Journal of Controlled Release 261 (2017) 4361

Contents lists available at ScienceDirect

Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Review article

Self-assembled amphiphilic core-shell nanocarriers in line with the modern MARK

strategies for brain delivery
Reham S. Elezaby, Heba A. Gad, Abdelkader A. Metwally, Ahmed S. Geneidi, Gehanne A. Awad
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: Disorders of the central nervous system (CNS) represent increasing social and economic problems all over the
Brain targeting world which makes the eective transport of drugs to the brain a crucial need. In the last decade, many strategies
Core-shell nanocarriers were introduced to deliver drugs to the brain trying to overcome the challenge of the blood brain barrier (BBB)
Micelles using both invasive and non-invasive methods. Non-invasive strategy represented in the application of nano-
Nanotechnology
carriers became very common. One of the most hopeful nanoscopic carriers for brain delivery is core-shell
nanocarriers or polymeric micelles (PMs). They are more advantageous than other nanocarriers. They oer small
size, ease of preparation, ease of sterilization and the possibility of surface modication with various ligands.

Abbreviations: 17-AAG, 17-Allylamino-17-demethoxy geldanamycin; 5-FU, 5-ourouracil; AAV, adeno-associated virus; ABC, ATP binding cassette; Ach, acetyl choline; AFM, atomic
force microscopy; AmB, amphotericin B; AMT, adsorbtive-mediated transcytosis; APN, aminopeptidase N; Apo, apolipoprotein; ATRA, all-trans retinoic acid; BBB, blood brain barrier;
BCECs, brain capillary endothelial cells; BCNU, bis-chloroethylnitrosourea; BCSFB, bloodcerebrospinal uid barrier; BMECs, brain microvascular endothelial cells; BS, bile salt; BVECs,
brain vascular endothelial cells; CA8, dendritic octamer of cholic acid; CAC, critical aggregation concentration; CD, choline derivatives; CDs, cyclodextrins; cl-micelles, cross linked
micelles; CIMS, core-inversible micelles; CMC, critical micelle concentration; CMT, carrier-mediated transport; CNS, central nervous system; CPPs, cell penetrating peptides; CPT,
captothecin; cRAD, cyclic-arginine-alanine-aspartic acid; CREKA, cysteine-arginine-glutamic acid-lysine-alanine; cRGD, cyclic Arginine-Glycine-Aspartic acid; CRM, cross reacting ma-
terial; CSF, cerebrospinal uid; CS-SA, stearic acid-grafted chitosan; CT, X-ray computed tomography; DACHPt, (1,2-diaminocyclohexane) platinum(II); DHA, dehydroascorbic acid; DiR,
near-infrared uorescent dye; DL, drug loading; DLS, dynamic light scattering; DOM, domperidone; DOPE, dioleolyphosphatidyl ethanolamine; DOX, doxycycline; DPA, 3,30-dithio-
dipropionic acid; DPc, dendrimerphthalocyanine; DSPE, distearoylphophatidyl ethanolamine; DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene
glycol); DT, diphtheria toxin; DTR, diphtheria toxin receptor; DTSSP, 3,3-dithiobis(sulfosuccinimidylpropionate); DTX, docetaxel; ED, 1,2-ethylenediamine; EDC, 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimidehydrochloride; EE, encapsulation eciency; EPR, enhanced permeability and retention eect; FA, folic acid; GBM, glioblastoma multiforme; GLUT,
glucose transporter; GLUT-1 scFv, GLUT1 antibody single chain fragment variable; GTA, glycidyltrimethylammonium chloride; HIV, human immunodeciency virus; HRP, horseradish
peroxidase; HSA, human serum albumin; HSVtk, herpes simplex virus thymidine kinase gene; IAs, inhaled anaesthetics; ITZ, itraconazole; LAT1, large neutral amino-acid transporter type
1; LCST, lower critical solution temperature; LINCL, late infantile neuronal ceroidlipofuscinosis; LRP, lipoprotein receptor-related protein; Lt, lactoferrin; LTG, lamotrigine; LtR, lacto-
ferrin receptor; mAbs, monoclonal antibodies; Mal, maliemide; MAN, aminophenyl--d-mannopyranoside; mPEG-b-P(Glu), poly(ethyleneglycol)-b-poly(L-glutamic acid); mPEG-b-PCL-b-
PPEEA, monomethoxy poly(ethylene glycol)-block-poly(-caprolactone)-block-poly(2-aminoethyl ethylene phosphate); mPEG-OA, monomethoxy poly(ethylene glycol)-oleate; mPEG-
PAHy-GTA, methoxy poly(ethylene glycol)-,-polyasparthydrazide-glycidyltrimethylammonium; m-PEG-PDMAEMA, methoxy poly(ethylene glycol)-poly(2-(dimethylamino)ethyl
methacrylate; mPEG-St, mPEGylated starch; MRI, magnetic resonance imaging; MRP, multidrug resistant protein; MRSA, methicillin-resistant Staphylococcus aureus; NGR, Asn-Gly-Arg
peptide; NHS, n-hydroxysuccinimidyl; NIR, near-infrared; NPs, nanoparticles; OX26, an anti-TfR antibody; PC, phosphatidylcholine; PCL-b-p(NIPAAm-co-DMA), poly(-caprolactone)-
block-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide); PCL-PEEP, poly(-caprolactone)-block-poly(ethyl ethylene phosphate); PDI, polydispersity index; PEG, poly(ethylene
glycol); PEG-b-Chol, poly(ethylene glycol)-block-cholesterol; PEG-b-P(NIPAAm-co-NHPAAm), poly(ethylene glycol)-block-poly(N-isopropylacrylamide-co-N-(4-hydroxyphenethyl)acry-
lamide); PEG-b-PCL, poly(ethylene glycol)-block-poly(-caprolactone); PEG-b-PLA, poly(ethylene glycol)-block-poly(lactic acid); PEG-b-PLL, poly(ethylene glycol)-block-poly(L-lysine);
PEG-b-PMA, poly(ethylene glycol)-block-poly(methacrylic acid); PEG-PAE-API, poly(ethylene glycol)-poly(b-amino ester)-1-(3-aminopropyl) imidazole; PEG-PBLA, poly(ethylene glycol)-
poly(b-benzyl-L-aspartate; PEGPGluPPhe, poly(ethylene glycol)-poly(glutamic acid)-poly(phenylalanine); PEG-pLys-pPhe, poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-phenylala-
nine); PE-mPEG, 1,2 distearoyl-snglycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)); PEO, polyethylene oxide; P-gp, P-glycoprotein; PHEA-EDA-PLA, ,-poly((N-hy-
droxyethyl)-DL-aspartamide)-ethylenediamine-poly(lactic acid); pHPMA-co-pLMA, poly(N-(2-hydroxypropyl)-methacrylamide)-poly(lauryl methacrylate); PIC, polyion complex; PLA-b-
PDMAEMA, poly(lactic acid)-poly(dimethylaminoethyl methacrylate; PMs, polymeric micelles; pNIPAAm, poly(N-isopropyl acrylamide); PPO, polypropylene oxide; PS80, polysorbate 80;
PTX, paclitaxel; R3V6, 3-arginine and 6-valine; R7L10, 7-arginine and 10-leucine; RAFT, reversible addition-fragmentation chain transfer; RES, reticuloendothelal system; RMT, receptor-
mediated transcytosis; ROP, metal-free organo catalyticpolymerization; RT, radiation therapy; RVG, rabies virus glycoprotein; SACDs, sodium alginate cholesterol derivatives; SA-g-
pNIPAAm, sodium alginate-graft-poly(N-isopropylacrylamide); SAH, subarachnoid hemorrhage; SDS, sodium dodecyl sulphate; siRNA, small interfering RNA; SMA, poly(styrene-co-
maleic anhydride); SPIONs, superparamagnetic iron oxide nanoparticles; sPMI, stapled peptide antagonist of both MDM2 and MDMX; T7, His-Ala-Ile-Tyr-Pro-Arg-His peptide; Tat, trans-
activating transcriptional activator; Td, tryptophan derivative; TEER, transendothelial electrical resistance; TEM, transmission electron microscope; Tf, transferrin; TFF3, trefoil factor 3;
TfR, transferrin receptor; TGN, TGNYKALHPHNG peptide; Tjs, tight junctions; TMZ, temozolomide; TPGS, D-alphatocopheryl polyethylene glycol succinate; UV, ultra-violet; VEGF-
siRNA, vascular endothelial growth factor-small interfering RNA; YC1, 5-cholestane-24-methylene-3,5,6,19-tetraol; Zot, zonulaoccludens toxin

Corresponding author.
E-mail address: reham_samir@pharma.asu.edu.eg (R.S. Elezaby).

http://dx.doi.org/10.1016/j.jconrel.2017.06.019
Received 17 March 2017; Received in revised form 16 June 2017; Accepted 20 June 2017
Available online 23 June 2017
0168-3659/ 2017 Elsevier B.V. All rights reserved.
R.S. Elezaby et al.
Hence, the aim of this review is to discuss modern strategies for brain delivery, micelles as a successful delivery
system for the brain and how micelles could be modied to act as magic bullets for brain delivery.
1. Introduction
Diseases can aect both brain and spinal cord causing central ner-
vous system (CNS) neurological or psychiatric disorders. CNS diseases
are numerous and can be classied under three categories [1,2]. The
rst category includes neurological infectious diseases caused by bac-
teria, viruses and fungi such as meningitis, encephalitis, Lyme en-
cephalopathy, CreutzfeldJakob and cerebral toxoplasmosis. The
second category includes neurological disorders such as Alzheimer's
disease, Parkinson's disease, epilepsy, seizures, dementia, depression,
multiple sclerosis, mononeuropathy and polyneuropathy. The last ca-
tegory involves brain tumors and physical injuries such as cerebral
tumors, thrombosis, embolism, hemorrhage and vasculitis. CNS dis-
orders represent an increasing problem both socially and economically
all over the word with regard to both the number of patients and the
cost of therapy.
So far the CNS disorders are responsible for almost 11% of disease
burden - as they aect 1.5 billion people - and about 1% of deaths
worldwide [3]. Hence, ecient treatment of CNS diseases became a
crucial issue. Despite the presence of relatively high blood ow to the
CNS (total surface area of 20 m2 and a total length of 400 miles), the
eective targeting presents a great problem [4].
The two main barriers hinder the brain delivery are the bloodbrain
barrier (BBB) and the bloodcerebrospinal uid barrier (BCSFB), with
the rst one regarded as the most important obstacle to the brain
parenchyma [57]. Generally, invasive strategies were oftenly used [7].
Lately, non-invasive strategies involving nanocarriers delivery became
the most targeted strategy [8]. Polymeric micelles (PMs) as self-as-
sembled amphiphilic core-shell nanocarriers are among the promising
nanoscopic carriers [9,10]. During the last decade, numerous studies
utilized PMs as an ecient drug delivery system for brain delivery
[1113]. In this review, we will highlight the modern strategies for
brain delivery. Likewise, an in-depth focus on PMs as promising na-
nocarriers for brain targeting will be upraised. The review will em-
phasize at the end the use of PMs as magic bullets in brain ther-
apeutics.
Fig. 1. Schematic illustration of blood brain barrier.
Reprinted from [16] after permission of Elsevier.
44
Journal of Controlled Release 261 (2017) 4361
2. Challenge of the BBB
Being of selective nature, BBB oers to the brain protective func-
tions against noxious circulating substances, while at the same time
aording needed nutrients for an adequate brain function [5]. It is
physiologically made up of three layers; the rst is the inner endothelial
cell layer forming the capillary wall and containing tight junctions
(Tjs); the second is the basement membrane and nally the third layer
composed of pericytes and astrocytic feet processes lying on the base-
ment membrane [2] (Fig. 1). These whole anatomical and physiological
properties generate the concept of BBB.
Tjs, acting as a physical barrier, are formed and regulated by claudins
(e.g. claudins 1 and 5), occluding, annexin-1 and junction adhesives,
which are protein molecules linked together by zonulaoccludens and
cingulin to the beta-actin cytoskeleton. They are lling the gap within
brain vascular endothelial cells creating the tight junctions. This will
not only reduce the paracellular movement of substances, but also will
contribute to the high transendothelial electrical resistance (TEER)
of > 1500 cm2 [2,8].
Astrocytes and pericytes play essential role in maintaining BBB
function in healthy CNS and following injuries. Astrocytes main roles
are in the healthy CNS and include blood ow regulation, nutrients
provision and assistance of extracellular ion balance. Moreover, fol-
lowing traumatic injuries, it has a fundamental eect on the reforma-
tion of the damaged brain and spinal cord [5,14]. Pericytes, responsible
for growth factors secretion or changes in the extracellular matrix, are
important in the maturation, renovation and conservation of the vas-
cular system. Besides, their participation in molecules delivery across
the BBB and vascular permeability control is well dened [5,15].
The concept of biochemical barrier is established from the occupancy
of many receptors, transporter proteins and eux pumps that permit
the passage of certain vital substances into the brain and prevent the
others [17]. Actually, during physiological and pathological processes,
BBB is included in several functions like tissue homeostasis, brinolysis
and coagulation, adjustment of vascular tone, blood cell movement and
activation, preservation of neuronal environment and vascularization of
normal neoplastic tissues [5].
R.S. Elezaby et al. Journal of Controlled Release 261 (2017) 4361

3. Transport across the BBB

The last two decades have witnessed many researchers focusing on

the deep understanding of dierent transport pathways across the BBB
[18]. As illustrated in Fig. 2, there are dierent transport routs through
which molecules can cross the BBB.
Passive diusion of substances can be divided into paracellular and
transcellular. The paracellular pathway across the tiny spaces in be-
tween cells always provides a pathway for small water soluble mole-
cules. In contrast to other cell membranes, brain paracellular pathway Fig. 3. Schematic illustration of modern strategies for brain delivery.
is virtually absent due to presence of Tjs. The transcellular pathway is
available for molecules, which fulll certain criteria such as [1921]: i-
gp) [29], ATP binding cassette (ABC) transporter [30,31] and multidrug
molecular weight < 400500 Da; ii- unionized compounds; iii- log P
resistant protein (MRP) [32].These account for forcing out a vast ma-
value close to 2; iv- maximum number of cumulative hydrogen bonds in
jority of biologically active molecules.
molecule equal ten; and v- no anity to eux pumps. However, only
Finally, the cell-mediated transcytosis [33] is a recent dened route
few molecules meet these criteria and are capable to bypass the BBB
depending on monocytes and leukocytes to cross the BBB. Drug mole-
transcellulary by dissolving in lipid plasma membrane such as alcohol
cules or carriers can be uptaken by the inammatory cells and cross
and steroid hormones. A paradigm rarely encountered in drug mole-
through the BBB where inammatory cells are taking the function of
cules [21,22].
Trojan horses [34]. Cell-mediated transcytosis is distinctive for being
Carrier-mediated transport (CMT) involves some transporter proteins
practically suitable for both any type of molecules and particulate de-
on the BBB that bind to certain substrates followed by conformational
livery systems.
changes at the protein and transportation of the substance across the
So, the prohibited eects of the BBB on various important drugs
membrane. The most common transporter proteins are the glucose
delineated several modern strategies for eective drug transport to the
transporters (GLUT). There are ve members of the sodium-in-
brain.
dependent glucose transporters (GLUT1-5). Each GLUT transports glu-
cose, galactose, mannose, 2-deoxyglucose and 3-O-methylglucose to the
CNS [17]. The large neutral amino-acid transporter type 1 (LAT1), is
4. Modern strategies for brain delivery
also responsible for transportation of > 10 amino acids across the BBB
[18].
Multi-discipline approaches have lately emerged to elevate drug
Receptor-mediated transcytosis (RMT) [23] provides a mean for se-
concentrations within the brain. As shown in Fig. 3, these advances are
lective uptake of macromolecules via the reaction with their selective
based either on the route of delivery, modulation of drug chemistry or
receptors such as transferrin receptor (TfR) [24], insulin receptor [25],
BBB physiology or the use of nanocarriers. Herein we will discuss
lipoprotein receptor-related proteins (LRP1, 2) [26], and diphtheria
briey these access roads.
toxin receptor [27].
Adsorptive-mediated transcytosis (AMT) [28], involves an electro-
static interaction between a solute molecule carries a positive charge
4.1. Approaches based on the route of delivery
and the plasma membrane which carries a negative charge.
Eux pumps or transporters are the primary cause for drug expul-
To circumvent the BBB, scientists tried to overcome the barriers
sion from the brain. They are considered the main restriction for the
either by invasive routes through direct injection into the brain such as
retention of a large number of therapeutics in the brain. The most well
intracerebral, intraventricular and intrathecal injections or non-in-
characterized eux pumps or transporters are the P-glycoprotein (P-
vasively using the intranasal route [5].

Fig. 2. Transport routes across the blood brain barrier.

45
R.S. Elezaby et al.
4.1.1. Invasive routes
Intracerebral (intraparenchymal) delivery necessitates direct drug
injection into the parenchymal space of the brain through cranium
holes [35]. Therapeutic agents are administered as bolus or infusion via
intrathecal catheters [36,37]. Likewise, the intraventricular route im-
plicates direct injection into the cerebral ventricle, providing rapid drug
distribution in high concentrations, into ventricles and subarachnoidal
area within the brain, therefore highly applicable in meningioma and
metastatic cells of cerebrospinal uid (CSF) [38]. In comparison to the
intracerebral delivery, the absence of interconnection of this route with
the interstitial uid is an asset [39]. However, the ependymal brain
exterior is subjected to large drug amounts, which may provoke sub-
ependymal astrogliatic reaction.
Though less invasive than intracerebral or intraventricular admin-
istration, intrathecal route (direct into the cistern mana of the brain)
doesn't allow drug accumulation deeply in the brain parenchyma which
is greatly imperative for drug sustainment. Spinal and disseminated
meningeal diseases, but not glioblastoma, are conveniently treated by
this route [40,41].
However, all direct approaches suer from infection hazards, high
neurosurgical cost, need of experts and low eciency in diuse tumors,
metastasis, Alzheimer's disease, epilepsy, etc.
4.1.2. Non-invasive route
Recently, the intranasal route became successful for both systemic
and brain delivery. The nose is divided into two nasal cavities via the
septum, with a total surface area of 150 cm2 for nasal epithelium and a
total volume of 15 ml [42]. Nasal cavities are further subdivided into
three distinct areas namely the nasal vestibule, respiratory and olfac-
tory regions. As shown in Fig. 4, systemic drug absorption occurs via
the respiratory region either transcellularly or paracellularly. CMT and
other transcytosis pathways are also available [43]. Direct brain ab-
sorption takes place through the olfactory region by diverse mechan-
isms, embracing transcellular, paracellular, olfactory and trigeminal
neural pathways [43]. Many recent projects targeted the olfactory area
intranasally for direct brain delivery [4450].The convenience of the
intranasal route resides in [43]: i- non-invasive nature, lacking risks of
infection or disease transmission, ii-facile self-administration, iii- high
surface area of absorption, iv- fast onset of action, v- a direct path to the
brain bypassing the BBB. On the other side, its limitations are: i- small
volume (25200 l) for administration, so limited for drugs of low dose,
ii- individual variability, iii- low pH of nasal cavity (5.56.5 in adults),
iv- mucocilliary clearance, v- enzymatic degradation by nasal cyto-
chrome P-450/peptidases/proteases (pseudo rst pass eect).
4.2. Approaches based on drug chemistry
These rely on performing chemical drug modication allowing its
transfer across the BBB. Chemistry may increase drug hydrophobicity to
facilitate paracellular transport or attach a certain moiety to the drug
molecule to help in their brain uptake. Chemical changes embrace the
preparation of prodrugs, chimeric peptides and cationic peptides.
4.2.1. Prodrugs
Prodrug is a chemically modied form of the drug that has neither
biological toxicity nor activity. Although the concept of prodrugs for
brain delivery was introduced many decades ago [51], yet they are the
center of many researches till now. Peura et al. [52] used prodrug ap-
proach to increase cationic dopamine brain uptake by LAT1 receptors
by synthesizing three amino acid prodrugs of dopamine. They observed
that the phenylalanine promoiety of the prodrug had greater binding
ability for LAT1 and hence higher brain uptake in comparison to those
having an alkyl amino acid promoiety. Recently, Placzek et al. [53]
developed ester prodrugs of sobetirome, the selective thyromimetic that
trigger myelin repair to be used in demyelinating diseases such as
multiple sclerosis. They found that ethanolamine ester 11 resulted in
46
Journal of Controlled Release 261 (2017) 4361
more sobetirome brain distribution in comparison to unmodied one.
4.2.2. Chimeric peptides
It was used since many decades ago to deliver peptides through the
BBB [54,55]. It depends on covalent binding of a non-transportable
peptide to a peptide vector which is able to pass the BBB by RMT or
AMT. The conjugated vector may be endogenous peptides [56],
monoclonal antibodies (mAbs) [57], or modied proteins [58], etc.
Chimeric peptides dier from prodrugs in being biologically active so
they don't need any extra biological steps to start their action. Pardridge
et al. coupled beta-endorphins (non- transportable peptides) to cationic
albumin -which pass the BBB by AMT- via disulphide linkage [54,55].
They found that beta-endorphin-cationized albumin chimera was ra-
pidly embraced by the brain; conversely, the BBB uptake of native beta-
endorphin was negligible. Recently, Condamine et al. [59] have studied
the structural-activity relationship of some chimeric peptides obtained
from peptide E and beta-endorphin with and without various spacers.
They deduced that the length and physicochemical properties of spacers
had a minimal eect on the analgesic potency.
4.2.3. Cationic peptides
This technique depends on increasing the positivity of the poly-
peptide chain to pass the BBB by AMT pathway [60]. The free carboxyl
groups of the polypeptide (e.g., IGF-I, IGF-II, NGF) may be cationized
using polylysine (PLL), hexamethylenediamine, diazomethane or PLL
with cleavable ester bonds [61]. Protamine, histone, avidin and catio-
nized polyclonal bovine immunoglobulin are examples on cationic
peptides with a profound ability to cross the BBB [61].
4.3. Approaches based on the physiology of the BBB
The noticed increased BBB permeability in pathological conditions
such as ischemic stroke, cerebral edema and inammatory disorders
encouraged the scientists to similarly use the same gated enhancement
of drug permeation [62]. Meanwhile, Achareya et al. showed increased
BBB permeability after administration of inhaled anaesthetics (IAs), like
sevourane and isourane [63]. Subsequently, scientists succeeded to
open the Tjs temporarily using dierent stimuli: chemical, physical or
biological. Such phenomenon is known as BBB leakage or CNS dis-
ruption.
4.3.1. Chemical stimuli
These are hyperosmolar solutions injected intra-arterially, causing
shrinkage of endothelial cell accompanied by Tjs opening. Mannitol
hyperosmolar solution is the most commonly used. Foley et al. deliv-
ered adeno - associated virus (AAV) vectors to the brain using mannitol
[64]. The AAV vector encodes the human CLN2 gene, the gene of in-
terest in Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). They
Fig. 4. Schematic illustration showing the fate of drug after intranasal delivery.
R.S. Elezaby et al.
reported that the mannitol volume was a determining factor that af-
fected the degree of BBB disruption. The combination of mannitol and
stem cell was reported by Gonzales et al. to enhance the therapeutic
eects in adult stroke and neonatal cerebral palsy [65]. Substances like
alkylglycerols [66,67], Lysophosphatidic acid [68], Cyclodextrins (CDs)
[69], surfactants like sodium dodecyl sulfate (SDS) [70], and poly-
sorbate-80 (Tween-80) [71], endocannabinoids [72] revealed also BBB
permeability modication.
4.3.2. Physical stimuli
Ultrasound [73], microwave [74] and electromagnetic radiations
[75] are considered as physical stimuli for tight junctions opening in
the BBB.
4.3.3. Biological stimuli
They are exemplied by zonulaoccludens toxin (Zot) which was
found to reversibly promote - in a reversible concentration-dependent
manner - the Tjs opening [76]. Similarly, inammatory stimuli and
vasoactive compounds such as histamine [77], bradykinin [78] and its
analogues such as labradimil [79] were found to act as transient Tjs
openers.
Although being eective in enhancing drug passage across the BBB,
opening of Tjs may reinforce the crossing of noxious pathogens. Besides
and more seriously, blood plasma protein leakage may occur producing
astrocytes toxicity, a condition known as astrogliosis [80]. Therefore, to
ensure a safe and ecient use of this approach in brain therapeutics, it
deemed necessary to accurately estimate the time and extent of brain
exposure to such stimulations.
4.4. Approaches based on nanotechnology
CNS drug delivery has found appealing promises in nanotechnology.
Due to their nano size range, they can overthrow the restraints of
conventional drug delivery to access the brain [7]. An ideal nanocarrier
for CNS delivery should fulll the following criteria:
Particle size < 200 nm [81]. However, some researches preferred a
size < 100 nm [7].
Long circulating, controlled-release particles with low im-
munogenicity, good biocompatibility and bioavailability, averting
eux transport.
Protects the therapeutic cargo from enzymatic degradation.
Preferably, attached to a BBB-targeting moiety.
4.4.1. Optimization of nanoparticles blood circulation time
The proximity of the nanoparticles (NPs) to their target organ for
long time will ensure maximum eciency. After oral or intravenous
administration, the NPs circulating in the blood suer from two main
challenges viz. renal clearance and phagocytosis.
NPs with size (< 25 nm) are immediately cleared from blood by
kidneys [82].On the other hand, increasing size will result in re-
ticuloendothelial system (RES) clearance due to protein adsorption.
Actually, Fang et al. found that the 80 nm particles had slower blood
clearance compared t0 the larger nanoparticles (171 and 243 nm) due
to fewer protein adsorptions [83].
NPs with particle size (< 100 nm) can pass BBB without active
targeting. Sonavane et al. showed that gold nanoparticles with particle
size 15 and 50 nm could pass BBB, but those with size of 200 nm
couldn't [84]. On a similar basis, Shilo et al. demonstrated a great re-
liance of the intracellular uptake of gold NPs on size [85]. They com-
pared four sizes viz. 20, 50, 70 and 110 nm, and found that the size of
70 nm was the optimum. Consequently, the range of 25100 nm is
optimum for crossing BBB, avoiding renal clearance and phagocytosis.
Zeta potential (), an expression of the net total charge on the
particle's outside surface, is an important physical factor controlling its
pharmacokinetics and biodistribution [8,86]. After opsonization in
47
Journal of Controlled Release 261 (2017) 4361
fresh mouse serum, RAW 264.7 murine macrophages were found to
engulf NPs with high negative or positive surface charges more ade-
quately [87]. Likewise, the same group found that there was a direct
proportionality between the magnitude of positive surface charge and
the hemolytic activity and cytotoxicity against RAW 264.7cells;
whereas negatively charged NPs were devoided from any eect. In the
blood stream, cationic nanoparticles undergo opsonization, i.e. they
adsorb, on their surface, the protein opsonin from the blood stream
forming a corona [86]. Such opsonized NPs are cleared from the
circulation by the RES very fast [86].
Meanwhile, increasing surface hydrophobicity facilitates phago-
cytes uptake and hemolysis [88]. The PEGylation as a very common and
popular example on stealth technology has been applied to all nano-
carriers to solve this problem [8991]. This technology provides the
coating of nanoparticles with neutral, hydrophilic, highly exible PEG
polymer chains imparting protection against plasma protein opsoniza-
tion. Grafting PEG to a polymer (e.g., PEG-PLA and PEG-PLGA) or in-
cluding it as a main fragment of the copolymer (e.g., poloxamers) has
the same eects [92]. Coating the NPs with poloxamers, a PEG con-
taining surfactants, was also adequate for extending the circulation
time [93]. The diminished opsonization provided by PEGylation was
found to be due to steric hindrance as well as charge shielding [94,95].
Recently, it was reported that PEGylated NPs showed an enhanced
extracellular bioavailability due to the escape from brain cell popula-
tions (immune and non-immune) [96]. However, a PEGylated NPs
prepared with long and dense PEG chains suered from a reduced ef-
ciency due to the prohibited interaction with target cells [97,98]. The
removal of PEG coating after reaching the target site have been the
work of various authors [99,100]. Generally, minimal PEG coverage is
required to maximize circulation times. Although PEGylation is the
most common strategy applied for stealth technology, other strategies
are available. For example, the use of various stealth lipids contained in
liposomes to suppress complement-dependent phagocytosis [101]. Al-
though not fully understood, the authors suggested that the suppression
of complement-induced phagocytosis induced by lipids occurs at a step
after liposome opsonization [101].
4.4.2. NPs for brain delivery
Several classes of nanocarriers were applied for brain delivery
during the last decades such as liposomes [102,103], solid lipid nano-
particles [104,105], polymeric nanoparticles [106,107], polymeric
micelles [108,109], cubosomes [110], nanospheres [111,112], den-
drimers [113,114], cyclodextrins [115,116], drug polymer conjugates
[117], gold nanoparticles [118,119], silver nanoparticles [120], carbon
nanotubes [121,122] and quantum dots [123,124]. Many review arti-
cles discussed the dierent classes of nanocarriers for brain delivery
before [8,125132].
Despite the fact that NPs can pass BBB owing to their small particle
size, many researches pinpoint on specic delivery of NPs to CNS de-
pending on their conjugation to a particular ligand specic to cross the
BBB via active targeting, e.g. of ligands specic to certain pathways
include:
Ligands specic for CMT: The most commonly used ligands for this
pathway are the ligands that target glucose transporters such as
glucose and dehydroascorbic acid (DHA) [133,134] that target the
GLUT1 and aminophenyl--d-mannopyranoside (MAN) [135,136]
which targets GLUT1 and GLUT3.
Ligands specic for RMT: this pathway is very common and is the
most targeted pathway. Many receptors are present on the surface of
BBB. Here are examples on the receptors found on the BBB with
their targeting ligands:
1. Insulin receptors: Amyloid -peptide [137], HIRMAb [138]
2. Transferrin receptor (TfR): Transferin (Tf) [139,140] or its anti-
bodies (e.g. 8D3 [141] OX26 [142] and R17 [143])
3. Lactoferrin receptors (LfR): Lactoferrin (Lf) [144]
R.S. Elezaby et al.
4. LRP1 and LRP2 receptors: Polysorbate 80 (PS80) [145,146],
lactoferrin (Lf) [147149], apolipoproteins (ApoA, E or B)
[150152] and angiopeps family (specially angiopep 2)
[153,154].
5. Diphtheria toxin receptor (DTR): Cross reacting material (CRM
197) which is the benign mutant of diphtheria toxin (DT) [155].
6. Acetyl choline (Ach) receptors: Rabies virus glycoprotein (RVG)
[108].
Ligands specic for AMT: Cell penetrating peptides (CPPs) [156]
which are small peptides of < 30 amino acids. They dier from each
other in the peptide chain length, sequence of amino acids and the
net charge on the peptide chain. Their positive charge makes them
attracted to the negative charge on the cell membrane followed by
transcytosis. Examples of CPPs applied for brain delivery include
lectin [157,158], odorranalectin [110,159], penetratin [160,161]
and trans-activating transcriptional activator (Tat) [119,162].
Ligands specic for inhibition of eux pumps: An ecient strategy
for drugs having anity to any of the eux pumps. Poloxamers
were commonly found to block P-gp by interacting with the cell
membrane, and uidizating it; inhibiting of P-gp ATPase activity;
and attenuating cellular ATP [163,164].
Ligands specic for cell-mediated transcytosis: Cyclic Arginine-
Glycine-Aspartic acid (cRGD) was used by Jain et al. to target in-
tegrins expressed on monocytes and neutrophils [165]. The in-
ammatory cells carried liposomes loaded with diclofenac to the
brain eciently. Qin et al. formulated negatively charged nanosized
liposome loaded with trefoil factor 3 (TFF3) [166] for treatment of
depression. In-vitro studies proved that their negative charge en-
abled their success transfer inside the bound monocytes across the
BBB. This approach doesn't require nanosized carriers and impedes
one of the routes by which pathogens intrude the brain.
A special class of NPs which arised in the last decade as a really
promising nanocarrier system for brain delivery is polymeric micelles
(PMs) [1113]. In the upcoming section, we will discuss PMs as na-
nocarriers, its history with brain delivery, its advantages over other
nanoparticles and its applications in brain delivery. The modications
performed on micelles to be in line with modern strategies for ecient
brain delivery will be focused.
4.4.3. Micelles as a type of nanocarries: history
PMs are nanoscopic carriers of amphiphilic copolymers formed by
their self-assembly in an aqueous medium [167] presenting their hy-
drophilic shell or corona to the medium, while their core is composed
from their hydrophobic portion (Fig. 5). The aggregation occurs in
aqueous medium when the amphiphilic block copolymer concentration
surpasses what's called critical aggregation concentration (CAC) or
critical micelle concentration (CMC) [168]. For entropy considerations,
the hydrophobic segments of the amphiphilic copolymer should ar-
range themselves away from water molecules. This will be
48
Journal of Controlled Release 261 (2017) 4361
accompanied by their association together with the formation of core -
shell micellar structure [169]. PMs were rst introduced as drug de-
livery vehicles by Helmut Ringsdorf in the early 1980s [170]. However,
it was used for the rst time for brain delivery in 2008 by Liu et al. [11].
Table 1 summarizes the studies using PMs for brain delivery since that
time.
4.4.3.1. Advantages of PMs as nanocarriers for brain delivery. PMs are
more advantageous than other nanocarriers for brain delivery.
Generally, it has been shown that PMs enjoy the following advantages:
1) Small size (10100 nm), which is in the ideal range to pass BBB.
2) Ease of preparation and handling (just by self-assembly).
3) Ease of incorporation of hydrophobic drugs.
4) High encapsulation eciency (EE %) of hydrophobic drugs.
5) Ease of sterilization by ltration due to small size [171].
6) The hydrophilic shell prevents non-specic interactions with plasma
proteins or uptake by RES (inherited stealth property).
7) Possibility of surface modication by various ligands for ecient
brain targeting.
4.4.3.2. Classication of PMs. PMs can be classied on a dierent basis
as follows:
A) Classication based on the type of copolymer used
PMs can be classied as polymer-polymer micelles or polymer-lipid
micelles. In polymer-polymer micelles, both the core and shell are
formed of polymers. These polymers may be of c-c chains (Table 1) as
PLA, PLGA, PEG, Poloxamer etc., or it may be of polypeptide backbone
as poly-glutamic acid [172], poly-L-phenylalanine (PPhe) [133] and
poly-asparthydrazide [108]. In polymer-lipid micelles, the corona is
formed of hydrophilic polymer mostly PEG while the core is formed of
lipid. The commonly used lipids were synthetic phospholipids such as
dioleolyphophatidyl ethanolamine (DOPE) and distearoylphophatidyl
ethanolamine (DSPE) [173]. Cholesterol [11] and oleic acid [174] were
also used (Table 1).
B) Classication based on the type of interaction between drug and
polymer
PMs can be classied on this basis to conventional micelles; drug
conjugated micelles and polyion complex (PIC) micelles. In conven-
tional micelles, no interaction exists between drug and copolymer.
Hydrophobic drugs are easily included in micellar cores by direct
mixing. A technique only suitable for PMs with soft cores (i.e. with a Tg
lower than the temperature used for drug loading) such as pluronic
micelles. On the other hand, PMs with solid cores (crystalline or with a
Tg higher than the temperature used for drug loading) need more
complex techniques for loading drugs; examples include poly(ethylene
Fig. 5. Schematic illustration of polymeric micelles struc-
ture and formation.
R.S. Elezaby et al. Journal of Controlled Release 261 (2017) 4361

Table 1
Summary of studies discussing micelles for brain delivery.

Drug Disease Copolymer Method of preparation Ref.

ATRA GBM PEG-chitosan Simple addition [187]

Ciprooxacin Encephalitis PEG-b-Col Dialysis [11]
PHEA-EDA-PLA Dialysis [175]
Zolmitriptan Migrain Pluronic F127 + PEG-400 and vitamin E-TPGS Simple addition [188]
Coumarin PCL-PEEP Dialysis method [189]
GBM PEG-b-PLA Co-solvent evaporation [190]
PTX GBM PEG-b-PLA Thin lm hydration [191]
Flurbiprofen Alzheimer PHEA-EDA-PLA Dialysis method [192]
PTX GBM DSPE-PEG Thin lm hydration [173]
siRNA Cancer mPEG-b-PCL-b-PPEEA Co-solvent evaporation [184]
DIR PEG-b-PLA Thin lm hydration [193]
Coumarin PEG-b-PCL W/O emulsion [194]
HSA PEG-PAE-API Simple addition [183]
Doxurobicin Cancer CS-SA Dialysis [195]
AmB Meningoencephalitis PE-PEG Thin lm hydration [196,197]
PTX GBM PCL-PEEP + PEG-PCL Dialysis [12,171]
Plasmid DNA mPEG-PDMAEMA Direct addition [198]
DACHPt GBM mPEG-b-P(Glu) Dialysis [172]
17-AAG GBM Pluronic P123/F127 Thin lm hydration [164]
Efavirenz HIV Pluronic F127 + poloxamine T904 Simple addition [185]
Flurbiprofen Alzheimer PEG-b-PLA Co-solvent evaporation [199]
BCNU and VEGF-siRNA GBM R3V6 Direct addition [200]
miR-21 inhibitor and doxorubicin GBM PLA-b-PDMAEMA Co-solvent evaporation [201]
siRNA and captothecin GBM PEG-b-PCL Thin lm hydration [202]
Captothecin GBM PEG-b-PCL W/O emulsion [203]
Curcumin Cancer mPEG-OA Co-solvent evaporation [174]
YC1 Neuroprotection SACDs Co-solvent evaporation [177]
GBM DSPE-PEG Thin lm hydration [204]
Doxorubicin GBM Pluronic105 Dialysis [205]
WIN55, 212-2 Neuropathic pain SMA Co-solvent evaporation [178]
Clonazepam Epilepsy BS: sodium glycocholatehydrate + soya PC Thin lm hydration [206]
LTG Epilepsy Pluronic P123 Thin lm hydration [163]
Pluronic P123/F127 [207]
Docetaxel Cancer PEG-b-PLA Thin lm hydration [109]
ITZ Intracranial fungal infections PEG-pLys-pPhe Dialysis [133]
siRNA Neurodegenerative diseases mPEG-g-PAHy-GTA Simple addition [108]
SPIONs GBM PEG-b-PCL Co-solvent evaporation [208]
Curcumin and HSVtk GBM R7L10 O/W emulsion [209]
Nimodipine SAH Pluronic F127 and P123 + PC Thin lm hydration [186,210]
sPMI GBM PEG-b-PLA Thin Film hydration [211]
Curcumin and rapamycin GBM PEG-b-PCL Dialysis [212]
Docetaxel Cancer TPGS Thin lm hydration [13]
DOM Psychosis pHPMA- co -pLMA Co-solvent evaporation [213]
TMZ GBM R7L10 O/W emulsion [214]
AmB Intracranial fungal infection PEG-b-PLA Co-solvent evaporation [215]
Myricetin GBM Pluronic P123/F68 + Chitosan Thin lm hydration [216]
Quercetin GBM Soy lecithin/Pluronic F68/Labrasol Thin lm hydration [217]
Doxorubicin GBM PEG-b-PCL Dialysis [218]
Doxorubicin and paclitaxel GBM Pluronics Thin lm hydration [219]
Carmustine GBM PEG-PLGA Thin lm hydration [220]
Gold and SPIONs GBM PEG-b-PCL O/W emulsion [221]
Curcumin and doxorubicin GBM PE-PEG Thin lm hydration [222]
Curcumin GBM PEG-b-PLA Thin lm hydration [223]
Clonazepam Status epilepticus Pluronic P123/L121 Thin lm hydration [224]
Myricetin GBM Pluronic P123/F68 Thin lm hydration [225]
Vinpocetine Cerebrovascular and cerebral degenerative diseases Pluronic P123 + PEG-PCL Thin lm hydration [226]

17-AAG: 17-Allylamino-17-demethoxy geldanamycin; AmB: amphotericin B; APN: aminopeptidase N; ATRA: all-trans retinoic acid; BCNU: bis-chloroethylnitrosourea; BS: bile salt; CS-SA:
stearic acid-grafted chitosan; DACHPt: (1,2-diaminocyclohexane) platinum(II); DiR: near-infrared uorescent dye; DOM: domperidone; DSPE-PEG: 1,2-distearoyl-sn-glycero-3-phos-
phoethanolamine-N-methoxy(polyethylene glycol); DTX: docetaxel; FA: folic acid; GBM: glioblastoma multiforme; HIV: human immunodeciency virus; HSA: human serum albumin;
HSVtk: herpes simplex virus thymidine kinase gene; ITZ: itraconazole; LTG: lamotrigine; mPEG-b-P(Glu): poly(ethylene glycol)-b-poly(L-glutamic acid); mPEG-b-PCL-b-PPEEA: mono-
methoxy poly(ethylene glycol)-block-poly(-caprolactone)-block-poly(2-aminoethyl ethylene phosphate); mPEG-OA: monomethoxy poly(ethylene glycol)-oleate; mPEG-PAHy-GTA:
methoxy poly(ethylene glycol)-,-polyasparthydrazide-glycidyltrimethylammonium; m-PEG-PDMAEMA: methoxy poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate;
PC: phosphatidylcholine; PCL-PEEP: poly(-caprolactone)-block-poly(ethyl ethylene phosphate); PEG: poly(ethylene glycol); PEG-b-Chol: poly(ethylene glycol)-block-cholesterol; PEG-b-
PCL: poly(ethylene glycol)-block-poly(-caprolactone); PEG-b-PLA: poly(ethylene glycol)-block-poly(lactic acid); PEG-PAE-API: poly(ethylene glycol)-poly(b-amino ester)-1-(3-amino-
propyl) imidazole; PEG-pLys-pPhe: poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-phenylalanine); PE-mPEG: 1,2 distearoyl-snglycero-3-phosphoethanolamine-N-(methoxy(polyethylene
glycol); PHEA-EDA-PLA: ,-poly((N-hydroxyethyl)-DL-aspartamide)-ethylenediamine-poly(lactic acid); pHPMA-co-pLMA: poly(N-(2-hydroxypropyl)-methacrylamide)-poly(lauryl me-
thacrylate); PLA-b-PDMAEMA: poly(lactic acid)-poly(dimethylaminoethyl methacrylate); PTX: paclitaxel; R3V6: 3-arginine and 6-valine; R7L10: 7-arginine and 10-leucine; SACDs:
sodium alginate cholesterol derivatives; SAH: subarachnoid hemorrhage; siRNA: small interfering RNA; SMA: poly(styrene-co-maleic anhydride); SPIONs: superparamagnetic iron oxide
nanoparticles; sPMI; stapled peptide antagonist of both MDM2 and MDMX; TMZ: temozolomide; TPGS: D-alphatocopheryl polyethylene glycol succinate; VEGF-siRNA: vascular en-
dothelial growth factor-small interfering RNA; YC1: 5-cholestane-24-methylene-3,5,6,19-tetraol

49
R.S. Elezaby et al. Journal of Controlled Release 261 (2017) 4361

glycol)-poly(lactic acid) (PEG-PLA), poly(ethylene glycol)-poly(-ca- C) Classication based on the number of copolymers incorporated in
prolactone) (PEG-b-PCL) and poly(ethylene glycol)-poly(b-benzyl-L-as- PMs fabrication
partate) (PEG-PBLA) micelles. Incorporating drugs into conventional
micelles can be done by ve dierent methods: On this basis, PMs can be classied into purebred micelles and
mixed or hybrid micelles (Table 1). Purebred micelles are composed of
(1) Dialysis method: it is an extensively used method [11,175] done by only one type of amphiphilic copolymers. On the other hand, for mixed
preparing a drug solution in a water miscible organic solvent in or hybrid micelles, there is a combination of more than one type of
which the copolymer is soluble too, followed by dialysis against copolymers [171,185]. Mixing of more than one copolymer during
water. During dialysis, water as a non-solvent will replace the or- micelle preparation aims always at improving their physicochemical
ganic solvent provoking the hydrophobic blocks to self- assemble properties. Mixing pluronic F127 with pluronic P123 produced PMs
into the micelle cores followed by drug encapsulation. Micelles with enhanced kinetic stability due to the long polyethylene oxide
diusion out is prohibited by the semi-permeable membrane while (PEO) chains of Pluronic F127, counteracting the stacking of cylindrical
unloaded free drug will be removed. aggregates of hydrophobic polypropylene oxide (PPO) chains of
(2) Emulsion method (O/W or W/O): PMs are formed due to the eva- Pluronic P123 [164]. Mixing these micelles with phosphatidylcholine
poration of a volatile water immiscible organic solvent, in which (PC) lowered CMC and multiplied blood micellar stability as noticed by
the drug is dissolved, after the formation of an emulsion due to the Baselious et al. [186].
vigorous mixing with the aqueous solution [176]. The copolymer
may be present in the organic or aqueous phase according to its 4.4.3.3. Eect of physicochemical properties of PMs on its behavior in brain
solubility. delivery.
(3) Solvent evaporation method (i.e., thin lm evaporation or solvent
casting method): a drug-impregnated polymer lm is formed due to A) Critical micellar concentration
heat and vacuum aided evaporation of a volatile organic solvent
containing both components [163,164]. The drug loaded PMs are As we mentioned before PMs are formed when the copolymer
formed thereafter due to lm hydration accompanied with vigorous concentration reaches a certain value called CAC or CMC. The CMC
shaking. aects greatly the thermodynamic stability of PMs due to the extreme
(4) Co-solvent evaporation method: similar to dialysis method both dilution eect of the circulation after IV injection. If micellar con-
drug and polymer are dissolved in a water miscible organic solvent centration drops below the CMC, the encapsulated drug will be early
[177,178]. Water is added to this solution with vigorous shaking released into the circulation before getting to its target site with the
causing organic phase evaporation, forming the drug-loaded mi- possibility of drug precipitation inside the blood vessels [167]. Gen-
celles. erally, the blood stability improves as the CMC value decreases.
(5) Freeze-drying method: a freeze-dryable organic solvent such as tert- For CMC determination denite concentrations of pyrene (hydro-
butanol is used in this method to dissolve polymer and drug [179]. phobic uorescent probe) are mixed with serial copolymer dilutions by
After water addition, the mixture is freeze-dried then reconstituted ultrasonication to ensure complete pyrene lodging into the hydrophobic
with aqueous media. micro-domains [177]. The uorescence spectra are recorded on a
uorometer at room temperature. The intensity ratios of its rst peak in
A drug covalently bonded to one segment of the copolymer to build water (I1) to its third peak in the hydrophobic medium (I3), obtained
the hydrophobic core of a PM forms what's known as conjugated mi- from the pyrene emission spectra, are plotted versus the logarithmic
celles [180]. To achieve a high drug load a linear dendritic drug- function of polymer concentrations [192]. The intersection of the hor-
polymer conjugates modied with choline derivate (CD) was synthe- izontal tangent with that of the curve at the inection is taken as the
sized then self-assembled into PM [181]. Eight molecules of doxor- CMC value.
ubicin (DOX) were attached to PEG, which was linked to CD. The CD-
PEG-DOX8 micelles showed higher cellular uptake and antitumor ac- Table 2
tivity against glioma in comparison to the non-modied conjugate. CMC values for some of copolymers used for brain delivery.
Likewise, a water soluble PEG-PLA-PTX micelles were prepared by
Copolymer CMC Ref. Copolymer CMC (mg/ Ref.
Wang et al. after conjugation of paclitaxel (PTX) with PEG-b-PLA [182]. (mg/L) L)
They were tested in C6 glioma model in Wistar rats. After tail injection,
the micelles could cross the BBB and showed an enhanced therapeutic mPEG-OA 30 [174] PHEA-EDA- 25 [192]
PLA
eect when compared to Taxol.
SACDs [177] PEG2000- 107 [167]
PIC or micelleplex depends on electrostatic interaction between the PLA700
drug and the copolymer with no covalent bond formation. A very (Ch pretreated with 0.8 PEG2000- 22
popular example on PIC was reported between negatively charged triphosgene) PLA1000
nucleic acids and proteins and positively charged amphiphilic copoly- (Ch not pretreated 2 PEG2000- 12
with triphosgene) PLA1300
mers [183,184]. Huo et al. prepared a novel PIC micelles from PEG-
CS-SA 65 [195] PCL-PEEP 0.51 [189]
polyasparthydrazide-glycidyltrimethylammonium (mPEG-g-PAHy- PEG-b-Col 6.2 [11] DSPE-PEG700 28 [227]
GTA) copolymer to deliver siRNA to the brain [108]. PAHy was the Tat-PEG-b-Col 7.8 DSPE- 30.85
core, while PEG corona shielded the surface charges of the micelles for PEG2000 17.39
stability and prolonged circulation. The negative charged siRNA inter- DSPE-
PEG5000
acted with the positive charged GTA connected to PAHy. The PIC mi- Pluronic F127 35.228 [228]
celles decorated with RVG (a 29 amino acid peptide with the sequence Pluronic P123 25.3
(YTIWMPENPRPGTPCDIFTNSRGKRASNGC) - as a specic binder for Pluronic P105 40.3
Ach receptors expressed by neuronal cells - were easily internalized in
CS-SA: stearic acid-grafted chitosan; DSPE-PEG: 1,2-distearoyl-sn-glycero-3-phos-
the neuro2a cells. In neuro2a cells, Huo et al. proved that cellular up-
phoethanolamine-N-methoxy(polyethylene glycol); mPEG-OA: monomethoxy poly(ethy-
take of the RVG-decorated micelles was 1.52.6 times that of the un-
lene glycol)-oleate; PCL-PEEP: poly(-caprolactone)-block-poly(ethyl ethylene phos-
modied micelles. By this study, Huo et al. introduced an interesting phate); PEG-b-Chol: poly(ethylene glycol)-block-cholesterol; PEG-b-PLA: poly(ethylene
siRNA carrier to the brain delivery. glycol)-block-poly(lactic acid); PHEA-EDA-PLA: ,-poly((N-hydroxyethyl)-DL-asparta-
mide)-ethylenediamine-poly(lactic acid); SACDs: sodium alginate cholesterol derivatives;

50
R.S. Elezaby et al.
Table 2 shows that CMC is greatly aected by hydrophilic and hy-
drophobic chain length as well as the ratio between them, copolymer
type and ligand conjugation [167]. Generally, CMC value reduces as the
hydrophobic block length increases [167].
B) Size, shape and zeta potential
Crossing the BBB by NPs has been known to be particle size de-
pendent. Dynamic light scattering (DLS), atomic force microscopy
(AFM) and transmission electron microscope (TEM) are used to de-
termine and assist PMs particle size [189,205]. Those in the range of
10100 nm are optimally uptaken by the brain. Micellar particle size
depends on many factors viz.; copolymer, hydrophilic/hydrophobic
segment's ratio, drug loading, drug/polymer ratio, ligand conjugation
and density. Generally, particle size increases after drug loading due to
expansion of hydrophobic core volume accommodating the drug mo-
lecules [205]. Leyva-Gomez et al. showed that particle size increased
from 12.4 1.8 to 16.5 1.4 nm after drug loading [206]. Con-
versely, Mao et al. observed a decrease in particle size of micelles from
60.3 0.3 to 47.7 0.4 nm after drug loading ascribable to the very
high interaction between the siRNA and copolymers [184]. In a further
study, Zhang et al. showed that particle size increased from
33.31 0.61 to 87.85 2.32 nm after Tf conjugation [171]. How-
ever, Niu et al. showed no PMs size increase after conjugation with
glucose or folic acid (FA) because of their small size [205]. Moreover,
Mu et al. observed a particle size increase from 22.2 1.5 to
111.3 17.2 nm after raising the feeding ratio from 5% to 20% [199].
The shape of PMs can be examined using TEM [108,177,198,205]
and AFM [13,133,189]. Almost in all studies for brain targeting, mi-
celles were found to be spherical in shape with no aggregates
[133,198].
Zeta potential is also an important parameter for longer blood cir-
culation. It is measured depending on DLS using zetasizer. It is aected
by the type of copolymer, drug loading, ligand conjugation and ligand
density. Chung et al. showed that zeta potential increased from
27.4 4.5 to 12.6 1.6 mV upon conjugation of CREKA pep-
tide to the surface of PEG-PE micelles [204]. Miura et al. showed non-
signicant zeta potential variation upon increasing in cRGD conjuga-
tion ratio from 5% to 20% on the surface of poly(ethyleneglycol)-b-poly
(L-glutamic acid) (PEG-b-P(Glu)) micelles [172].
C) Encapsulation eciency and drug loading
Micelles are generally separated from unentrapped drug by ltra-
tion using 0.22 m nylon lter [164,173,193,205]. To calculate en-
capsulation eciency (EE%) and drug loading (DL%), three methods
are reported: the rst involves the mixing of an aliquot of the ltrate
with organic solvent to dissolve the micelles and calculate the released
drug [11,211]. However, this method is not accurate in calculating DL
as it doesn't consider the micelles yield. In the second method which is
considered to be the most accurate, the ltrate is subjected to freeze
drying followed by organic solvent addition to dissolve the micelles
powder [171,189,199]. Finally, a third method stated only in one study
by Sonali et al. in which they removed the water in the ltrate via
evaporation by rotary evaporator under high temperature and reduced
pressure to form a lm [13], which was then dissolved in an organic
solvent. EE % and DL % are then calculated using to the following
equations:
Weight of drug in micelles
EE% = 100
Weight of the feeding drug
Weight of drug in micelles
DL% = 100
Weight of the micelles
Almost in all studies, PMs showed high EE% for hydrophobic drugs.
Factors aecting EE% and DL% of certain drugs include both
51
Journal of Controlled Release 261 (2017) 4361
formulation and process related factors. The formulation-related de-
terminants include the type of copolymer, percentage of grafting of
hydrophilic and hydrophobic segments and the drug/polymer feeding
ratio. While those associated to the process comprise method of pre-
paration and conditions of the preparation including solvent nature, its
ratio to aqueous phase and so on.
Huo et al. showed that increasing PEG grafting degree on COOH-
PEG-g-PAHy-GTA copolymer decreased the encapsulation of siRNA
[108]. Flexible PEG chains could shield the positive charge on the
polymer which decreased its interaction with siRNA. Niu et al. used
dierent drug DOX to polymer ratios viz. 2/200, 4/200, 6/200, 8/200
and 10/200 [205]. The increase in DOX content, up to a certain limit,
raised the EE% from 18.0% to 45.3%. The initial increase in drug
concentrations enhanced drug diusion from the organic to the aqu-
eous phase with an increased tendency to reside in the core. However,
drug amounts surpassing a certain limit precipitate before micelle for-
mation, which lowered the EE% signicantly. When studying the level
of the organic phase (DMSO), they found that both EE% and DL% de-
creased with higher DMSO concentrations due to micelles disruption
induced by DMSO. Besides, DMSO enhanced DOX solubilization in the
aqueous phase hindering its diusion into the micellar core. The au-
thors recommended a ne-tuning of the organic solvent amounts during
PM preparation.
D) Cytotoxicity and hemolysis assays
Cytotoxicity test is performed on in-vitro BBB model to examine the
toxicity of the formulation on brain cells [12,189,191,198]. Using MTT
assay, Shao et al. tested the cytotoxicity of AmB-incorporated PE-PEG
micelles against brain capillary endothelial cells (BCECs) [197]. The
percentage of viable cells was determined using the following equation:
Viability% = (ABst ABsc) 100
where Abst and Absc are the absorbances of drug-treated and control
wells, respectively.
Hemolysis test is performed to examine the damaging eect of mi-
cellar solution on RBCs. It is very critical if the copolymer or ligand is
used for the rst time. Zhang et al. performed this test on dierent
concentrations of blank PMs and their Tf modied analogs on isolated
erythrocytes from SD rat blood using a positive control (Triton X-100)
[171]. After centrifugation, the supernatant was analyzed for he-
moglobin content and the percentage of hemolysis was calculated as
follows:
Hemolysis% = (A s A 0) (A100 A 0) 100
where As, A0and A100 are the respective absorbances of the sample,
unlysed sample treated with saline and fully lysed sample treated with
deionized water. All copolymers and ligands listed in (Tables 1 and 4)
are safe for brain delivery.
E) Stability of PMs
Both shelf and serum stability should be tested for PMs. Shelf sta-
bility is performed by leaving PMs at 4 C followed by examination of
the particle size, zeta potential and EE% at specied time intervals.
Zheng et al. showed that negligible changes in particle size and EE%
occurred in PMs at 4 C after 15 days, 30 days and 60 days [177].
However, PMs can't retain its stability > 60 days, and that's why lyo-
philization is very common just after the preparation [164,174]. Serum
stability test is performed in conditions mimicking the physiological
conditions (i.e. PBS 7.4, 37 C, in presence [108] or absence of serum
[192] with moderate stirring). Sarpietro et al. performed the test
through a 48 h period in PBS pH 7.4 [192] and found non-signicant
dierence in the particle size or PDI. Additionally, Huo et al. performed
the test in presence of 50% fetal bovine serum for 24 h [108] and
conrmed the sequence integrity of siRNA.
R.S. Elezaby et al.
F) In-vitro drug release
Studies are generally done using dialysis method where 12 ml of
micellar solution is introduced in pre-swollen dialysis bag and im-
mersed in a buer from which samples are withdrawn at predetermined
time intervals [13,177]. Generally, a sustained drug release was usually
obtained preceded by an initial burst phase. The release proles depend
mainly on the type of copolymer used. PEG-PLA copolymer achieved
sustained release of PTX up to 72 h [191]. In another study, 65% of
lamotrigine was released from Pluronic P123 during the rst 4 h
[163].Similarly, Sonali et al. and Zhan et al. reported the same beha-
viors with docetaxel from Tf receptor-targeted vitamin E TPGS micelles
and PTX from PEG-PLA micelles, respectively [13,191].
In addition, drug type, pH and ligand conjugation have also high
inuence on drug release. Dierent authors showed that the release of
DOX was commonly fastened by the decrease in pH [205,229,230]. An
observation which was likely due to its 30-NH2 group protonation and
azomethine bond breakdown in its dimer, which increases its solubility
in water [205]. In addition, Sonali et al. showed that decoration of PMs
surface with Tf slowed the drug release kinetics, in comparison to non-
targeted micelles [13]. They also showed that the increase in the ligand
concentration extended the release sustainment. The t50% (time at
which 50% of the drug was released) of the formulations were 12, 16,
24 and 48 h for the unconjugated micelles, DTX-TPGS-Tf1, DTX-TPGS-
Tf2 and DTXTPGS-Tf3, respectively where DTX-TPGS-Tf1(contains
50 mg of TPGS-Tf and 100 mg of TPGS), DTXTPGS- Tf2 (contains 75 mg
of TPGS-Tf and 75 mg of TPGS) and DTX-TPGS-Tf3 (contains 100 mg of
TPGS-Tf and 75 mg of TPGS). On the opposite side, Zhang et al. showed
that Tf conjugation didn't aect release behavior [12]. Likewise, Mu
et al. showed that aptamer conjugation didn't destroy the integrity and
compactness of micelles and didn't aect the release [199].
G) Cellular uptake and in-vivo study
Cellular uptake is performed on in-vitro BBB models to study the
ability of crossing the barrier in-vivo [8]. McCarthy et al. described two
types of models; primary cell models utilizing brain microvascular en-
dothelial cells (BMECs or BMVECs) or also called BCECs. The second
suggested model was immortalized cell lines isolated from mice, rats or
humans. After proper incubation of cells, micellar solutions application
and drug quantitation is performed. Table 3 shows a comparison be-
tween the two types and examples used with micelles for brain delivery.
As shown, most studies used primary cell lines due to their similarity
with in-vivo conditions. In-vivo study and pharmacokinetic analysis
should follow cellular studies [173,205].
4.4.3.4. Limitations of PMs.
A) Delivery of hydrophilic drugs
Table 3
Comparison of dierent in-vitro BBB models used with micelles for brain delivery.
Primary cell models
Denition They are isolated from blood vessels of dierent species of animals such as rats, mice, calves, pigs
and human
Advantages 1- High TEER values
2- With various receptors and enzymes resembling in-vivo conditions
Disadvantages 1- Their procurement is a challenge
2- Short time use
3- Exhibit batch to batch variation
Examples BMECs [12,133,171,173,189,196198,205]
BMECs: brain microvascular endothelial cells
52
Journal of Controlled Release 261 (2017) 4361
As we mentioned before, PMs consist of hydrophilic shell and hy-
drophobic core. They encapsulate hydrophobic drugs such as paclitaxel,
docetaxel, vincristine and many other hydrophobic drugs eciently in
their cores (Table 1). They are not suitable for encapsulation and de-
livery of hydrophilic drugs. However, Huang et al. prepared core-in-
versible micelles (CIMs) for delivery of hydrophilic drugs [231]. They
developed a novel biocompatible linear-dendritic amphiphilic co-
polymer viz. PEG5kCA8 which is composed of polyethylene glycol (PEG
5000) and a dendritic octamer of cholic acid (CA8). This amphiphilic
copolymer was found to self-assemble into micelles in aqueous solution
for ecient delivery of hydrophobic drugs and into CIMs selectively in
certain apolar solvents with a PEG shell and a hydrophilic core for ef-
cient delivery of hydrophilic drugs. Despite the trials of applying PMs
for hydrophilic drugs delivery, so far PMs are used mainly for ecient
hydrophobic drugs delivery.
B) Shelf stability
PMs like all nanoparticles have poor shelf stability. They tend to
aggregate with time leading to an increase in the particle size [232].
Undoubtedly, this particle growth will aect the physicochemical,
pharmacodynamic and pharmacokinetic properties of the PMs. Lyo-
philization is a popular and common solution for this problem. Many
studies subjected PMs to lyophilization just after the preparation to
keep it in the dry solid form with high stability [164,186]. Lyophili-
zation can be performed in absence or presence of cryoprotectants such
as glucose, sucrose, maltose, mannitol and trihalose. Cesur et al. could
successfully prepare freeze-dried DSPE-PEG2000 micelles without the
addition of any cryoprotectants [233]. They found that particle size and
encapsulation eciency didn't change signicantly before and after the
lyophilization process. However, Moretton et al. showed that the ad-
dition of a cryoprotectant to PCL-PEG-PCL ower-like PMs was a must
to prevent the particle size growth during lyophilization [234].
C) Serum stability
The main challenge of PMs is the possibility of premature release of
drugs in the circulation due to the high dilution [167]. The premature
drug release results in inability to deliver a sucient amount of the
drug to the targeted site, increased systemic side eects and the pos-
sibility of precipitation of the drug in the blood vessels. Many ap-
proaches were performed to increase the serum stability of PMs which
are discussed in details in 4.4.3.6 Section.
Despite all the aforementioned limitations, PMs were able to reach
the clinical studies for treatment of dierent types of cancer [235].
4.4.3.5. Applications of PMs in brain delivery.
A) Drug delivery
Immortalized cell lines
They have been immortalized through various techniques
1- Easily subcultured
2- Used for number of passages without changing their
properties.
1- Lower TEER values
2- Higher paracellular permeability in comparison to
primary cell models
b.END3 [195,199],
Neuro2a [108,175],
hCMEC/D3 [206]
R.S. Elezaby et al.
As shown in Table 1, many studies utilized PMs as promising suc-
cessful nanocarriers for the transportation of dierent therapeutic
agents in dierent diseases across the BBB. Obviously, PMs have
especially many applications in brain cancer treatment adopting the
dierent techniques of controlled dissociation and active targeting.
Many review articles discussed the advances and applications of PMs in
cancer [236240]. Although a signicant number of PMs formulations
for treatment of cancer reached the clinical trials phase, none of them
was for brain cancer [235,241].
B) Imaging
Earlier disease detection, assessment of therapy and disease condi-
tions have been widely improved using magnetic resonance imaging
(MRI), nuclear imaging, and X-ray computed tomography (CT) [235].
Zhou et al. developed optical MRI bifunctional targeted PMs for glioma
to determine its location before and during tumor resection operation
[208]. The PMs were prepared from PEG-b-PCL encapsulating hydro-
phobic superparamagnetic iron oxide nanoparticles (SPIONs). The PMs
were decorated with Lf which is a glioma- targeting ligand, in addition
to the modication with Cy5.5 which is a near-infrared uorescent
probe. The glioma was obviously delineated due to Cy5.5 and Lf en-
sured high tumor specicity. Hence, good imaging properties were of-
fered by the newly synthesized contrast agent combining both magnetic
and uorescent properties. Finally the remarkable hydrophobicity of
PCL15000 in the used PEG-b-PCL made it possible to follow the brain
tumor after surgery or radiation even after the decrease of the nano
probe concentration in the brain. Similarly, Sun et al. prepared mixed
SPIONs and gold micelles (GSMs) utilizing PEG-b-PCL copolymer for
both imaging and treatment of glioblastoma [221]. They found that the
combination between GSMs and radiation therapy (RT) resulted in ~ 2-
fold increase in double-stranded DNA breaks density in tumor cells.
They also showed that MRI but not CT was sensitive enough to detect
and delineate borders of the tumor after GSMs administration and ac-
cumulation.
4.4.3.6. Micelles in line with the modern strategies for brain targeting.
A) Controlled dissociation PMs
It is well known that PMs are easily dissociated when diluted in the
blood causing an early drug release. Accordingly, many approaches
were developed to prevent this premature dissociation, e.g. cross linked
PMs, stimuli responsive PMs and cross linked- stimuli responsive PMs.
1- Cross linked PMs
Cross-linked micelles (cl-micelles) are prepared to prevent PMs de-
stabilization [242]. It can be hypothesized that cl-micelles behave as
nanogels [242,243]. They can be prepared by:
i- Cross linking the shell
This was done by Li et al. with the PMs prepared from poly(styrene-
b-butadiene-b-styrene) copolymer. They used 1,6-hexanediamine as a
linker and produced clearly more stable micelles than the original ones
[244]. However, intermicellar cross linking is a common drawback of
this method, which would require the preparation at high dilution, a
fact which decreases the process eciency [242].
ii- Cross linking the core
Core crosslinking was reported to have a dual action: rst avoidance
of PMs destabilization upon dilution and second the cross-linked core
would control the burst release frequently encountered in PMs [245].
Talelli et al. summarized the most widely used methods for preparing
53
Journal of Controlled Release 261 (2017) 4361
core cl-micelles as follows: 1- radical polymerization: used for poly-
merizable groups containing PMs; 2- the addition of a bifunctional
crosslinker: used for reactive groups containing PMs; and 3- disulphide
bridges: used for thiol groups containing PMs [246]. In their study,
Oberoi et al. prepared cisplatin core cl-micelles using poly(ethylene
glycol)-b-poly(methacrylic acid) (PEG-b-PMA) where cross linking was
performed using 1,2-ethylenediamine (ED) and 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimidehydrochloride (EDC) in presence of CaCl2.
The obtained cl-micelles were a nanogel assembly with a particle size
ranging from 100 to 200 nm with a narrow PDI. The drug incorporation
reached 42% and the tuned cross linking controlled the drug release
eciently [243]. Desale et al. used the same bifunctional cross linker
for the preparation of PMs with multicompartment morphology to in-
clude both the hydrophilic cisplatin and the hydrophobic PTX. The
amphiphilic poly(ethylene glycol)-poly(glutamic acid)-poly(phenylala-
nine) (PEGPGluPPhe) triblock copolymer was used [247]. Wu et al.
were able to synthesize a phenol-containing amphiphilic diblock co-
polymer poly(ethylene glycol)-block-poly(N-isopropylacrylamide-co-N-
(4-hydroxyphenethyl)acrylamide)(PEG-b-P(NIPAAm-co-NHPAAm)) by
reversible addition-fragmentation chain transfer (RAFT) polymeriza-
tion of NHPAAm. This was followed by conjugation of the phenolic
derivatives using horseradish peroxidase (HRP) in presence of H2O2.
Finally, cl-micelles with cross linked core were self - aggregated at
~37 C [248].
2- Stimuli responsive PMs
i- Temperature-responsive PMs
The amphiphilic copolymers with thermo-responsive blocks, are
smart copolymers that respond to temperature change by decreasing
their aqueous solubility, disrupting the micelles and provoking drug
release [238]. Most of the studies dealt with thermoresponsive poly-
mers presenting with lower critical solution temperature (LCST) or
cloud point (CP) [235]. Below the LCST and due to hydrogen bonds, the
polymer is clear aqueous solutions. While, above this temperature the
polymer solution developed into a cloudy one (due to polymer pre-
cipitation). Higher temperature causes hydrogen bonds cleavage driven
by an energetically more favorable situation rendering water less ar-
ranged with higher entropy. The polymer LCST regulation is usually
done by incorporating either hydrophilic or hydrophobic monomers for
higher or lower temperatures, respectively [235]. One of the most
widely used thermoresponsive polymer with LCST of 32 C is poly(N-
isopropyl acrylamide) (PNIPAAm). pNIPAAm can form either the core
of PMs with PEG as the corona [249,250] or the shell with PCL as the
hydrophobic core [251,252]. Recently, Yu et al. used divalent metal
ions such as (Ba+ 2, Zn+ 2, Co+ 2) to graft sodium alginate with
pNIPAAm. The cross-linked alginate formed the core, while the corona
was made of PNIPAAm chains [253]. At 37 C, the hydrophobic
PNIPAAm chains helped in micellar dissociation with 5-uorouracil (5-
FU) release. However, no drug release was detected at 25 C.
ii- pH-responsive PMs
In normal extracellular matrix and blood, pH maintains at about
7.4, while the tumor microenvironment shows acidic pH (~ 6.5) due to
low oxygen supply in the intercellular environment [254]. The en-
dosomal and lysosomal pHs are still lower ranging from 4.5 to 6 [254].
Numerous studies have tried to prepare pH responsive PMs to deliver
drugs to tumors, endosomes or lysosomes [235,255257]. At acidic pH,
the carboxylate groups of orthoester, acetal, hydrazone, cis-acotinyl,
imine and oximeare easily protonated. Hence their covalent bonds
break down releasing the drug in acidic environment only [255]. Cao
et al. prepared pH-sensitive PMs for DOX delivery from PEG-b-PCL
containing citraconic amide as pH-sensitive bond (PEG-pH-PCL) [258].
The drug was released faster in the endosomes pH 5.5.This was
R.S. Elezaby et al. Journal of Controlled Release 261 (2017) 4361

visualized by confocal laser microscopy and ow cytometry measure-
ments
iii- Redox-responsive PMs
Due to the abundance of glutathione (GSH) tripeptide in-
tracellularly, the intracellular uids are known to be in a less oxidized
state than the extracellular uids. This will create a redox potential
[255]. Amphiphilic copolymers with disulphide linkage will be reduced
at low redox potential and hence will be redox responsive [255]. Hence,
contrary to pH-responsive micelles, redox-responsive micelles destabi-
lize and release drugs in the cytoplasm not in endosomes or lysosomes.
The PMs core with a disulphide linkage will be cross-linked and when
present intracellularly drug release is initiated by the drop in redox
potential. Shoe et al. prepared itraconazole redox- responsive PMs from
poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-phenylalanine) (PEG-
pLys-pPhe) for treatment of intracranial infections [133].The segment
of pPhe oered the hydrophobic core for hydrophobic drug itracona-
zole. The amino groups of pLys segments could react with a cross-
linking agent containing disulphide bonds viz. 3,3-Dithiobis(sulfo-
succinimidylpropionate) (DTSSP) to produce a shell cl-micelles. The
prepared redox-responsive PMs were able to shield the leakage of drug
in the circulation with enhanced drug release intracellulary.
iv- pH and redox dual responsive PMs
PMs can be designed to be responsive to both change in the pH and
redox potential. Teo et al. prepared diblock copolymers of PEG and
polycarbonate containing both disulphide bonds and carboxylic acid
groups (on the same monomer unit) via metal-free organo cataly-
ticpolymerization (ROP) of functional cyclic carbonate, and employed
it to form pH and redox dual-responsive micelles [254].
v- Photo- responsive PMs
Photo-responsive PMs are prepared from light-sensitive groups
containing copolymers, included in the core, corona, or at their inter-
face [238]. Upon exposure to the specic light stimulus which may be
at the region of ultra-violet (UV), visible or near-infrared (NIR), the
photo-responsive polymers undergo structural modication which
causes sudden drug release. For example, azobenzene group and its
derivatives undergo reversible photoisomerization from trans to cis and
from cis to trans upon exposure to UV (300380 nm) and visible
(420490) light stimuli respectively. This phase change resulted in PMs
assembly disruption due to the more hydrophobicity of the trans-azo
bond than the cis-azo bond [259261]. Due to its high skin absorption
UV light cannot inltrate the deep body tissue. Hence, it is only used for
topical treatment. On the other side, NIR can go deep into the body up
to 10 cm, helped by the low absorption in the NIR region (650900 nm)
of hemoglobin, water and lipids [235]. Additionally, healthy tissues can
be severely damaged due to the high absorbance of UV light by many
biological molecules [262]. Cabral et al. developed two groups of PMs,
one encapsulate captothecin (CPT), an antitumor drug, while the other
encapsulate dendrimer phthalocyanine (DPc) [262]. The CPT-PMs were
formed by a disulphide bond conjugation between a thiolated-camp-
tothecin derivative (CPT-DP) and a thiolated poly(ethylene glycol)-poly
(glutamic acid) (PEG-b-P(Glu-DP)) block copolymer. As mentioned
before, the disulphide bond is stable in blood and cleaved specically in
the intracellular reductive environment. DPc is a photosensitizer which
stimulates reactive oxygen formation when exposed to suitable wave-
lengths. DPc-PMs were prepared from poly(ethylene glycol)b-poly(L-
Lysine) (PEG-b-PLL). PMs were able to accumulate at the tumor site by
enhanced permeability and retention eect (EPR) and entered the cell
by endocytosis. The presence of DPc-PMs in the endosome allows a
selective photo-damage of the endosomal membrane upon photo-irra-
diation releasing the CPT-PMs in the cytosol. CPT is released in the
cytosol upon the reduction in the redox potential. Huang et al. used NIR
irradiation to enhance the cellular uptake of nely tuned temperature-
responsive PMs [263]. They prepared PMs from poly(-caprolactone)-
block-poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (PCL-b-

Table 4
History of surface modication of micelles for brain targeting.

Targeting ligand Pre- or post-conjugation Targeted moiety Targeted pathway Ref.

Tat Pre-conjugation ve charge on epithelial cells AMT [11,194,202,203]

PS80 Pre-conjugation LRP1 and LRP2 RMT [175,192]
Angiopep-2 Pre-conjugation LRP RMT [193]
NGR Pre-conjugation APN RMT [173]
cRGD Pre-conjugation v3 and v5 integrins RMT [109,191,211,218,219]
Angiopep-2 Post-conjugation LRP RMT [196,197]
Tf Post-conjugation TfR RMT [171,190]
Tf Post-conjugation TfR RMT [12]
cRGD Pre-conjugation v3 and v5 integrins
cRGD Post-conjugation v3 and v5 integrins RMT [172]
TGN Pre-conjugation NA RMT [198]
FB4 aptamer Post-conjugation TfR RMT [199]
DHA Pre-conjugation GLUT1 CMT [133]
Glucose and FA Pre-conjugation GLUT1 CMT [205]
FA-receptors RMT
Lt Pre-conjugation LfR RMT [177]
CREKA Pre-conjugation Fibrin deposited in tumors EPR [204]
RVG Post-conjugation Ach receptors RMT [108]
Lt Post-conjugation LfR RMT [208]
PS80 Post-conjugation LRP1 and LRP2 RMT [186]
Tf Pre-conjugation TfR RMT [13]
Td Pre-conjugation LAT1 CMT [207]
OX26 Pre-conjugation TfR/CD71 RMT [215]
T7 Pre-conjugation TfR RMT [220]
GLUT-1 scFv Post-conjugation GLUT1 CMT [222]

Ach: acetyl choline; AMT: adsorbtive- mediated transcytosis; APN: aminopeptidase N; CMT: carrier-mediated transport; cRGD: cyclic Arginine-Glycine-Aspartic acid; DHA: dehy-
droascorbic acid; FA: folic acid; GLUT-1 scFv: GLUT1 antibody single chain fragment variable; LAT1: large neutral amino-acid transporter type 1; LRP: lipoprotein receptor-related
protein; Lt: lactoferrin; LtR: lactoferrin receptor; NGR: Asn-Gly-Arg peptide; OX26: an anti-TfR antibody; PS80: polysorbate 80; RMT: receptor- mediated transcytosis; RVG: rabies virus
glycoprotein; T7: His-Ala-Ile-Tyr-Pro-Arg-His peptide; Tat: trans-activating transcriptional activator; Td: tryptophan derivative; Tf: transferrin; TfR: transferrin receptor; TGN:
TGNYKALHPHNG peptide.

54
R.S. Elezaby et al.
p(NIPAAm-co-DMA)), with nely tuned compositions of p(NIPAAm-co-
DMA) for desirable LCST. NIR irradiation increased PMs and cell
membranes interactions due to the hydrophilic to hydrophobic transi-
tion of p(NIPAAm-co-DMA) shells. The cellular uptake of the PMs and
therapeutic benets of loaded anticancer drugs were dramatically
promoted.
3- Cross linked-stimuli responsive PMs.
PMs starting drug release upon subjection to stimuli were also cross
linked for a higher stability upon dilution. pH and redox sensitive core
cl-micelles encapsulating DOX were prepared by Zhang et al. [245].The
hydrophobic moiety and the amino group of DOX enabled its tight and
high core encapsulation in cl-micelles formed from PEG as the shell
while the core contained (n-butyl methacrylate) and carboxylic acid
groups (methacrylic acid), by both hydrophobic and ionic interactions.
The long circulation was ascertained by the PEG shell. Moreover, the
core was cross linked via disulphide linkage to beat the noxious eect of
CMC rise, resulting from the presence of the hydrophilic carboxylic acid
groups inside the micellar core. After entering the cancer cells, a high
drug release rate was possible due to: rst, the reduction of the PMs
disulphide cross linkages in the existence of high amount of in-
tracellular glutathione; second, the low pH environment protonation of
the carboxylate groups breaking the bond with the DOX amino group.
Recently, a novel redox-responsive core cl-micelles was prepared by Wu
et al. using a cross linker with a disulphide bond, 3,30-dithiodipro-
pionic acid (DPA), with mPEGylated starch (mPEG-St) copolymer
[264]. The authors concluded that core-cross linked redox-responsive
mPEG-St-DPA micelles have high abilities for cancer therapy.
B) Active targeted PMs
In addition to their various advantages, the propensity of PMs
decoration with ligands able to target the brain gives them a special
appeal. In the last decade, the two principle brain targeted pathways
were RMT and CMT pathways. Both pre- and post-formulation con-
jugation strategies were applied. Table 4 summarizes the surface
modications on the micellar surface in the last decade for brain de-
livery.
1- Micellar targeting for RMT pathway
Angiopep-2 - a member of angiopeps peptide family obtained from a
natural occurring protein - proved to cross the BBB at LRP-1 better than
other proteins such as aprotinin and transferrin. Being entangled in
several cellular processes, LRP-1 has been used as a therapeutic
gateway to the brain. The fact of being over-expressed in malignant
astrocytomas, especially in glioblastomas encouraged scientists to use it
for brain cancer therapy [193]. Shen et al. synthesized cysteine an-
giopep-2 (TFFYGGSRGKRNNFKTEEYC) and conjugated it to mal-PEG-
PLA copolymer via simple one-step chemistry (click chemistry) ap-
plying the pre-conjugation strategy [193]. Shen et al. showed very
rapid brain uptake of angiopep-2 conjugated PMs immediately after IV
injection in comparison to the unmodied PMs. On the same chemical
basis, Shao et al. conjugated angiopep-2 to mal-PEG-PE copolymer
applying post-conjugation strategy [196]. The interaction between an-
giopep-2 and mal-PEG portion of any copolymer is simple click chem-
istry too. A stable thioether conjugate was formed between the thiol
group in angiopep-2 and the mal-PEG. Simply, angiopep-2 is added to
mal-copolymer or to mal-conjugated micelles in phosphate buer saline
at pH = 7.27.4 with stirring for 12 h in darkness at room temperature.
Unconjugated angiopep-2 was then removed by dialysis [193,196].
Fig. 6 shows the general scheme for interaction between maliemide
moiety (usually attached to PEG) and the thiol group of any ligand,
which is very popular in ligand conjugation.
Mal-PEG-b-poly-L-glutamic acid PMs were conjugated by Miura
55
Journal of Controlled Release 261 (2017) 4361
et al. with cRGD based on the same chemistry [172]. v3 and v5
integrins are generally over expressed on endothelial cells of tumor
angiogenic vessels and glioblastoma cells have been selectively targeted
by cRGD [172]. The ecacy of a platinum anticancer drug was found
highly enhanced when loaded in 20% cRGD-linked micelles in contrast
to non-targeted PMs.
PepTGN, a specic peptide capable of binding to a specic brain
protein, was prepared by phage display techniques and introduced
during the last ve years [21,198,265270]. TGN is a 12-amino acid
peptide, TGNYKALHPHNG which when conjugated to PEG-PLGA na-
noparticles proved signicant superiority in crossing the BBB [21].
Qian et al. conjugated TGN simply to mal-PEG through its cysteine end
[198]. The decorated polyplexes enhanced signicantly the BCECS
uptake and higher cell uorescence intensity was evident in YOYO-1-
labeled TGN-PEG-PDMAEMA/DNA polyplexes in comparison to the
unmodied polyplexes [198].
Another simple technique of conjugation, that depends on N-hy-
droxysuccinimidyl PEG (NHS-PEG) rather than mal-PEG was presented
in many projects [173]. A stable amide bond was obtained due to the
reaction between the terminal amino group of the cyclic Asn-Gly-Arg
(NGR) and NHS-PEG [173] (Fig. 6). A twice higher cellular uptake of
the modied micelles with a signicant inhibition in the cell pro-
liferation was proven when compared to the unmodied micelles
[173]. Additionally, Mu et al. conjugated FB4 aptamer to PEG-PLA
micelles via this technique [199]. They showed that the surface mod-
ication increased the cellular uptake in bEND5 by a 2.2-fold.
Tf is another popular ligand eectively trancytosed across the BBB
via binding TfR. Zhang et al. conjugated Tf to mal-PEG-PCL copolymer
[171]. According to the general scheme described before (Fig. 6),
briey, Tf was activated with 2-iminothiolane hydrochloride (ITH) at a
molar ratio 1:20. The thiolated Tf was then puried, and extent of
thiolation was determined [12,171]. Both cell and brain uptake proved
almost a two-fold increase in the tagged micelles than their unmodied
counterpart. The TfR mediated transcytosis was further conrmed by
inhibited cellular uptake in the presence of free Tf [171].
Thereafter, Zhang et al. conjugated PTX to cRGD to obtain cRGD-
PTX conjugate (RP) which was encapsulated into Tf modied PCL-PEEP
polymeric micelles (TRPM). The study proved an enhanced cellular
uptake in BMECs (2.4-fold in comparison to unmodied micelles) with
a specic superior accumulation of the drug in mice brain glioma tumor
and peritumor areas following intravenous injection [12].
Lf is a multifunctional protein of Tf family. Lf proved to be more
advantageous for brain targeting than Tf due to its much lower plasma
concentration than the saturation value of its receptors, ability to allow
more chemicals across the BBB and participation in the brain lesions
pathogenesis [177]. Besides, its receptors (LfR) expression was higher
Fig. 6. General scheme for interaction between A) Mal reagent and a thiol group B) NHS
reagent and an amine group.
R.S. Elezaby et al.
Fig. 7. An illustration of the trip of magic bullet PMs after IV injection to its targeted
location in the brain.
in patients with acute stroke and Parkinson's disease [271,272]. Zheng
et al. conjugated Lf to sodium alginate-cholesterol derivatives (SACDs)
simply by reaction with activated NHS-SACDs [177]. The Lf conjugated
PMs of the cholesterol derivative showed higher brain uptake in mice
than the unmodied micelles.
2- Micellar targeting for CMT pathway.
D-Glucose and other brain energy supply hexoses cross the brain via
GLUT1. DHA, an oxidized form of ascorbic acid, is one a GLUT1 sub-
strates which is rapidly reduced to ascorbic acid once its uptake by the
brain. Ascorbic acid itself isn't one of GLUT1 substrates, which will
guarantee its presence in a high concentration in the brain. Hence, DHA
enjoys an eective one-way ux across the BBB compared with D-
glucose, which suers from bidirectional uxes.
Shoa et al. conjugated DHA to poly(ethylene glycol)-b-poly(L-ly-
sine)-b-poly(L-phenylalanine) (PEG-pLys-pPhe) to construct active tar-
geted PMs [133]. Propargylic DHA was linked to the terminus of N3-
PEG-b-PLL- PPhe via a click reaction of the copper (I)-catalyzed azi-
dealkyne. In comparison to unmodied micelles, these PMs exhibited
improved cellular uptake by 6 fold in-vitro and 1.48 in-vivo [133].
3- Dual targeting PMs
Another very interesting approach in brain targeting is the dual
targeting. In this approach, nanoparticles are decorated by two types of
ligands; one ligand targets a special pathway to cross the BBB while the
other targets a certain location in the brain. Niu et al. conjugated both
glucose and FA to Pluronic P105 PMs designed for DOX delivery named
(GF-DOX). Glucose was added to target the CMT pathway via
GLUT1while FA was added to target brain tumor cells per se [205].
After 8.0 h GF-DOX showed a 2-fold increase in the uptake of C6 cells,
in comparison to unmodied micelles or micelles decorated with glu-
cose only, encouraging such approach for successful brain targeting.
Dual targeting strategy was introduced dierently in some other
researches, where PMs were decorated by one ligand that targets a
specic brain pathway while the other ligand - which targets specic
receptors in the brain - was conjugated to the drug itself. As mentioned
before, Zhang et al. applied this strategy for brain targeting where they
conjugated PTX to cRGD followed by encapsulation into Tf modied
PCL-PEEP micelles.
4.4.3.7. PMs as magic bullets for brain delivery. PMs are very promising
nanocarriers for brain delivery, which can be modulated to behave as
56
Journal of Controlled Release 261 (2017) 4361
magic bullets. The expression of magic bullet for brain delivery means
that the delivery system reaches the BBB, directly, without any
instability problems; escaping any immune reaction, overcomes the
challenge of BBB, readily uptaken by the brain and reaches the aected
area to exert its pharmacological action. PMs can be modulated to
behave as magic bullets as follows:
1) The small particle size (10100 nm) and the hydrophilic shell (helps
also in shielding of charge) of PMs help in escaping from the RES.
2) Core or shell cross-linking or stimulus responsive would help in
avoidance of instability upon circulatory dilution.
3) Decoration of the PMs surface with a ligand that targets a certain
pathway in the brain helps in the BBB passage.
4) Decoration of the PMs surface with a ligand - or being conjugated to
the drug - that targets special tissues or receptors in the brain im-
proves the eciency and minimizes the side eects.
As shown in Fig. 7, if the PMs preparation combines all the previous
criteria, they will act as brain magic bullets that deliver therapeutic
agents directly to the desired location in the brain.
5. Conclusion
Numerous strategies have been adopted to trigger dierent ap-
proaches to overcome limited brain transport. Nevertheless, nano-
technology remains of paramount signicance for CNS delivery. The
wide use of dierent nanocarriers has led to a wide array of techniques
for improving brain targeting. PMs with a controlled, targeted and
smart drug release would help in the production of the so called magic
bullets for brain delivery. Dissociation controlled PMs which are de-
corated with multiple ligands that target a certain brain pathway and a
certain area inside the brain was already tried. Collectively, this will
open a new era for friendly non-invasive treatment of CNS diseases.
Besides, the shift from initial pragmatic delivery ways towards tailored
multifunctional PMs would oer more eective treatment of mental
and neurobiological disorders, which account for 11% of disease
burden all over the world.
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