FA-BSA-SPIO NPs were effectively
SPIO NPs
Human brain internalized for MRI imaging and
coated with N.A. FA 339
tumor intracellular visualization after FITC labeling
BSA
in the targeted U251 cells.
Ischemic Thromobosonolysis therapy provided stimuli
Nanogel Upa N.A. cerebral responsive release at the clot site and 335
stroke improved efficacy
Glycosylation of A7R peptide enhanced
Glycosylated
Nanodisk PTX Glioma endothelial cells uptake and brain 342
A7R
accumulation of nanodisk.
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5.3 Metal-based nanoformulations

Gold, silver and iron oxide NPs are metal-based inorganic nanoformulations with tunable surface
characteristics such as variable shape, surface functionalization and surface plasmon resonance
phenomenon. They are widely used nanovehicles with bench of theranostic and clinical

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applications in neurodegenerative disorders and brain tumor therapy. Gold NPs (AuNPs) were

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implied for neuroprotection in Parkinson’s disease. Parkinson’s disease is accompanied by the
over-expression and aggregation of α-Syn. AuNPs were explored as a nonviral gene vector to

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carry siRNA targeting and down regulating the genes responsible for α-Syn expression. AuNPs
were modified with chitosan, and conjugated with plasmid DNA (pDNA) and nerve growth

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factor to design a composite through electrostatic adsorption and photochemical immobilization
for gene therapy of Parkinson’s disease. Composite transported pDNA into cells through
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receptor mediated endocytosis and inhibited p12 cells apoptosis. Further, experiments based on
in vitro and in vivo models elucidated that the composite with MPP + significantly reduced the
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expression of α-Syn in p12 cells than cells treated with MPP + alone [328]. Multifunctional
AuNPs would have bright future in the therapeutic delivery of neuropharmaceuticals potent for
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cerebral disorders therapy. Silver NPs (AgNPs) with the size range of 1-100 nm have diverse
contributions in the field of biomedicine. It was suggested that AgNPs may reside in astrocytes
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abundantly found in the brain and induce the dose dependent hormesis response by MUDENG
gene. AgNPs induced hormesis of astroglioma cells and an increased expression of MuD was
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observed. Hormesis inducing effect of AgNPs at low dose and involvement of MuD could be
beneficial to exploit AgNPs in glioma targeted therapy [329]. Park et al. elucidated the cytotoxic
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potential of AgNPs in human neuroblastoma cells. They reported that Ag nanocarriers entered
the blastoma cells, and altered mitochondrial functions and brain-derived neurotrophic signaling
mechanism. Cytotoxic effects of AgNPs were resulted from the production of ROS, destruction
of DNA and leakage of lactate dehydrogenase [330]. AgNPs may induce neuroinflammation and
toxicity to brain tissues. At neuron level, AgNPs altered the expression of genes encoding
transporters which are involved in regulation of lipid and cholesterol level and maintaining the
structure of cell membrane [331]. One study reported that prenatal exposure to AgNPs led to
abnormal behaviors and cognitive dysfunctions at adulthood [332]. AgNPs are not safe for
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lactating mothers and may be harmful at the developmental stages of CNS. They were detected
in fetuses’ brain after administering to lactating dams [333].

5.4 Nanogel

Nanogels are three dimensional cross linked networks of hydrophilic polymeric materials
capable of encapsulating small therapeutic agents, macromolecules, and peptide and protein

T
based drugs without affecting their three dimensional structure. Their unique features of softness,

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amphiphillicity with tunable size, charge and porosity, and high hydrophilicity, drug loading and
biocompatibility made them superior over other drug delivery vehicles [334]. They can be

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actively targeted at specific site by surface modification or passively targeted owing to their
small size in nanometer range. Nanogels have been widely implied in neurological disorders and

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glioma targeted delivery. Stroke is life threatening disease and the efficacy of thrombolytic
agents especially urokinase-type plasminogen activator (uPA) is limited by the short circulation
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half life. Administration of thrombolytics with ultrasonic mediation could achieve higher
efficacy than alone. Furthermore, ultrasound mediated nanogel delivery of thrombolytic agents
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could provide stimuli responsive release at the clot site. In this context, nanogels with polymer
matrix responsive to ultrasound were encapsulated with uPA and their efficiency was evaluated
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in rat model of ischemic stroke. Intervention of thrombolytic loaded nanogels with ultrasound
resulted in an improved effect of uPA in ischemic stroke model than free drug and ensured the
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safety profile of sonothrombolytic nanocarriers [335]. Sonothrombolysis therapy of cerebral

ischemic stroke based on nanogels is expected to be translated into clinical practice in the near
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future. Poly (N-isopropylacrylamide (PNIPAM) nanogels as novel nanostructure were

constructed to tackle the BBB and attain donepezil brain level for extended period of time. Zebra
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fish model was established to evaluate the trans-BBB delivery of nanogels. The resultant small-
sized nanogels had high drug loading and their drug release behavior was thermoresponsive.
Donepezil encapsulated nanogels released the drug slowly to enter the brain of zebra fish and
confirmed the better safety of nanogels delivery compared with free drug [336].

6. Conclusion

BBB is a major hindrance in the successful brain uptake of neuropharmaceuticals, leaving the
high risk patients of neurological disorders and brain tumors untreated. Currently investigated
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invasive and non invasive approaches achieved some success in targeting the brain with
limitations. Endogenous cells such as macrophages, astrocytes, exosomes and stem cells have
been investigated as carriers of neurotherapeutics for immune therapy and treatment of
neuroinflammation in neurological disorders. Various clinical trials based on acive or passive
immunotherapy, stem cell therapy and clearance of Aβ plaque, etc. are in ongoing phase and
hopeful for positive outcomes. In recent decades, researchers have been able to design
nanoformulations of plain or crosslinked polymer, lipid or inorganic materials with hydrophilic,

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hydrophobic or amphiphillic nature, variable surface properties, loading hydrophilic or

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hydrophobic drug contents and decorated with multifunctional ligands to tackle the biological

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barriers, and improve the safety and efficacy of brain therapeutics. Theranostic potential of
nanoformulations has also been exploited, helpful in diagnosis and better management of GBM

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and neurological disorders. Low drug loading and encapsulation efficiency, large scale
manufacturing challenges, serum instability, off-target side effects and toxicity of polymeric
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material and surfactants are the major drawbacks of nanoformulations hindering their entry to
clinical trials. Keeping in view the severity of brain disorders, highly efficacious, BBB
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permeable and potent therapeutic agents delivered via innovative technology based
nanoparticulate systems are urgently demanded with no peripheral toxicity and safety profile.
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Acknowledgements
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This work is supported by the Major Research Project of Shandong Province, P.R.China
(No.2018GSF118004).
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