pharmaceutics

Review
Encapsulation, Release, and Cytotoxicity of Doxorubicin
Loaded in Liposomes, Micelles, and Metal-Organic
Frameworks: A Review
Mihad Ibrahim 1 , Waad H. Abuwatfa 1,2 , Nahid S. Awad 1 , Rana Sabouni 1,2 and Ghaleb A. Husseini 1,2, *

1 Department of Chemical Engineering, College of Engineering, American University of Sharjah,

Sharjah P.O. Box 26666, United Arab Emirates; g00056692@[Link] (M.I.);
g00062257@[Link] (W.H.A.); nawad@[Link] (N.S.A.); rsabouni@[Link] (R.S.)
2 Materials Science and Engineering Program, College of Arts and Sciences, American University of Sharjah,
Sharjah P.O. Box 26666, United Arab Emirates
* Correspondence: ghusseini@[Link]; Tel.: +971-6-515-2970

Abstract: Doxorubicin (DOX) is one of the most widely used anthracycline anticancer drugs due
to its high efficacy and evident antitumoral activity on several cancer types. However, its effective
utilization is hindered by the adverse side effects associated with its administration, the detriment to
the patients’ quality of life, and general toxicity to healthy fast-dividing cells. Thus, delivering DOX to
the tumor site encapsulated inside nanocarrier-based systems is an area of research that has garnered



colossal interest in targeted medicine. Nanoparticles can be used as vehicles for the localized delivery

 and release of DOX, decreasing the effects on neighboring healthy cells and providing more control
Citation: Ibrahim, M.; Abuwatfa, over the drug’s release and distribution. This review presents an overview of DOX-based nanocarrier
W.H.; Awad, N.S.; Sabouni, R.; delivery systems, covering loading methods, release rate, and the cytotoxicity of liposomal, micellar,
Husseini, G.A. Encapsulation, and metal organic frameworks (MOFs) platforms.
Release, and Cytotoxicity of
Doxorubicin Loaded in Liposomes,
Keywords: doxorubicin; liposomes; micelles; metal-organic frameworks (MOFs)
Micelles, and Metal-Organic
Frameworks: A Review.
Pharmaceutics 2022, 14, 254.
[Link]
1. Introduction
pharmaceutics14020254
Cancer refers to uncontrolled cell division due to DNA mutations. It occurs when cells
Academic Editors: Donatella Paolino,
are induced to over-proliferate, caused by the permanent activation of proto-oncogenes in
Ana Isabel Fraguas-Sánchez and
upregulated oncogenes [1]. Throughout their normal life cycle, cells divide controllably,
Cristina Martin-Sabroso
differentiate, and eventually die through the programmed cell death mechanism known as
Received: 30 November 2021 apoptosis. However, mutations perturbing the ordinary growth pathway of the cells can
Accepted: 16 January 2022 introduce behavioral changes on genetic and epigenetic levels, where these transformed
Published: 21 January 2022 cells exhibit different growth characteristics, including enhanced mobility and the decreased
Publisher’s Note: MDPI stays neutral
contact inhibition of growth mechanisms [2]. Some cancer cells incur tumor-initiating
with regard to jurisdictional claims in
abilities and can travel to other parts of the body through the bloodstream or lymph vessels
published maps and institutional affil- (metastasis) [3].
iations. Anticancer drugs are classified into three categories: chemotherapy, immunotherapy,
and hormonal therapy. These therapies can target tumor cells at the DNA, RNA, or protein
level [4–6]. Chemotherapy is the invasive anticancer therapeutic regimen used worldwide.
It was introduced by the German chemist Paul Ehrlich and has been used to treat cancer
Copyright: © 2022 by the authors. since the beginning of the 20th century [7–9].
Licensee MDPI, Basel, Switzerland. The formulations of antineoplastic agents were divided, according to their chemical
This article is an open access article structure and mechanism of action, into alkylating agents, antibiotics, antimetabolites,
distributed under the terms and topoisomerase inhibitors, and others [5–7,10–12]. The last decades have witnessed colossal
conditions of the Creative Commons efforts to discover novel chemotherapeutic drugs including, but not limited to, doxorubicin,
Attribution (CC BY) license (https://
epirubicin, pirarubicin, methotrexate, pemetrexed, 5-fluorouracil, paclitaxel, cisplatin, gem-
[Link]/licenses/by/
citabine, and others [3,10,13]. Chemotherapeutics are usually administered either orally or
4.0/).

Pharmaceutics 2022, 14, 254. [Link] [Link]

Pharmaceutics 2022, 14, x 2 of 32

Pharmaceutics 2022, 14, 254 2 of 29

rubicin, epirubicin, pirarubicin, methotrexate, pemetrexed, 5-fluorouracil, paclitaxel, cis-
platin, gemcitabine, and others [3,10,13]. Chemotherapeutics are usually administered ei-
ther orally or intravenously
intravenously to achieve systemic to achieve systemic
distribution, distribution,
thus maximizing thus maximizing
their their effect.
effect. Unfortunately,
the practicality of chemotherapy drugs is counteracted by their lack of selectivity,lack
Unfortunately, the practicality of chemotherapy drugs is counteracted by their of se-
causing
lectivity, causing grave damage to healthy cells with high division
grave damage to healthy cells with high division rates like bone marrow, hair follicles, and rates like bone marrow,
hair follicles, and
gastrointestinal gastrointestinal
epithelia. In addition,epithelia. In addition, both and
both spermatogenesis spermatogenesis and oogen-
oogenesis processes are
esis processes
highly susceptible are to
highly susceptible
the cytotoxic sidetoeffects
the cytotoxic side effects of chemotherapeutics.
of chemotherapeutics.
Althoughsurgery
Although surgeryand and radiotherapy
radiotherapy areare
ideal ideal for treating
for treating localized
localized solid solid
cancers,cancers,
their
their successful use is hampered in many cases where cancer
successful use is hampered in many cases where cancer has metastasized to other organs has metastasized to other
organs
in in the Thus,
the body. body. chemotherapy
Thus, chemotherapy is commonly
is commonly offeredoffered to patients
to patients at advanced
at advanced can-
cancer
cer stages.
stages. It isItaissystemic
a systemic therapy
therapy thatthat targets
targets rapidly
rapidly dividingcells
dividing cellsby byemploying
employingagents agents
that interfere
that interfere withwiththeir
theirlife
lifecycle
cycleandandreplication
replicationmechanisms.
mechanisms. ItIt isis advantageous
advantageous over over
conventionaltherapies
conventional therapiesbecause
becauseititcan canact actthroughout
throughout the
the whole
whole body,
body, thusthus targeting
targeting can-
cancer
cer cells
cells that that metastatically
metastatically spread spread far from
far from the primary
the primary tumortumor
site and site and helping
helping to shrink to shrink
bulky
tumors that might
bulky tumors that otherwise
might otherwisebe unresectable [14]. From
be unresectable [14].the
From 1960s
theonwards, the success
1960s onwards, of
the suc-
chemotherapeutic
cess of chemotherapeutic drugs indrugstreating various various
in treating advanced cancers cancers
advanced led to the leddevelopment
to the develop- of
the
mentso-called adjuvant therapy,
of the so-called adjuvantintherapy,
which chemotherapeutics
in which chemotherapeutics are combined arewith surgery
combined to
with
eradicate
surgery to potential
eradicate cancer dissemination
potential or recurrence [15,16].
cancer dissemination Sometimes,
or recurrence chemotherapy
[15,16]. Sometimes,
combined
chemotherapy with radiotherapy
combined with is radiotherapy
more effectiveisthan more either modality
effective alone. modality alone.
than either
Among
Among the the most
most widely
widely used used class
classof ofchemotherapeutics
chemotherapeutics are are anthracycline
anthracycline agents,agents,
which are prevalently successful in achieving short-time cancer
which are prevalently successful in achieving short-time cancer progression inhibition, progression inhibition,
high
highresponse,
response,and andimproved
improvedsurvivalsurvivalratesrates[17].
[17]. Anthracyclines
Anthracyclines are are the
thefirst
firstanti-tumor
anti-tumor
antibiotics approved by the Food and Drug Administration
antibiotics approved by the Food and Drug Administration (FDA), among which (FDA), among which is Doxoru-
is Dox-
bicin (DOX),
orubicin one one
(DOX), of theoffirst effective
the first anthracycline
effective anthracycline antibiotic cytotoxic
antibiotic drugs
cytotoxic discovered.
drugs discov-
Itered.
wasItisolated from from
was isolated the red thepigment-producing
red pigment-producing soil bacterium;
soil bacterium; Streptomyces
Streptomyces peucetius
peuce-
via mutagenic treatment [18]. DOX (Figure 1) is extensively
tius via mutagenic treatment [18]. DOX (Figure 1) is extensively used to treat lymphomas, used to treat lymphomas,
leukemia,
leukemia,as aswell
wellas asovarian,
ovarian,breast,
breast,small
small cell
cell lung,
lung, stomach,
stomach, and and liver
liver cancers
cancers [4,7,19,20].
[4,7,19,20].

[Link]
Figure Thechemical
chemicalstructure
structureof
ofDOX
DOX[7].
[7].

Likemost
Like mostanthracycline
anthracycline antibiotics,
antibiotics, DOX DOX oxidizes
oxidizes andand inhibits
inhibits thethe activity
activity ofof topoi-
topoi-
somerase II,
somerase II, an enzyme
enzymeresponsible
responsiblefor forDNA
DNAtranscription
transcription andandreplication,
replication, generating
generating un-
stable, highly
unstable, highlyactive free
active radicals
free radicalsthatthat
damage
damage the the
DNA DNAandandcause cell cell
cause death [4,7,19].
death Alt-
[4,7,19].
hough it is
Although anan
it is effective
effectiveanti-tumor
anti-tumoragent,
agent,itsitsside
sideeffects
effectsare
aremost
most evident
evident on cells exhibit-
exhibit-
inghigh
ing highdivision
divisionrates,
rates,such
suchas ashair
hairfollicles
folliclesandandthethegastrointestinal
gastrointestinaltracttractlining;
lining;thus,
thus,hair
hair
loss,
loss,digestive
digestivetract
tractulcerations,
ulcerations,vomiting,
vomiting,nausea,
nausea, and and diarrhea
diarrhea areare all
all common
common complica-
complica-
tions/side
tions/side effects [21].
[21]. Also, it has been known known to to stimulate
stimulate myelosuppression
myelosuppressionand andinduce
induce
cardiotoxicity
cardiotoxicityby bythe
theupregulation
upregulation ofofapoptosis
apoptosis receptors
receptors in cardiomyocytes
in cardiomyocytes [22,23]. Clinical
[22,23]. Clin-
investigations by Von
ical investigations by Hoff et al. et
Von Hoff [24][Link] 7.5% 7.5%
[24] showed cardiomyopathy
cardiomyopathy occurrences associated
occurrences asso-
with
ciatedcumulative DOX doses
with cumulative DOX of 550
dosesmg/m of 2550
. Thus,
mg/m cumulative doses of 400–500
2. Thus, cumulative dosesmg/m 2 are
of 400-500
generally
mg/m2 areemployed
generallyfor free administration
employed once every three
for free administration once weeks to prevent
every three weekscardiotoxic
to prevent
effects.
cardiotoxic effects. In addition, DOX is a vesicant and can cause blisteringifand
In addition, DOX is a vesicant and can cause blistering and necrosis extravasation
necrosis if
occurs at the time
extravasation of drug
occurs at theadministration [14]. Moreover, [14].
time of drug administration drugMoreover,
resistance drug
in theresistance
tumor cells in
is another problem that limits the clinical use of DOX [19,25,26]. In general, the toxicity of
chemotherapeutic drugs limits their effective dosage threshold. In most cases, a therapeutic
Pharmaceutics 2022, 14, x 3 of 32

Pharmaceutics 2022, 14, 254 3 of 29

the tumor cells is another problem that limits the clinical use of DOX [19,25,26]. In general,
the toxicity of chemotherapeutic drugs limits their effective dosage threshold. In most
dose
cases,ofa the chemotherapy
therapeutic dose ofdrugs capable of efficiently
the chemotherapy killingofthe
drugs capable tumor cells
efficiently is highly
killing the tu-
toxic to theisfast-growing
mor cells normal
highly toxic to cell and, therefore,
the fast-growing normal cannot
cell and,betherefore,
given to patients
cannot be(Figure
given2).
to
Instead,
patients a(Figure
lower dosage capable
2). Instead, of destroying
a lower considerable
dosage capable numbers
of destroying of tumor cells
considerable with
numbers
lower toxic
of tumor effects
cells withonlower
normal cells
toxic is utilized
effects [14]. cells is utilized [14].
on normal

Figure2.2. Dose
Figure Dose responsive
responsive curvecurve for
for chemotherapy
chemotherapy drugs
drugs kills
kills both
both tumor
tumor cells
cells and
and healthy
healthy cells.
cells.
The therapeutic dose (A) is close to the toxic dose (B). It is not safe to give this drug at a therapeutic
The therapeutic dose (A) is close to the toxic dose (B). It is not safe to give this drug at a therapeutic
[Link]
dose. safedose
dose(C)
(C)is is chosen
chosen forfor administration
administration to patients.
to patients. Reproduced
Reproduced withwith permission
permission from from
[14],
[14], Elsevier, 2008.
Elsevier, 2008.

Followingthe
Following thetreatment
treatment with
with chemotherapy
chemotherapy drugs,
drugs, tumor
tumor cells
cells show
show slower
slower replica-
replica-
tion times compared to healthy cells, which can regenerate at controlled
tion times compared to healthy cells, which can regenerate at controlled replication rates. replication rates.
This led to the development of the intermittent administration
This led to the development of the intermittent administration of chemotherapy whichof chemotherapy which
allowed the
allowed the total
total regeneration
regeneration of of normal
normal tissues
tissuesbutbutnot
notthat
thatofoftumor
tumortissues,
tissues,leading
leading to
reducing
to reducing butbut
notnot
eliminating
eliminatingthethetoxicity of these
toxicity drugs
of these [27].[27].
drugs Chemotherapeutics
Chemotherapeutics are asso-
are
ciated withwith
associated other challenges
other thatthat
challenges hinder theirtheir
hinder use,use,
including:
including:
(a) Limited
(a) Limitedsolubility
solubilityininaqueous
aqueoussolutions:
solutions: most
most of of
thethe chemotherapy
chemotherapy drugs
drugs are hydro-
are hydropho-
phobic.
bic. Thus, Thus, solvents
solvents are used
are used to solubilize
to solubilize thesethese
drugs,drugs,
whichwhich increases
increases their tox-
their toxicity.
icity. specific targeting of the cancer cells, i.e., high toxic dosages, are delivered to
(b) Poor
(b) healthy
Poor specific
as welltargeting
as cancerofcells.
the cancer cells, i.e., high toxic dosages, are delivered to
healthycells
(c) Cancer as well
canas cancerresistance
develop cells. to chemotherapy drugs, a phenomenon known as
(c) multi-drug
Cancer cellsresistance (MDR).
can develop This results
resistance in minimal cell
to chemotherapy deatha and
drugs, the expansion
phenomenon knownof
drug-resistant tumors. (MDR). This results in minimal cell death and the expansion
as multi-drug resistance
of drug-resistant tumors.
2. Nanoparticles as Drug Delivery Systems (DDS)
2. Nanoparticles as Drug to
Due to the detriment Delivery Systems
the patients’ (DDS)
quality of life and the potential lethality of some
of theDue
sideto
effects associatedtowith
the detriment conventional
the patients’ chemotherapy,
quality novel
of life and the drug delivery
potential lethalitysystems
of some
(DDS) aim to reduce the adverse side effects and enhance the specificity
of the side effects associated with conventional chemotherapy, novel drug delivery sys-of chemother-
apeutic drugs.
tems (DDS) aimInto1964, Cheng
reduce initiatedside
the adverse the effects
use of and
encapsulation
enhance theofspecificity
enzymes and other
of chemo-
biologically active materials in semipermeable vesicles and tested their use
therapeutic drugs. In 1964, Cheng initiated the use of encapsulation of enzymes and other to suppress
the growth ofactive
biologically lymphosarcoma
materials inin a mice model vesicles
semipermeable [28,29]. and
This tested
approach
theirhas
useevolved and
to suppress
been extensively extended into nanomedicine, biotherapeutics, blood
the growth of lymphosarcoma in a mice model [28,29]. This approach has evolved and substitutes, drug
delivery, enzyme/gene therapy, cancer therapy, nanoparticles, liposomes, bioencapsulation,
been extensively extended into nanomedicine, biotherapeutics, blood substitutes, drug
regenerative medicine, nanobiotechnology, and nanotechnology [30]. Earlier studies on
the transformed cancer cells showed that they had adopted many biochemical strategies
Pharmaceutics 2022, 14, x 4 of 32

Pharmaceutics 2022, 14, 254 delivery, enzyme/gene therapy, cancer therapy, nanoparticles, liposomes, bioencapsula- 4 of 29
tion, regenerative medicine, nanobiotechnology, and nanotechnology [30]. Earlier studies
on the transformed cancer cells showed that they had adopted many biochemical strate-
gies
to to support
support their uncontrolled
their uncontrolled growth growth
[31,32]. [31,32].
A deep A understanding
deep understanding of the of the
key key en-
enzymes
zymes and antagonistic pathways of synthesis and catabolism
and antagonistic pathways of synthesis and catabolism involved in tumor progression involved in tumor progres-
sion resulted
resulted in theindevelopment
the development of “targeted
of “targeted therapy”.
therapy”. TargetedTargeted
DDS DDS are nanoplatforms
are nanoplatforms that
that incorporate nanoparticles (NPs) as drug delivery
incorporate nanoparticles (NPs) as drug delivery vehicles, ranging in size vehicles, ranging in size
from from
1 nm 1 nmto
to 1000
1000 nmnm [33,34].
[33,34]. These
These include,
include, but but
are are not
not limitedto,to,gold
limited goldNPs,
NPs,dendrimers,
dendrimers,polymeric
polymeric
nanogels,micelles,
nanogels, micelles,metal-organic
metal-organicframeworks
frameworks(MOFs), (MOFs),liposomes,
liposomes,and andquantum
quantumdots. [Link]
synthesis routes of these NPs vary and are generally divided into
synthesis routes of these NPs vary and are generally divided into chemical and biological chemical and biological
ways,where
ways, wherethe thelatter
latterare
arepreferred
preferredas asthey
theyarearesafer
saferand
andinnocuous
innocuous[35]. [35].
Utilizing these nanocarriers for the remote delivery
Utilizing these nanocarriers for the remote delivery of appropriate dosages of appropriate dosages to tar-
to targeted
geted anatomical
anatomical sites under
sites under controlled
controlled release release conditions
conditions can overcome
can overcome the the shortcomings
shortcomings of
of traditional
traditional treatment
treatment approaches.
approaches. TheThe localized
localized accumulation
accumulation of nanoparticles
of the the nanoparticles at the at
the neoplastic
neoplastic site site is mainly
is mainly achieved
achieved by by passive
passive and/or
and/or activetargeting
active targetingroutes.
[Link] Inpassive
passive
targeting,the
targeting, theintrinsic
intrinsic features
features of of the
the tumor
tumor neovasculature
neovasculature beneficially
beneficially provide
provide fenestra-
tions where the NPs accumulate. As a tumor undergoes rapid,
tions where the NPs accumulate. As a tumor undergoes rapid, chaotic growth, it exhibits chaotic growth, it exhibits a
a disorganized
disorganized vascular
vascular networkand
network andbecomes
becomeshypoxichypoxicdue dueto toinsufficient
insufficient oxygen
oxygen supply.
Tumor
Tumorcells
cellscan
cansecrete
secrete growth
growth factors
factorsto induce
to induce vascularization
vascularization to resolve this hypoxia
to resolve this hypoxiaand
get
andnutrients from neighboring
get nutrients from neighboring healthy cells bycells
healthy a process referredreferred
by a process to as angiogenesis [35,36].
to as angiogenesis
The newly
[35,36]. Theformed capillitial
newly formed endothelium
capillitial in tumor
endothelium in tissues is disorganized
tumor tissues and leaky
is disorganized and
with
leakyimproper lymphatic
with improper drainage
lymphatic and inadequate
drainage and inadequatetransport phenomena
transport mechanisms
phenomena mecha-
(Figure 3) [37]. 3)
nisms (Figure Consequently, the tumor
[37]. Consequently, site suffers
the tumor from abnormal
site suffers from abnormal molecular and fluid
molecular and
dynamics. These features allow the NPs to extravasate into the
fluid dynamics. These features allow the NPs to extravasate into the tumor’s interstitium tumor’s interstitium [38]
and
[38] accumulate
and accumulate insideinside
the diseased tissues,
the diseased in a phenomenon
tissues, in a phenomenon referred to as the
referred to enhanced
as the en-
permeability and retention
hanced permeability (EPR) effect.
and retention (EPR) effect.

Figure3.3. (A)
Figure (A)aanormal
normalvascular
vascular network
network where
where the
the vessels
vessels are
are parallel-aligned
parallel-aligned next
next to
to each
each other;
other;
(B) a tumoral vasculature with chaotic defective arrangement. Adapted with permission from [37],
(B) a tumoral vasculature with chaotic defective arrangement. Adapted with permission from [37],
Oxford University Press, 2006.
Oxford University Press, 2006.
Yet,designing
Yet, designingDDS DDSwith
withcomplete
completedependence
dependence on onpassive
passivetargeting
targeting has
hassignificant
significant
limitations, such
limitations, such as
as the
the possible
possible accumulation
accumulation of of the
the NPs
NPs in in the
thespleen
spleenand
andliver
liverasasthese
these
organs have fenestrated vasculature as well as the inability of the NPs
organs have fenestrated vasculature as well as the inability of the NPs to sufficiently to sufficiently pen-
etrate deep enough through the complex tumoral network due to
penetrate deep enough through the complex tumoral network due to heterogeneities inheterogeneities in struc-
ture [39].[39].
structure Thus, the development
Thus, the developmentof systems thatthat
of systems incorporate
incorporate bothboth
passive andand
passive active tar-
active
geting mechanisms became imperative. Since tumor cells overexpress
targeting mechanisms became imperative. Since tumor cells overexpress receptors that receptors that par-
ticipate in in
participate growth
growth and survival
and survivalpathways,
pathways,suchsuchreceptors
receptorsmakemakepromising
promising active
active targets.
targets.
To this end, nanocarriers could be conjugated to natural ligands to target
To this end, nanocarriers could be conjugated to natural ligands to target these receptors these receptors
leadingto
leading totheir
theiraccumulation
accumulationand andinternalization
internalizationby bythe
thecancer
cancercells
cells[40].
[40].
This review will discuss three types of nanocarriers: liposomes,
This review will discuss three types of nanocarriers: liposomes, micelles, micelles, and
and metal-
metal-
organic frameworks (MOFs) delivering the antineoplastic agent
organic frameworks (MOFs) delivering the antineoplastic agent DOX to treat differentDOX to treat different
types of solid cancers, including their DOX loading techniques, encapsulation efficiency,
and their ability to eradicate tumors.
Pharmaceutics 2022, 14, x 5 of 32

Pharmaceutics 2022, 14, 254 5 of 29

types of solid cancers, including their DOX loading techniques, encapsulation efficiency,
and their ability to eradicate tumors.

3.
3. DOX
DOX Delivery
Delivery Systems
Systems Based
Based on
on Liposomes
Liposomes
Bangham
Bangham and and co-workers
co-workers usedused multilamellar
multilamellar bilayer lipid microspheres and lipo-
somes
somes asas models
models of of biological
biological membranes
membranes for for basic
basic research
research inin the
the 1960s
1960s[41,42].
[41,42]. ItIt was
was
Gregoriadis’
Gregoriadis’ group
group that
that first
first used
used liposomes
liposomes as as drug
drug carriers
carriers in
in the
the 1970s
1970s [43].
[43]. Later,
Later, thethe
multilamellar
multilamellaronion-like
onion-like microspheres
microsphereswere weredeveloped
developedinto into submicron
submicron dimension
dimension bilayer
bilayer
lipid
lipidvesicles
vesicles(Figure
(Figure4).4).
TheThehydrophilic heads
hydrophilic of the
heads of phospholipids
the phospholipids are directed outwards,
are directed out-
whereas the hydrophobic tails are directed inwards. Cholesterol is usually
wards, whereas the hydrophobic tails are directed inwards. Cholesterol is usually added added to the
phospholipids to increase
to the phospholipids the mechanical
to increase rigidityrigidity
the mechanical of liposomes [44,45].[44,45].
of liposomes Liposomes
Liposomeshave
gained immense
have gained interestinterest
immense as drugascarriers becausebecause
drug carriers of their biocompatible, biodegradable,
of their biocompatible, biode-
non-toxic,
gradable, and targetable
non-toxic, andnature. Moreover,
targetable [Link] deliver athey
Moreover, highdeliver
drug-to-lipid
a high ratio and can
drug-to-lipid
also
ratiobe functionalized
and to avoid rapidto
can also be functionalized elimination
avoid rapid from the body from
elimination [46]. the body [46].

Figure4.
Figure 4. Structure
Structureof
ofliposomes.
liposomes. Reproduced
Reproducedfrom
from[46],
[46],IntechOpen,
IntechOpen,2014.
2014.

For the
For the encapsulation
encapsulation of of lipophilic
lipophilic DOX
DOX into
into liposomes,
liposomes, two loading routes (i.e., pas-
sive and
sive and active)
active) are
aregenerally
generally usedused (Figure
(Figure 5).
5). One
One ofof the
themost
mostcommon
commonpassivepassiveloading
loading
methods is
methods is based
based onon the
the thin-film
thin-film hydration
hydration method,
method, where
where thethe drug
drug solution
solution isis added
added
during
during thethe liposome
liposome formation
formation process,
process, either
either while
while the
the film
film isis being
being formed
formed or or during
during
the
the hydration step. step. ToToenhance
enhancetrapping
trappingefficiency,
efficiency,thethe drug
drug is typically
is typically added
added during
during the
the
filmfilm formation
formation of of liposomescontaining
liposomes containingnegatively
negativelycharged
charged(acidic)
(acidic) lipids [44,45].
[44,45]. The
The
dried
dried lipids are initially
initiallyrandomly
randomlyorientated,
orientated,butbut upon
upon exposure
exposure to an
to an aqueous
aqueous solu-
solution,
tion, the water causes the molecules to self-organize into a bilayer
the water causes the molecules to self-organize into a bilayer configuration to minimize configuration to min-
imize
entropicentropic interactions.
interactions. In the In the presence
presence of hydrophilic
of hydrophilic and/or and/or
hydrophobichydrophobic
drugs indrugs
solu-
in solution,
tion, some ofsome of themolecules
the drug drug molecules would passively
would passively partitionpartition and accumulate
and accumulate in the cores in
the cores
and/or theand/or
shells the
of theshells of the liposomes.
liposomes. Although thisAlthough
method this
is method
simple, itis yields
simple, it yields
very small
very
drugsmall drug encapsulation
encapsulation [Link].
In 1983, DOXInwas 1983, DOX was
passively passively
entrapped entrapped
in the bi-layerin of
the bi-layer of positively (phosphatidylcholine-cholesterol-stearyl
positively (phosphatidylcholine-cholesterol-stearyl amine) and negatively (phosphatidyl-amine) and negatively
(phosphatidylcholine-cholesterol-phosphatidylserine)
choline-cholesterol-phosphatidylserine) charged liposomes. charged Theliposomes.
drug wasThe drugtowas
added the
added to the phospholipids before film formation [47]. One day
phospholipids before film formation [47]. One day after DOX loading, the mean diametersafter DOX loading, the
mean diameters
of positive of positive
and negative and negative
non-filtered non-filtered
multilamellar multilamellar
liposomes liposomes
were between were nm
500-600 be-
tween 500-600 nm and 1 µm, respectively. The maximum loading
and 1 μm, respectively. The maximum loading capacities ranged between 60-75 mmol capacities ranged be-
tween 60-75 mmol DOX/mol phospholipid for the negative and
DOX/mol phospholipid for the negative and approximately 55 mmol DOX/mol phospho- approximately 55 mmol
DOX/mol
lipid for the phospholipid for the positive,
positive, non-filtered, non-filtered,
non-sonicated non-sonicated
liposomes [47]. liposomes [47].
On the other hand, the size stability and release profiles were determined over an
extended period. The filtered and sonicated/centrifuged negatively-charged liposomes
were stable during the entire storage period and maintained average diameters of 270 and
120 nm, respectively. However, the sonicated/centrifuged positively-charged liposomes
were not stable and had a size of 120 nm with a standard deviation of 40 nm over 2 weeks of
storage. The negative filtered liposomes released only 10% of DOX during 75 days, whereas
40% was released over 20 days from the negative sonicated/centrifuged liposomes. For the
Pharmaceutics 2022, 14, 254 6 of 29

Pharmaceutics 2022, 14, x release from positive sonicated/centrifuged liposomes, the baseline was set after 126 days,
of 32
and the cumulative release was ~30% after 48 days [47].

Figure
Figure 5.
5. Two
Two approaches
approaches for
for DOX
DOX loading
loading into
into liposomes:
liposomes: passive
passiveand
andactive.
active. (A) before “pasive”
and (B) before “active”.
On the other hand, the size stability and release profiles were determined over an
extended
Otherperiod.
passiveThe filtered
loading and sonicated/centrifuged
methods negatively-charged
include the freezing-and-thawing techniqueliposomes
(FAT).
were stable during the entire storage period and maintained
The liposomal solution undergoes a series of freezing and thawing processes, causing average diameters of 270 andthe
120 nm, respectively.
interlayer distance within However, the sonicated/centrifuged
the liposomes to increase [48]. As positively-charged
a result, ice crystals liposomes
cause
were not holes
transient stableandandpores
had atosize formof within
120 nmthe with a standard
liposomal deviation
structures, of 40 nm
allowing forover 2 weeks
passive drug
of storage. The
entrapment. negative
However, thisfiltered
methodliposomes
yields 5–20% released
loading only 10% ofand
efficiency DOX during
requires 75 days,
heavy post-
whereas
processing 40% was released
to remove the excessoverdrug.20 days from the reverse-evaporation
Likewise, negative sonicated/centrifuged
method can lipo- load
drugs with
somes. efficiencies
For the release fromup topositive
50%. Still, the formulations lackliposomes,
sonicated/centrifuged reliable inthe vivo drug retention
baseline was set
and often
after suffer
12 days, andfrom
therapid cargo release
cumulative releaseunder
was ~30%physiological conditions.
after 48 days [47]. Thus, occurrences
of “burst
Otherrelease”,
passive where
loading a large
methods fraction
includeof thetheentrapped drug is abruptly
freezing-and-thawing released
technique due
(FAT).
to poor
The association
liposomal solutionandundergoes
retention in the vesicles,
a series combined
of freezing with the
and thawing relatively
processes, low drug-
causing the
to-lipid entrapment
interlayer distance withinratio, typically
the liposomes do not toexceed
increase 0.05%
[48].(w/w), making
As a result, iceit crystals
imperative causeto
divert to other
transient holes loading
and pores techniques
to form [48].within Moreover, the high
the liposomal variability
structures, and dependence
allowing for passive of
the encapsulation efficiency on operational factors like
drug entrapment. However, this method yields 5–20% loading efficiency and requiresthe drug’s solubility, nanocarriers’
chemistry,
heavy size, and preparation
post-processing to remove method deemed
the excess active
drug. loading methods
Likewise, more preferable
the reverse-evaporation
and more commonly used for the stable entrapment of DOX into
method can load drugs with efficiencies up to 50%. Still, the formulations lack reliable in liposomes.
vivo Ondrug the other hand,
retention and active methods,
often suffer fromalso rapidknown
cargoasrelease
remoteunderloading methods, employ
physiological condi-
pH gradients between the internal acidic core buffer of blank
tions. Thus, occurrences of “burst release”, where a large fraction of the entrapped drug liposomes and the external
buffer
is containing
abruptly released DOXdueat toneutral physiological
poor association conditions.
and retention Under
in the such combined
vesicles, conditions,with the
drug molecules incubated with the formed liposomes can
the relatively low drug-to-lipid entrapment ratio, typically do not exceed 0.05% (w/w), permeate selectively through
the lipids
making transmembrane,
it imperative to divert where theyloading
to other becometechniques
protonated intra-vesicularly
[48]. Moreover, the high [45]. varia-
Work
by Bally and colleagues [49] investigated the effects of creating
bility and dependence of the encapsulation efficiency on operational factors like the drug’s a membrane potential by
altering the pH of the intraliposomal solution from a 300
solubility, nanocarriers’ chemistry, size, and preparation method deemed active loadingmM citrate buffer at pH 4.0 to
a HEPESmore
methods buffer at pH 7.4and
preferable on the
more entrapment
commonlyof DOX
used forand biogenic
the stable amines inofliposomes.
entrapment DOX into
High encapsulation
liposomes. efficiencies of up to 98% were achieved, with a drug-to-lipid ratio of
0.3 (w/w%) corresponding to 400 nmol DOX/µmol phospholipid (~260 mM internal con-
On the other hand, active methods, also known as remote loading methods, employ
centration). Also, retention times (T50 ) significantly increased from 1 h to 30 h in passively
pH gradients between the internal acidic core buffer of blank liposomes and the external
versus actively DOX-loaded formulations, respectively. The proposed “citrate” method
buffer containing DOX at neutral physiological conditions. Under such conditions, the
resulted in highly stable liposomes with remarkably enhanced drug retention and release
drug molecules incubated with the formed liposomes can permeate selectively through
characteristics, with the added benefit of cost-effectiveness. A commercially available
the lipids transmembrane, where they become protonated intra-vesicularly [45]. Work by
formulation of liposomal DOX, Myocet® , is loaded using the same approach (Figure 6).
Bally and colleagues [49] investigated the effects of creating a membrane potential by al-
The Myocet® liposomes (~150 nm) are made from egg phosphatidylcholine and cholesterol
tering the pH of the intraliposomal solution from a 300 mM citrate buffer at pH 4.0 to a
HEPES buffer at pH 7.4 on the entrapment of DOX and biogenic amines in liposomes.
High encapsulation efficiencies of up to 98% were achieved, with a drug-to-lipid ratio of
 0.3 (w/w%) corresponding to 400 nmol DOX/μmol phospholipid (~260 mM internal con-
centration). Also, retention times (T50) significantly increased from 1 h to 30 h in passively
versus actively DOX-loaded formulations, respectively. The proposed “citrate” method
resulted in highly stable liposomes with remarkably enhanced drug retention and release
Pharmaceutics 2022, 14, 254 characteristics, with the added benefit of cost-effectiveness. A commercially available7 of for-29
mulation of liposomal DOX, Myocet , is loaded using the same approach (Figure 6). The
®

Myocet® liposomes (~150 nm) are made from egg phosphatidylcholine and cholesterol at
a ratio
at of of
a ratio 55/45
55/45 (mole/mole
(mole/mole %). As
%).aAs
result of the
a result of pH
the gradient,
pH gradient, DOXDOX molecules diffuse
molecules and
diffuse
accumulate
and accumulate in the inliposomal corescores
the liposomal and form a complex
and form a complexwith with
the citrate anions.
the citrate The drug
anions. The
entrapment
drug entrapmentefficiencies (>95%)
efficiencies and and
(>95%) drug-to-lipid
drug-to-lipid ratioratio
(~0.27) exceeded
(~0.27) exceeded thethetheoretical
theoreti-
predictions
cal predictions as the bundled
as the bundledfibers complexes
fibers complexesallowed
allowed higher DOX
higher DOXloading beyond
loading beyondits so-
its
lution solubility
solution solubility limits.
[Link]
TheDOX-citrate
DOX-citratecomplexes
complexesresembled
resembledthe theappearance
appearanceof of coffee-
coffee-
bean liposomes, where the linear bundles prevented prevented drug drug leakage
leakage andand premature
premature release.
release.
The formulation also exhibited prolonged circulation times and improved in vivo tissue
distribution compared to free DOX [50]. According According to Li et al. [51], 99% of DOX loaded
into citrate
citrate buffered
buffered liposomes
liposomesappears
appearsininthe
theform
formofof fiber
fiber aggregates.
aggregates. TheThe threshold
threshold for
for forming
forming these these fibrous-like
fibrous-like DOX-citrate
DOX-citrate structures
structures can be canasbelowas as
low~20asmM~20 internal
mM internal
DOX
DOX concentration.
concentration. As the AsDOX
the DOX concentration
concentration increases,
increases, the fibers
the fibers beginbegin cross-linking
cross-linking via
via cit-
citrates and then pack into bundles. Kanter and co-workers [52]
rates and then pack into bundles. Kanter and co-workers [52] demonstrated that the My- demonstrated that the
Myocet liposomal
ocet liposomal formulation
formulation effectively
effectively reducedthe
reduced theeffects
effectsofofmyocardial
myocardial degeneration,
degeneration,
commonly
commonly associated
associatedwith withanthracycline-based
anthracycline-basedtreatments.
[Link]
Histological analysis
analysis ononbeagle
bea-
dogs treated
gle dogs withwith
treated conventional DOXDOX
conventional versus thosethose
versus treated with with
treated Myocet (single
Myocet i.v. adminis-
(single i.v. ad-
tration every every
ministration 3 weeks; 8 cycles;
3 weeks; cumulative
8 cycles; dose =dose
cumulative 12 mg/kg) showed
= 12 mg/kg) that the
showed latter
that thegroup
latter
did not suffer from any anthracycline-induced cardiotoxicity, while
group did not suffer from any anthracycline-induced cardiotoxicity, while the first group the first group had
lesions of myocardial degeneration.
had lesions of myocardial degeneration.

Figure 6. (A) DOX loading via citrate transmembrane gradient method, (B,C) confocal images of
Figure 6. (A) DOX loading via citrate transmembrane gradient method, (B,C) confocal images of DOX-
DOX-loaded liposomes showing the coffee-beans liposomes appearance where DOX-citrate com-
loaded liposomes showing the coffee-beans liposomes appearance where DOX-citrate complexes
plexes appear in rod, circular and U-shaped structures. A,B are adapted with permission from [50],
appear in2001.
Elsevier, rod, circular and U-shaped
C is adapted structures.
with permission fromA,B are Elsevier,
[51], adapted with
[Link] from [50], Elsevier,
2001. C is adapted with permission from [51], Elsevier, 1998.
Along the same lines, Haran et al. [53] generated a pH gradient across the liposomes
Along the same lines, Haran et al. [53] generated a pH gradient across the liposomes
using ammonium sulfate salt. The liposomes initially encapsulated a 300 mM ammonium
using ammonium sulfate salt. The liposomes initially encapsulated a 300 mM ammonium
sulfate solution at pH 5.5 in a pH 7.4 external buffer. The higher concentration of ammo-
sulfate solution at pH 5.5 in a pH 7.4 external buffer. The higher concentration of am-
nium ions on the inside led to the diffusion of the neutral ammonia molecules, and with
monium ions on the inside led to the diffusion of the neutral ammonia molecules, and
every molecule diffusing from the core, a proton was left behind. Due to salting-out effects
with every molecule diffusing from the core, a proton was left behind. Due to salting-out
and the acidification of the intraliposomal compartment, high fractions of DOX can accu-
effects and the acidification of the intraliposomal compartment, high fractions of DOX
mulate in the liposomes in an aggregated form. Liposomes loaded using this technique
can accumulate in the liposomes in an aggregated form. Liposomes loaded using this
exhibit a prolonged
technique stable storage
exhibit a prolonged period
stable beyond
storage period6 months
beyondbecause
6 monthsofbecause
the gelation
of theeffects
gela-
of DOX with the sulfate salt, which inhibit membrane re-permeation. Alyane
tion effects of DOX with the sulfate salt, which inhibit membrane re-permeation. Alyane et al. [54]
examined the stability and release behavior of liposomes loaded with DOX via the
et al. [54] examined the stability and release behavior of liposomes loaded with DOX via ammo-
nium
the sulfate transmembrane
ammonium method. Liposomes
sulfate transmembrane comprisedcomprised
method. Liposomes of hydrogenated egg yolk
of hydrogenated
phosphatidylcholine
egg (HEPS), cholesterol,
yolk phosphatidylcholine and DSPE-PEG2000
(HEPS), cholesterol, at a molar
and DSPE-PEG2000 at ratio of 185:1:15
a molar ratio of
showed an encapsulation efficiency exceeding 90%, with a drug-to-lipid
185:1:15 showed an encapsulation efficiency exceeding 90%, with a drug-to-lipid ratioratio of 1:20
of
◦
1:20 (w/w). Upon incubation at 37 C for 24 h in culture media and PBS, the liposomes
retained 98% and 90% of the encapsulated drug, respectively, showing minimal leakage
under the stated conditions. When the incubation medium was changed to phosphate
buffer at pH 5.3, the maximum release achieved was 37%. The pH responsiveness of the
liposomes suggested their suitability for controlled release in acidic compartments (i.e.,
tumor tissues) as they would keep their cargo intact under physiological conditions. The
Pharmaceutics 2022, 14, 254 8 of 29

study also revealed that encapsulating DOX in the liposomes decreased the agent’s uptake
by rat myocardial H9C2 cells, suggesting the decreased cardiac toxicity of the formulation.
Flow cytometry and MTT assay analysis showed that the toxic effects of DOX were reduced
when compared against that of free DOX, up to 20 h of incubation time. After 20 h, the
liposomal formulation became more toxic to the cells than the free one [54].
DOXIL® is a commercially available PEGylated liposomal DOX formulation that
uses the ammonium sulfate gradient method to encapsulate the drug. An in vivo study
by Sakakibara et al. [55] investigated the performance of DOXIL® , conventional DOX-
liposomes, and free DOX on the treatment of human lung tumors. The DOXIL® liposomes
were composed of hydrogenated soy phosphatidylcholine, cholesterol, PEG-DSPE, and dl-
a-tocopherol in a molar ratio of 56.1:38.2:5.5:0.2. In contrast, the non-PEGylated liposomes
had phosphatidylglycerol, phosphatidylcholine, cholesterol, and DL- α -tocopherol in a
molar ratio of 1:4:3:0.02. To assess and compare the different treatment groups (dosage:
1.5 mg/kg) of anti-tumor activity, human lung tumor xenografts were engrafted into the
gonadal fat pad of severe combined immunodeficient (SCID) mice. The tumor eventually
metastasized from the primary site into the mice’s peritoneal cavity (i.e., liver, lung). It
was concluded that the PEGylated formulation successfully suppressed the primary tumor
growth and arrested metastasis in the peritoneal cavity, while the free DOX was only able to
stop the growth of the primary tumor without significant effects on preventing the spread.
The PEGylated liposomes showed 5-folds higher circulation times than conventional lipo-
somes, while the uptake by vital organs like the spleen was reduced. Also, a significant
increase in extravasation and accumulation of the PEGylated formulation was observed,
as considerable amounts were detectable at the tumors after 1 week following adminis-
tration [55]. Another study [56] compared the in vivo pharmacokinetic performance of
free DOX with PEGylated and non-PEGylated liposomal formulations. Interestingly, the
PEGylated liposomal DOX clearance rate decreased by 100-folds (Cl = 0.023 L/h), and its
half-life (t1/2 = 83.7 h) was prolonged by 8-folds, compared to free DOX (Cl = 25.3 L/h,
t1/2 = 10.4 h). Moreover, the distribution volume decreased significantly from 364 L to
139 L to 3.0 L in the free DOX, non-PEGylated, and PEGylated liposomal DOX, respectively.
This conclusion demonstrated that PEGylation prevents premature drug release and that
most of it remains entrapped without leakage.
Fritze et al. [57] introduced another DOX remote loading method based on a phosphate
((NH4 )2 HPO4 ) transmembrane gradient, using different ammonium and sodium salts. The
liposomes were composed of egg phosphatidylcholine (EPC) and cholesterol in the molar
ratio 7:3. After hydrating the liposomes with 300 mM salt solutions at neutral pH, they
were incubated with [Link] for 12 h at 7 ◦ C to achieve a drug-to-lipids ratio of 1:3
(mol/mol). The sizes of liposomes containing DOX dissolved in different ammonium and
sodium salts, as well as their encapsulation efficiencies (EE%), are summarized in Table 1.
Results showed that loading did not significantly alter the size of the liposomes, and
those loaded with the ammonium salts gradients showed higher EE than the sodium salts.
Synergistic effects are suggested when using ammonium salts as they act as a reservoir for
donating free protons when DOX is internalized and protonated in the acidic interior of
the liposomes. Thus, intraliposomal protonation and DOX precipitation led to increased
encapsulation efficiencies. Further analysis was conducted to assess the impact of varying
the intravesicular ammonium concentration on liposome size and EE. DOX was dissolved
in 10 mM isotonic HEPES buffered saline (HBS), 50, 100, 200, and 300 mM ammonium
phosphate at pH 7.2. The sizes of the DOX-loaded liposomes were found to be 84 ± 0.4,
102 ± 1.4, 114 ± 3.6, 95 ± 1.5, and 92 ± 1.6 nm, respectively, with EE of 2.81, 23.52, 61.03,
83.35, 97.98%. Increasing the ammonium ion concentration augmented the effects of drug
protonation and intravesicular acidification, which led to increased DOX diffusion across
the transmembrane gradient. The least efficiency (<5%) was observed when incubating in
HBS, which contains no phosphate ions (no decrease in the intraliposomal pH level), while
the highest efficiency approaching 100% was observed at the highest concentration of the
ammonium phosphate (300 mM).
Pharmaceutics 2022, 14, 254 9 of 29

Table 1. Summary of the size and encapsulation efficiency of loaded liposomes achieved via differ-
ent salts.

Salt Gradient Size ± SD (nm) EE (%)

Ammonium Phosphate 129.3 ± 3.7 98
Ammonium Sulfate 129.2 ± 2.9 95
Ammonium Acetate 115.9 ± 1.0 77
Ammonium Citrate 114.9 ± 1.2 100
Sodium Phosphate 113.4 ± 1.6 52
Sodium Sulfate 111.8 ± 1.9 44
Sodium Acetate 113.4 ± 1.6 16
Sodium Citrate 151.7 ± 3.8 54

Another study [58] investigated the effect of varying the bilayer composition of DOX
entrapment and release efficiency. Three liposomal formulations were tested, which in-
cluded non-thermosensitive (NTS) liposomes composed of L-α-phosphatidylcholine (PC),
thermosensitive (TS) liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and
distearoylphosphatidylcholine (DSPC), lyso-thermosensitive (LTS) liposomes composed
of DPPC, DSPC, and 1-palmitoyl-2-lyso-glycero-3-phosphocholine (P-lyso-PC). All three
formulations contained small amounts of cholesterol. The encapsulation efficiency of
DOX dissolved in 0.9% NaCl solution was very low for TS and LTS liposomes (5.8 and
5.6%), while it was slightly higher (17.29%) for NTS liposomes because of the possibility
of controlling the temperature effectively during the loading (above the phase transition
temperature). In vitro drug release was modeled at controlled hyperthermia conditions
(41–42 ◦ C), and results showed that for a period of 5 h, 2%, 36%, and 54% DOX diffused
from NTS, TS, and LTS liposomes. The relatively lower encapsulation efficiency and higher
release performance of LTSL resort to the temperature-induced membrane instability when
operating at temperatures beyond the lipids transition temperature [58]. Generally, drug
loading via active loading depends on several factors besides the drug’s physiochemical
properties, like the extraliposomal medium condition, pH, conductivity, electrolytic activity,
loading duration, as well as operating temperatures [44].
Research efforts have also focused on the synergistic effects of using release trigger-
ing modalities, like ultrasound and pH, on the internalization and efficacy of DOX. Pitt
et al. [59] studied the in vivo performance of ultrasonically-triggered DOX-loaded lipo-
somes on BDIX rats bearing colonic carcinoma. The liposomes were comprised of soy
phosphatidyl choline, cholesterol, DSPE-PEG, and alpha-tocopherol combined in the molar
ratio 3:1:1:0.004, and DOX was entrapped via the ammonium sulfate gradient method.
Using sonication remarkably enhanced the DOX release kinetics and effects on mice. The
group treated with liposomes followed by ultrasound exposure (20 kHz for 15 min, once
for 4 weeks) showed significant regression in tumors growth to an immeasurable size by
the end of the treatment period. Likewise, another in vivo study [60] on mice bearing SCC7
murine squamous carcinoma cells showed that sonication using pulsed high-frequency
US (HFUS) enhanced the performance of the proposed liposomal drug delivery system.
Fluorescent spectrophotometry results proved that the mean DOX concentration in the son-
icated tumors was 124% more than in the control, which received the liposomal treatment
but without sonication. Work by Zhang et al. [61] synthesized pH-sensitive liposomes by
modifying their surfaces via the insertion of poly(2-ethyl-2-oxazoline)-cholesteryl methyl
carbonate (PEOZ-CHMC) copolymer. The performance of these liposomes was compared
to others grafted with PEG-DSPE chains. DOX hydrochloride was loaded into both types of
liposomes by the ammonium sulfate transmembrane gradient method. It was revealed that
functionalization with PEOZ-CHMC and PEG-DSPE had no effects either on the drug’s
encapsulation efficiency, which was 97.3 ± 1.4, or on the size, which was ~120 nm. In vitro
release done via dialysis showed that the release profile from PEOZ-CHMC-DOX lipo-
somes in PBS at pH 5.0 significantly surpassed that observed at pH 7.4, suggesting a strong
relationship between medium acidity and cargo release from the modified liposomes. More-
Pharmaceutics 2022, 14, 254 10 of 29

over, MTT assay results proved a direct relationship between the antiproliferative effects
of the PEOZ-CHMC-DOX liposomes and pH conditions. The pH-sensitive formulation
showed higher activity inhibition to the MCF-7 cells at lower pH; herein, pH can be used
as a triggering mechanism for drug release.
As mentioned, scheming highly efficient liposomal drug delivery systems with com-
plete dependence on passive targeting is hampered by limitations like the possible ac-
cumulation of the nanocarriers in the spleen and liver as these organs have fenestrated
vasculature and their incapability to sufficiently penetrate deep enough through the com-
plex tumoral network due to heterogeneities in structure [62]. Benefiting from the tumor
cells’ overexpression of receptors to aid in augmenting their growth and survival pathways,
such receptors make promising active targets. To this end, nanocarriers can be conjugated
to the ligands complementing these receptors to enhance DOX accumulation and internal-
ization by the cancerous cells. Xing and co-workers [63] examined the in vitro effects of
functionalizing DOX-loaded PEGylated liposomes with a DNA aptamer (Apt-Urn lipo-
somes) on MCF-7 cells. Flow cytometry results showed a 6.6-fold increase in drug uptake
and efficacy of the functionalized loaded liposomes as opposed to the conventional ones, as
the fluorescence response upon 4 h of incubation with the treatments corresponded to 93.6%
and 57.0%, in the cells treated with Apt-Urn and control liposomes, respectively. Moreover,
MTT assay results analyzed the cytotoxic effects of the Apt-Urn liposomes on cell viability.
The cells were treated with different liposomal concentrations followed by an incubation
period of 6 h, then re-cultured in fresh media, and the MTT test was done after 72 h. At a
liposomal DOX concentration of 500 nM, cells treated with the functionalized liposomes
exhibited a viability of 57.0 ± 6%, whereas cells treated with the control liposomes exhibited
a viability of 92.4 ± 9%, evidencing the superior localization and internalization of the
nanocarriers as a function of surface modification.
Yang [64] conducted a recent preclinical investigation on DOX-loaded liposomes
composed of EPC and cholesterol for treating head and neck cancer. The study compared
three treatment groups: free DOX, DOX-loaded liposomes, and DOX-loaded peptide-
conjugated liposomes. Results showed the dependence of the encapsulation efficiency
on the cholesterol content in the formulations, as it increased from 20% to 79% upon
the incorporation of cholesterol with EPC. Varying the EPC and cholesterol ratio yielded
different entrapment efficiencies ranging from ~48% to ~82%. Both liposomal formulations,
the functionalized and the conventional ones, showed sustained release profiles over a
period of 40 h, but the DOX-loaded peptide-conjugated liposomes had a faster in vitro
release behavior. In vivo analysis of the formulations’ efficacy was carried out on nude mice
bearing HSCC (human squamous cell carcinomas) xenograft. The tumor progression was
suppressed when treated with both liposomal formulations; however, the functionalized
liposomes arrested metastasis and increased the median survival time of the animals in that
group by 100%. The enhanced performance was due to the increased accumulation at the
tumor site, resorted to localized targeting by binding to the overexpressed surface-specific
receptors (Hsp47/CBP2) abundant on the head and neck cancer cells.
Another study [65] examined the performance of DOX-loaded liposomes, function-
alized with the monoclonal antibody Trastuzumab (TRA) for the enhanced targeting of
breast cancer cells overexpressing the Human Epidermal growth factor Receptor 2 (HER2).
The liposomes consisted of cholesterol, DPPC, and DSPE-PEG(2000)-NH2 at a molar ra-
tio of 30:65:5, respectively. DOX was loaded via the ammonium sulfate transmembrane
gradient method, yielding a drug-to-lipids ratio of 1:6. The cargo release from the li-
posomal formulations, conventional and TRA-conjugated, was triggered using pulsed
ultrasound. In vitro results showed synergistic effects on the DOX uptake and internal-
ization upon sonication and surface modification with TRA. Similarly, Chowdhury and
co-workers [66] designed an in vitro study where they tested different targeted (function-
alized with Aptamer-A6) and untargeted liposomal-DOX formulations for the localized
treatment of HER2-overexpressing breast cancers. Twelve liposomal formulations (F1-F12)
were prepared using the thin-film hydration method by varying the compositions of the
Pharmaceutics 2022, 14, 254 11 of 29

phospholipid mixtures. The liposome sizes slightly increased upon DOX loading and
ranged from 98.7 to 181.2 nm, with 74.9 to 94.1% entrapment efficiencies. The optimized
formulations for further complexation with Aptamer-A6 were determined based on the
smallest size to benefit from the EPR effect and the highest encapsulation efficiency. Formu-
lations 5 and 8 (10 mg, 150 mg, 40 mg, 40 mg, 20 mg, and 0.25 mg of DOX, POPC, DOTAP,
DOPE, DSPE-mPEG2000, and Mal-PEG, respectively) had sizes of 101.70 ± 14.04 nm and
98.7 ± 13.25 nm, respectively, and DOX entrapment efficiency of 92.8% and 94.1%, respec-
tively. These results suggest the statistical insignificance of the hydrophobicity and charge
(i.e., cationic) of the used phospholipids on the encapsulation efficiencies. However, the
presence of PEG chains and the charge of the liposomal complexes directly affect their
stability and integrity in long-term storage. Formulations with cationic lipids exhibited a
relatively smaller size and extended shelf-life lasting up to 10 weeks, and PEG prevented
liposomes from cross-linking and aggregating. Integrating Mal-PEG into the formulations
aided in linking the Aptamer-A6 at its amino-terminal. In vitro analysis was done on
SKBR3 and MCF7 cells, which overexpress HER2 on their surfaces. It was observed that
F5 was internalized the fastest and the most by both cell lines, as flow cytometry uptake
results showed the fluorescence intensities upon 2-h incubation to be 98.6% and 66.5%,
respectively. SKBR3 cells uptake of F5 compared to MDA-MB-231 cells (HER2 negative cell
line) was more by 1.79 times, substantiating the merit of targeted therapy. Table 2 presents
some studies involving liposomal DOX.
Pharmaceutics 2022, 14, 254 12 of 29

Table 2. Different liposomal-based DDS encapsulating DOX via the (NH4 )2 SO4 transmembrane gradient method.

Preparation
Method Target Cancer Functionalization Study Model Triggering Modality Findings Ref.

In vitro flow cytometry and Loaded decorated liposomes size~110 nm.

MTT analysis on MG63 Exhibited high encapsulation efficiency.
(estrogen overexpressing) cells Redox-sensitivity and Ex vivo analysis of the functionalized liposomes
Ethanol osteosarcoma Estrogen [67]
injection and LO2 (negative liver cells). glutathione showed more selective accumulation in tumor
Ex vivo imaging of MG63 responsiveness tissues compared to other vital organs, and in vitro
tumors extracted from Male results showed higher cytotoxicity towards
BALB/c nude mice. overexpressing cells.
Liposomes decorated with cleavable PEG chains
rapidly dissociated in the plasma. The
anti-CD19 moiety; PEG In vitro MTT assay pH-sensitive liposomes, targeting the CD19
Thin-film grafted by disulfide links In vivo model: Female BALB/c pH sensitivity epitope excessively abundant on
Lymphoma Cr Alt B/M mice bearing [68]
hydration B-lymphoma cells,
(mPEG-S-S-DSPE) Namalwa cells showed increased selective cytotoxicity towards
these cells, and enhanced release kinetics at lower
pH levels.
In vitro flow cytometry and Functionalization of DOXIL liposomes
Post-insertion; MTT assay on C-26 and significantly increased their size. The IC50 values
mixing with Cancer Stem Cells anti-CD44 monoclonal NIH-3T3 (non-tumor) cells.
(CSCs) antibody (mAb) N/A were lower on the C-26 cell line overexpressing [69]
preformed In vivo model: female BALB/c
DOXIL mice bearing C-26 colon CD44, while higher values were reported for the
carcinoma. negative cell line (NIH-3T3).

In vitro flow cytometry and Functionalization of liposomes with

MTT assay on MCF7/adr and Polymethacrylate derivatives increases their
Cationic
Solvent Various cancers Polymethacrylate H22 cells. N/A cellular internalization and antitumoral activity. [70]
evaporation In vivo model: ICR mice The in vivo results showed that four injections of
Eudragit RL100
bearing aggressive liver cancer the functionalized formulation led to tumor size
H22 cells. reduction by 60%.
In vitro cytotoxicity assay.
In vitro results showed that targeting significantly
Thin-film Metastatic lung CXCR4-antagonist cyclic In vivo model: C57BL/6 mice
hydration cancer bearing B16 human N/A decreased the IC50 while reducing metastasis and [71]
peptide (peptide R)
melanoma cells regression in tumor size growth.

In vitro specific uptake of HepG2 cells showed the highest uptake towards
HepG2, MCF-7, and the liposomes functionalized with GA alone, while
glycyrrhetinic acid (GA)
hepatocellular SMMC-7721 cells MCF-7 showed the highest affinity towards the
Film dispersion and peanut N/A [72]
carcinoma (HCC) In vivo model: male PNA functionalized liposomes. The dual-targeted
agglutinin (PNA) BALB/C-nu mice bearing liposomal formulation was most internalized by
SMMC-7721 xenografts. the SMMC-7721
Pharmaceutics 2022, 14, 254 13 of 29

As many liposomal DOX formulations have successfully translated to clinical appli-

cations, and others are still in the clinical testing phase, it is essential to compare their
performance and overall pharmacovigilance against the free drug. Table 3 presents a
comparison between phase III clinical trials results of two current commercially available
liposomal DOX formulations, namely Myocet® and DOXIL® , against conventional DOX.
Clinical studies have shown that DOXIL® reduced the constraints on the dose limits of
free DOX administration, in addition to reducing the cardiotoxic and myocardial dam-
age incidences even at high doses exceeding the 500 mg/m2 threshold [73,74]. Fukuda
et al. [75] presented the first large-scale study that compared the adverse effects (AEs)
associated with treatments of conventional, PEGylated (DOXIL® , Caelyx® , and LipoDox® ),
and non-PEGylated (Myocet® ) liposomal DOX formulations, based on data collected from
7,561,254 patient reports (from 2004 to 2015) retrieved from the Food and Drug Admin-
istration Adverse Event Reporting System (FAERS). The analysis was based on the top
30 AEs correlated with conventional DOX treatments, including nausea, diarrhea, vomiting,
anemia, cardiomyopathy, and cardiotoxicity. Figure 7 summarizes the findings, which
show the reporting odds ratios (RORs) of each treatment for each AE. ROR is a pharma-
covigilance index that reflects the chances of occurrence, detection, and prevention of AEs
of drug formulations. The lower the ROR, the more patient-friendly the formulation is. As
reported, all DOX treatments have been correlated with severities of myelosuppression,
cardiotoxicity, alopecia, nausea, and vomiting; however, both liposomal DOX formulations
show relatively better safety profiles than conventional DOX.
Nonetheless, PEGylated liposomal DOX had higher ROR for palmar-planar ery-
throdysesthesia (PPE), stomatitis, and mucositis than the non-PEGylated and conventional
formulations, though a complete understanding of these AE’s pathophysiology has not
yet been fully elucidated. A meta-analysis study was previously conducted by Rafiyath
et al. [76], which covered randomized controlled trials of treatments of different tumors
with conventional and liposomal DOX. The investigation analyzed data of 2220 patients in
total, of which 1108 and 1112 were treated with conventional and liposomal (PEGylated
and non-PEGylated) DOX, respectively. The results are generally aligned, evidencing that
the liposomal formulations show better therapeutic indications, but careful consideration
should be given to treating patients suffering from PPE with PEGylated DOX. In terms
of pharmacokinetics and biodistribution performance of conventional, non-PEGylated,
and PEGylated liposomal DOX, a preclinical evaluation by Tomkinson et al. [77] reported
that the area under the plasma concentration-time curve (AUC; µg hr/mL) was 4, 45,
and 900, respectively, and the elimination half-lives (hr) were 0.2, 2.5 and 55, respectively.
Although the prolonged circulation times of PEGylated formulations are preferable in
terms of therapeutic manifestations, several clinical trials [78,79] have associated that with
increasing the risk factors of PPE. The intricacy of correlating PEGylated liposomal DOX
toxicities with dose and pharmacokinetic parameters between pre- and clinical settings
calls for population pharmacokinetics guidance tools [77] to improve patient outcomes and
the overall quality, safety, and efficacy of the liposomal treatments [80].
Pharmaceutics 2022, 14, 254 14 of 29
Pharmaceutics 2022, 14, x 15 of 32

Figure 7. Summary of ROR of conventional, PEGylated, and non-PEGylated liposomal DOX in the
Figure 7. Summary of ROR of conventional, PEGylated, and non-PEGylated liposomal DOX in the
FAERS database. (High resolution image at [Link] ac-
FAERS database.
cessed (HighAdapted
on 3 Jan 2022). resolution
fromimage at [Link]
[75], PLOS, 2017.
accessed on 3 January 2022). Adapted from [75], PLOS, 2017.
Table 3. Phase III clinical trials findings of treatment with Myocet® and Doxil® against free DOX in
patients with
Table 3. Phase IIIbreast cancer
clinical [81–83].
trials findings of treatment with Myocet® and Doxil® against free DOX in
patients with breast
Therapeutic cancer
Survival Rate [81–83].
(SR) Progression-Free Survival Incidence of AEs
Formulation Phase
Indication All Presented Comparisons are Against Treatment with Free DOX
Myocet® Progression-Free
Formulation Phase TherapeuticFirst-year SR:Survival Rate (SR)4.3 vs. 3.6 months
Survival
Cardiac events: of AEs
Incidence
Indication 69% vs. 64% 13% vs. 29%
All Presented Comparisons are Against Treatment with Free DOX
Mucositis/stomatitis:
Cardiac events:
8.6% vs.13%
11.9%
vs. 29%
First-year SR: Mucositis/stomatitis:
Nausea/vomiting:
Myocet® 69% vs. 64% 4.3 vs. 3.6 months
8.6% vs. 11.9%
Metastatic 12.3% Nausea/vomiting:
vs. 20.3%
III
DOXIL® breast cancer Overall SR: 6.9 months vs. 7.8 months Cardiotoxic 12.3% vs. 20.3%
implications:
21 months vs. 22 Cardiotoxic
3.9% vs. 18.8% implications:
3.9% vs. 18.8%
III months
Metastatic breast cancer Vomiting:Vomiting:
19% vs.19%31%vs. 31%
Alopecia:
Overall SR: Alopecia:
20% vs. 66%
6.9 months vs. 7.8 Neutropenia:
DOXIL® 21 months vs. 22
months 20% vs.4% 66%
vs. 10%
months
PPE:
48% vs. 2%
Stomatitis:
22% vs. 15%
Mucositis:
23% vs. 13%
Pharmaceutics 2022, 14, 254 15 of 29

4. DOX Delivery Systems Based on Micelles

Micelles are amphiphilic colloidal block copolymers resulting from the self-assembly
of molecules. They are divided into two categories, i.e., low-molecular-weight surfactant
micelles and polymeric micelles. DOX was conjugated effectively to the terminal hydroxyl
group of PLGA fond in PLGA-b-PEG di-block copolymer micelles via a chemical reaction
and entrapped in the same micelle physically with sizes of 61.48 ± 7.17 and 58.21 ± 6.21 nm,
respectively. The loading efficiency and capacity of chemically conjugated DOX were
calculated to be 99.09 ± 1.81% and 2.18 ± 0.04, respectively. They were higher compared
to only 23.18 ± 3.18% loading efficiency and 0.51 ± 0.07% capacity for physically loaded
DOX. The release experiments were performed in phosphate-buffered saline (PBS) at 37 ◦ C.
Approximately 45% of chemically conjugated DOX was released during the first day. The
entire amount was delivered after 4 days, while physically entrapped DOX took longer to
release only 50% of DOX after 2 weeks. In vitro cytotoxicity of free DOX and DOX-PLGA-b-
PEG against HepG2 cells was examined. The loaded drug (IC50 = inhibitory concentration
to produce 50% cell death = 0.02 µg/mL) was found to be more toxic than the free drug
(IC50 = 0.3 µg/mL) [84].
A new pH-sensitive DOX-micelles delivery system was proposed by Bae and co-
workers [85]. Mixed micelles were prepared from poly(His-co-Phe (16 mole%))-b-PEG
(80 wt.%) blended with PLLA-b-PEG (20 wt.%), denoted as PHSM (20%), or folated PLLA-
b-PEG (20 wt.%), denoted as PHSM(20%)-f. The size of mixed micelles at neutral pH was
110 nm and reduced slightly at pH 6.5 and 6. On the contrary, it increased dramatically
to reach 900 nm at pH 5.5. This occurred due to the dissociation of PLLA-b-PEG from
the mixed micelles due to incompatibility with ionized poly(His-co-Phe). The drug en-
capsulation was carried out via dialysis, and the loading efficiency was 85% with 20 wt.%
drug loading content (DLC). The DOX release rate from PHSM (20%) was determined at
different pH environments under mechanical shaking (100 rev/min) at 37 ◦ C. As expected,
the slowest release was observed at neutral pH and started accelerating in acidic pH. After
10 h, approximately 25, 38, 75, and 85% were released at pH 7.4, 6.5, 6, and 5.5, respectively.
The complexity of polymerization, purification, and characterization of mixed micelles
limits their use.
In vitro cytotoxicity experiments were conducted for free DOX, folated, and non-
folated mixed micelles against ovarian A2780 wild-type cells with a drug concentration
of 1 µg/mL. The free DOX showed lower cell viability (between 17–20%) compared to
high viability percentages in the case of loading the DOX in PHSM(20%) without the folate
moiety at pH of 7.4, 7, 6.5, and 5.5, which reached up to approximately 80, 85, 75, and 30%,
respectively, whereas the same cell viability percentage of 18% was achieved at pH 5.5 of
free and loaded drug. The difference in the cell viability of folated micelles-free drug was
insignificant at all pH values. Another cell viability test was performed against a DOX-
resistant cell line (A2780/DOXR). Free DOX and DOX/m-PHSM (20%) exhibited very high
cell viability with percentages greater than 85%. Contrary to that, DOX/m-PHSM(20%)-f
showed around 15, 19, 21, 32, 50% at pH of 7.4, 7, 6.5, and 5.5, respectively. It was concluded
that conjugating active targeting folate molecules to the mixed micelles make them more
effective in killing ovarian A2780 wild-type and A2780/DOXR cell lines [85].
Hsieh et al. [86,87] reported loading neutralized DOX-HCL in triblock PEG-PCL-PEG
(EC220E) copolymers under mixing for 3 h. The empty and loaded micelles sizes were
85 ± 2 and 92 ± 7 nm, respectively. The loading content was 7.4%, with an efficiency
of 49%. An in vitro release study was performed at pH 7.4 and 5.4 at 37 ◦ C. During the
first 6 h, burst release with percentages of 20 and 25% at pH 7.4 and 5.4, respectively, was
observed (due to the diffusion of DOX located within the hydrophilic shell or near the
surface of micelles). Then, the accumulated drug delivery rate became relatively slow
to reach approximately 27% and 37% at pH 7.4 and 5.4, respectively, after 48 h. Finally,
the cytotoxicity of these treatments was evaluated using two human breast cancer cell
lines. The IC50 values of treating MCF-7 using free and encapsulated DOX were 0.031
and 0.218 µg/mL, respectively. When DOX-resistant cells (MCF-7/adr) were treated, both
Pharmaceutics 2022, 14, 254 16 of 29

free (IC50 = 4.68 µg/mL) and loaded DOX (IC50 = 5.96 µg/mL) showed somewhat similar
half-maximal inhibitory concentrations.
Moreover, DOX hydrochloride was loaded in one of the most frequently used am-
phiphilic tri-block copolymers in drug delivery applications, namely Pluronic® F127, grafted
with 5% and 10% O-succinyl chitosan [88]. The loading was carried out by mixing the drug
in 5, 7, and 10% (w/v) grafted micelles solutions at 250 rpm for 12 h in the dark. The mean
diameters of loaded micelles ranged between 34-40 nm. The encapsulation efficiencies
were calculated to be 73.69 ± 0.53 and 74.65 ± 0.44%. DOX release mechanism was studied
at 37 ◦ C in PBS at pH 7.5. During the first 24 h, burst release was observed with 39–42%
and 29–39% delivery for 5% and 10% nanoparticles. Then, the remaining amount was
released slowly to reach 85–90% and 73–86% after 22 days. From the in vitro experiments
against MCF-7 cells, it was found that the DOX loaded nanocarriers (IC50 ranged from 0.19
to 0.42 µg/mL) were more cytotoxic than Free DOX (IC50 = 0.67 µg/mL), while the empty
nanoparticles showed minimal toxic effects (IC50 ranged from 4.32 to 7.60 mg/mL) [88].
DOX was co-encapsulated with an optical fluorescence imaging agent, CdSe quan-
tum dots (QDs), in theranostic phospholipid-based micelles prepared to DSPE-PEG and
DSPC [89]. The average size of QDs-DOX micelles formulation was around 50 nm. The
release profile of 46 µg/mL loaded DOX from the QDs-DOX micelles was investigated
by incubating the QD-Dox micelles with 1% aqueous tween 80 (mimics the in vivo serum
conditions) at 37 ◦ C. Almost 50% of the DOX was released after 100 h, while the cumulative
release percentage reached 95% after seven days. Incubating free DOX and QD-Dox mi-
celles with HeLa cells for 24 h resulted in more than 65 and 50% cell survival, respectively,
at a drug concentration of 5 µg/mL [89].
Furthermore, 3-helix micelles were utilized to load DOX using the thin-film hydration
method [90]. The resulting micelles were loaded with 8 wt.% DOX and were 15 nm in size.
The release was investigated in PBS and in the presence of serum albumin (mimics the
conditions after intravenous injections) at 37 ◦ C and found to be slow, as only 11 and 12%
of DOX were recovered after 20 h, respectively. The results of in vitro cytotoxicity of free
DOX and loaded 3-helix micelles against PPC-1 (human line) and 4T1 (syngenic mouse
line) showed comparable cell viabilities. After 36 h incubation with PPC-1 at 5 µg/mL drug
concentration, both free and encapsulated DOX showed similar low cell viability. However,
the cytotoxic effect of micellar DOX was slightly higher compared to the free drug after
72 h of incubation. At low drug concentrations (1 µg/mL), free DOX was more effective
in killing the cells than micellar DOX. The incubation of 5 µg/mlL of the free drug and
DOX loaded in 3-helix micelles with 4T1 cells for 36 h resulted in low cell viability, while
all the cells were killed after 72 h of incubation. On the other hand, using the low drug
concentration of 1 µg/mL showed relatively high cell viability with more cytotoxic effects
of free DOX compared to the loaded DOX following 72 h of incubation [90].
Cross-linked (CL) and non-crosslinked (NCL) PEG-P(LL14- LA14), PEG-P(LL18-
CCA4/LA14), and PEG-P(LL18-CCA8/LA10) block micelles were used to load and release
DOX [91]. The sizes of NCL PEG-P(LL14- LA14) were 56.5, 77.6, and 87.5 nm with theoret-
ical DLC of 9.1, 16.7, and 23.1 wt.%. DOX-loaded cross-linked copolymers were slightly
smaller with sizes of 55.7, 66.9, and 78.2 nm and lower DLC of 6.6, 11.8, and 15 wt.%,
respectively. The loaded NCL PEG-P(LL18-CCA4/LA14) micelles had sizes of 44.9, 125.3,
and 199.5 nm with theoretical DLC of 9.1, 16.7, and 23.1 wt.%, respectively. These load-
ing contents were higher than those of the loaded CL micelles with sizes of 44.7, 94.2,
144.1 nm with DLC of 7.7, 13.3, and 15.9 wt.%, respectively. NCL and CL DOX-PEG-
P(LL18-CCA8/LA10) had sizes of 88.3 and 78.7 nm, respectively. The theoretical DLC of
non-crosslinked PEG-P(LL18-CCA8/LA10) was calculated to be 9.1 wt.%, while it was
6.4 wt.% for the cross-linked micelles [91].
Drug release kinetics from 20 µg/mL micelles were investigated at 37 ◦ C under shaking
(200 rpm) [91]. At neutral pH, CL PEG-P(LL14- LA14) and PEG-P(LL18-CCA4/LA14)
released only 20% of the drug in 24 h. In comparison, the corresponding NCL micelles
released 57.2 and 68.3%, respectively of the encapsulated DOX under the same conditions.
 CCA8/LA10) had sizes of 88.3 and 78.7 nm, respectively. The theoretical DLC of non-
crosslinked PEG-P(LL18-CCA8/LA10) was calculated to be 9.1 wt.%, while it was 6.4 wt.%
for the cross-linked micelles [91].
Drug release kinetics from 20 μg/mL micelles were investigated at 37 °C under shak-
Pharmaceutics 2022, 14, 254 ing (200 rpm) [91]. At neutral pH, CL PEG-P(LL14- LA14) and PEG-P(LL18-CCA4/LA14) 17 of 29
released only 20% of the drug in 24 h. In comparison, the corresponding NCL micelles
released 57.2 and 68.3%, respectively of the encapsulated DOX under the same conditions.
In an acidic environment (pH 5.0), the release from CL PEG-P(LL18-CCA4/LA14) was
In an acidic environment (pH 5.0), the release from CL PEG-P(LL18-CCA4/LA14) was
triggered to reach 30% (probably due to cleavage of the acid amide bonds between PLL
triggered to reach 30% (probably due to cleavage of the acid amide bonds between PLL
and CCA), whereas it was not affected significantly in the case of PEG-P(LL14- LA14). In
and CCA), whereas it was not affected significantly in the case of PEG-P(LL14- LA14). In
the presence of 10 mM glutathione (GSH), the release from CL PEG-P(LL18-CCA4/LA14)
the presence of 10 mM glutathione (GSH), the release from CL PEG-P(LL18-CCA4/LA14)
increased notably to reach 86.0% and 96.7% at pH 7.4 and 5.0, respectively, after 24 h. On
increased notably to reach 86.0% and 96.7% at pH 7.4 and 5.0, respectively, after 24 h. On the
the other
other hand,hand,
89.4%89.4% and 79.5%
and 79.5% of DOX ofwere
DOXreleased
were released
from CLfrom CL PEG-P(LL14-LA14)
PEG-P(LL14-LA14) copolymers co-
polymers under the same reductive conditions at pH 7.4 and 5.0,
under the same reductive conditions at pH 7.4 and 5.0, respectively. The addition of cis-1,2- respectively. The addi-
tion of cis-1,2-cyclohexanedicarboxylic
cyclohexanedicarboxylic acid (CCA) resulted acid (CCA)
in higherresulted
loadingin higher
and faster loading and After
release. faster
release. After a 48-h treatment of HeLa cells using CL PEG-P(LL18
a 48-h treatment of HeLa cells using CL PEG-P(LL18 CCA4/LA14) and CL PEG-P(LL14- CCA4/LA14) and CL
PEG-P(LL14-LA14),
LA14), the IC50 valuesthe wereIC5012.7
values
and were 12.7 andrespectively.
21.2 µg/mL, 21.2 μg/mL,Additionally,
[Link] Additionally,
incubation
the incubation of HepG2 with the same cross-linked micelles
of HepG2 with the same cross-linked micelles for 48 h resulted in 12.4 and 20.9 for 48 h resulted in 12.4
µg/mL, and
20.9 μg/mL, respectively. However, free DOX showed more effective
respectively. However, free DOX showed more effective cytotoxicity for both cell lines cytotoxicity for both
cell lines compared
compared to DOX loadedto DOX in loaded in CL[91].
CL micelles micelles [91].
Another DOX-micelles DDS
Another DOX-micelles DDS was reported bywas reported by Sui
Sui etetal.
al.[92].
[92]. They
Theyencapsulated
encapsulated the the
drug in
drug in two
two linear
linear [PEG-P(Glu)62
[PEG-P(Glu)62 and and PEG-P(Glu)62]
PEG-P(Glu)62] and and twotwo Y-shaped
Y-shaped copolymers
copolymers
[PEG-P(Glu26)2and
[PEG-P(Glu26)2 andPEG-P(Glu31)2]
PEG-P(Glu31)2]via viaananacid-labile
acid-labilehydrazone
hydrazone linker
linker using
using thethe dialy-
dialysis
sis method
method against
against PBSPBS(pH(pH 7.4,7.4,
10 10
mM) mM) andand deionized
deionized (DI)water.
(DI) [Link]
Figure88represents
representsthe the
structure of the loaded copolymers. The sizes of loaded PEG-P(Glu)26,
structure of the loaded copolymers. The sizes of loaded PEG-P(Glu)26, PEG-P(Glu)62, PEG- PEG-P(Glu)62,
PEG-P(Glu26)2,
P(Glu26)2, and PEG-P(Glu31)2
and PEG-P(Glu31)2 were measured
were measured to be ±
to be 149.9 149.9
3.7, ±231.4
3.7, 231.4
± 8.3,±141.3
8.3, 141.3
± 5.2,±
5.2, 165.6
and and 165.6
± 2.4± nm,
2.4 nm,
withwithDLC DLC of 9.92
of 9.92 ± 0.25,
± 0.25, 18.818.8 ± 0.18,
± 0.18, 16.216.2± ±0.12,
0.12,and
and18.2
18.2±± 0.45%,
0.45%,
[Link]
respectively. Therelease
releasepatterns
patternswerewereexamined
examinedat at55and
and7.47.4pHpHvalues.
[Link]
Approximately
70% of DOX was
70% wasreleased
releasedinin7272h h from
from thethe
Y-shaped
Y-shaped micelles,
micelles,whichwhich waswas higher thanthan
higher only
55% 55%
only released fromfrom
released the linear micelles.
the linear All micelles
micelles. All micellesshowedshowedsimilar release
similar at neutral
release pH,
at neutral
withwith
pH, less less
thanthan
45%45%of theof drug being
the drug released.
being To evaluate
released. the cytotoxicity
To evaluate the cytotoxicity of blank PEG-
of blank
PEG-P(Glu)26
P(Glu)26 and and PEG-P(Glu26)2,
PEG-P(Glu26)2, HeLa HeLacellscells
werewere incubated
incubated withwith the the polymeric
polymeric micelles
micelles for
for 24They
24 h. h. They
werewere not toxic
not very very attoxic at a concentration
a concentration up to 50upμg/mL.
to 50 µg/mL.
On the other On thehand,other
the
hand, the ICafter
IC50 values 50 values
72 h after 72 h incubation
incubation of DOX-conjugated
of DOX-conjugated PEG-P(Glu)26 PEG-P(Glu)26 and PEG-
and PEG-P(Glu26)2
P(Glu26)2
with HeLawith cellsHeLa
werecells were
0.063, 0.063,
0.517, and0.517, andμM,
0.673 0.673 µM, respectively.
respectively. In other In other
words,words,
PEG-
PEG-P(Glu26)2-DOX
P(Glu26)2-DOX werewere moremoretoxictoxic
thanthan PEG-P(Glu)26-DOX
PEG-P(Glu)26-DOX at theat same
the same concentrations.
concentrations. Ta-
Table 4 summarizes some preclinical and clinical studies on
ble 4 summarizes some preclinical and clinical studies on micellar-based DOX DDS, dis- micellar-based DOX DDS,
discussed
cussed in in [93].
[93].

[Link]
Figure Linear(left)
(left)and
andY-shaped
Y-shapedDOX
DOXloaded
loadedcopolymers
copolymers (right)
(right) Adapted
Adapted from
from [92].
[92]. MDPI,
MDPI, 2014.
2014.
Pharmaceutics 2022, 14, 254 18 of 29

Table 4. Summary of preclinical and clinical evaluations of some micellar-based DOX DDS. All presented comparisons are against treatment with free DOX.

Formulation Composition Features Preclinical Studies Clinical Trials

Phase I: patients with advanced solid tumors
Administered doses ranged from 5 to 90 mg/m2 , once every
3 weeks for six cycles
The maximum tolerated dose (MTD) was 70 mg/m2
The micellar formulation showed a similar toxicity profile to
In vitro free DOX
Enhanced activity against multidrug-resistant 11.5% of the patients had a partial or complete response to the
(MDR) cells micellar treatment
Pluronic® L61 Average size~30 nm Physical DOX
SP1049C In vivo The median time for disease progression: 17.5 weeks in 30.8% of
and L127 loading EE~8.2%
2-fold higher AUC (14.6 vs. 7.1 µg hr/mL) the patients
Lewis lung tumor growth in mice got Phase II: patients with advanced adenocarcinoma of the esophagus
arrested in more than 50% of 9 tumor models or gastroesophageal junction
Administered dose was 75 mg/m2 , once every 3 weeks for six cycles
Grade 3 or 4 neutropenia was observed in 62% of the patient
Median overall survival: 9.96 months
Median progression-free survival: 6.6 months
Phase III: approved
Phase I: patients with metastatic/recurrent solid tumors refractory
to conventional DOX chemotherapy
In vivo
Administered doses ranged from 6 to 67 mg/m2 , once every
29-fold higher AUC (120 vs. 4 µg hr/mL) in
Average size~40 nm 3 weeks
poly(ethylene mice bearing colon-26 carcinoma
DOX was covalently conjugated to MTD was 67 mg/m2
glycol)-b- 3.4-folds higher accumulation at the tumor
NK911 50% of the micelles’ carboxylic Grade 3 or 4 neutropenia was observed at doses of 50 mg/m2 with
poly(α,βaspartic site (1605 vs. 474 µg hr/mL)
groups as well as physically loaded AUC of 3.2 vs. 1.6 µg hr/mL
acid) effectively arrested the growth of sarcoma,
into the cores The maximum tolerated dose was 70 mg/m2/ and the
lung, colon and breast cancer in different
recommended dose for phase II trials was 50 mg/m2 to be
mouse models
administered once every 3 weeks
Phase II: currently undergoing
Average size~65 nm
In vivo
Modifications to the NK911
15-fold higher AUC (859 vs. 59 µg hr/mL) in
formulation by using pH
NC-6300 PEG-p(Asp-Hyd) mice bearing colon-26 carcinoma Phase I: -pending results
hydrolyzable linkers (hydrazone
4-folds higher accumulation at the tumor site
bonds) for the chemical conjugation
MTD (40 mg/kg vs. 10 mg/kg)
of DOX to the micelles
Pharmaceutics 2022, 14, 254 19 of 29

5. DOX Delivery Systems Based on Metal-Organic Frameworks (MOFs)

Recently, Metal-organic frameworks (MOFs) have attracted great interest among
scientists due to their unique physical and chemical properties (Figure 9). MOFs are
a new class of hybrid porous crystalline materials, known as coordination polymers,
consisting of metal clusters or metal ions connected by organic linkers to create one-, two-
or three-dimensional networks [94]. The flexible combination of organic-inorganic units
distinguishes MOFs from traditional porous materials and has enabled scientists to develop
thousands of new MOFs since its first discovery in 1989 [95,96].

Figure 9. Metal-organic frameworks structures.

Figure 9. Metal-organic frameworks structures.

For the past few decades, MOFs have been investigated and reported for several ap-
plications including, water purification [97,98], separation [99,100], gas storage [101,102],
catalysis [103,104], sensing [105,106] and energy [107,108]. On the other hand, MOFs have
gained an increasing attention in the biomedical field such as in imaging [109–111], drug de-
livery [112–115] and biological sensing [116,117] owing to their superior properties including
high surface area (i.e., ∼7000 m2 /g) [118], biocompatibility [119], wide range of pore size (i.e.,
2–50 nm) [120], tunable frameworks high porosity and low density (0.2 to 1 g/cm3 ) [121,122].
In particular, MOFs as drug delivery systems offer several advantages, for instance:
(1) high encapsulation capacity due to their ultrahigh porosity; (2) toxicity can be controlled
by choosing biocompatible metals and organic linkers [123,124]; (3) targeted delivery can be
achieved by alternating the surface structure of the MOFs through surface modification using
stimuli-responsive molecule or preforming post-synthetic surface modification [125,126].
Horcajada et al. [127] succeeded in encapsulating DOX in MIL-100(Fe) nanoparticles.
They achieved 8.5 wt.% loading capacity after 24 h of incubation, and this percentage
increased slightly to reach 9.1 wt.% when the impregnation was repeated. The release
mechanism was studied at 37 ◦ C in PBS under agitation. For 9.1 wt.% DOX payload, almost
half of the entrapped drug was released during the first day, while the remaining amount
was delivered at a very slow pace to complete after 13.5 days [127,128]. The IC50 values for
MIL-100(Fe) nanoparticles incubated with HeLa cells and J774 for 24 h were evaluated as
1100 ± 150, 700 ± 20 µg/mL, respectively [129].
Another example of the DOX-MOF delivery system was developed by Chakraborty and
co-workers using ZIF-7 and ZIF-8 [130]. DOX solution was added to the two ZIFs, and the
mixture was stirred for 48 h. The drug encapsulation percentages were 40% and 52% inside
ZIF-7 and ZIF-8, respectively. The release mechanisms of the loaded ZIFs were investigated
under different conditions. First, they were measured at different pH ranges (7.4, 6.0, and
5.0) in a time interval ranging from 0 to 2.5 h. The reason behind studying the release at acid
environment resides in the acidity of tumor microenvironments. ZIF-7 did not show any
release in the neutral and acidic environments, while ZIF-8 showed an insignificant increment
of fluorescence intensity at pH 7.4, and it started increasing to reach 4 times and 6 times above
its initial value at pH 6.0 and 5.0, respectively [130]. As expected, ZIF-8 showed an efficient
pH-sensitive DDS [131].
Pharmaceutics 2022, 14, 254 20 of 29

Additionally, the release behavior was observed by contacting the ZIFs with a biomimetic
membrane. Upon contact of ZIF-8 with 30 mM SDS micelles (above critical micelle con-
centration), the fluorescence intensity increased by 1.5 times within 1.5 h. When DMPC
liposomes were used, the release increased slightly (the fluorescence intensity increment
was only 1.1 times its initial value). In contrast, the initial intensity increased by approx-
imately 1.5 times in the presence of DMPC-DMPG (9:1) liposomes. The contact of ZIF-7
particles with SDS micelles showed almost 1.5 times increment in the fluorescence intensity
within a period of 3 h. Compared to ZIF-8, ZIF-7 showed relatively less intensity increment
when it contacted DMPC/DMPG (9:1) liposomes [130]. After a 24-h incubation of ZIF-8
particles with J774 and HeLa cells, the IC50 values for MIL-100(Fe) nanoparticles incubated
with HeLa cells and J774 for 24 h were 25 ± 1.0 and 100 ± 10 µg/mL, respectively [129].
Zheng and co-workers further proposed a novel method, called one-pot synthesis, to load
DOX in ZIF-8 during MOF preparation [132]. The encapsulation of DOX molecules was carried
out by mixing a DOX solution with the metal and organic precursors for 15 min, and the
maximum loading capacity obtained was found to be 20 wt.%. The loaded particles, DOX@ZIF-
8, did not show any release under neutral pH, while they released the drug for 7–9 days at pH
5.0-6.0 [132]. The IC50 values for MIL-100(Fe) nanoparticles incubated with HeLa cells and J774
for 24 h reached 1100 ± 150, 700 ± 20 µg/mL, respectively [129]. Mechanically downsized
gadolinium(III)-based MOF (MG-Gd-pDBI) was also used to encapsulate DOX [133]. After
stirring 0.33 mg/mL DOX solution with MOF particles for 24 h, the loading capacity was
calculated to be 5.0 wt.%, whereas it reached 12 wt.% when the DOX concentration was
increased to 2.0 mg/mL. Nearly 44% of the drug were released from 5 wt.% DOX loaded
MG-Gd-pDBI at pH 5.0 after 5 days. In contrast, the release was much slower (22%) at neutral
pH, and the entire release was achieved after 15 days. The IC50 value of 5 wt.% DOX loaded
MG-Gd-pDBI incubated with U 937 cells for 48 h was evaluated to be 75 µg/mL [133].
Recently, a unique DOX delivery system based on UMCM-1-NH–Py gated by carboxy-
latopillar[5]arene (CP5) has been designed [134]. The loading experiment was conducted
in two steps. First, UMCM-1-NH–Py particles were suspended in DOX hydrochloride-PBS
mixture for 12 h. Then, the capping agent (CP5) was added, and the whole mixture was
stirred for 48 h. At neutral pH, the loaded capped MOF did not release the DOX. However,
the release started increasing upon lowering the pH to 2.0 and reached 55% after 7.5 h.
Finally, in vitro cell viability was carried out for UMCM-1-NH-Py and CP5-capped UMCM-
1-NH-Py after incubation with normal human embryonic kidney (HEK) 293 cells for 24 h
at different concentrations. Both MOFs (capped and uncapped) showed low cytotoxicity as
the cell viabilities were still high (approximately 55% and 70% for UMCM-1-NH-Py and
CP5-capped UMCM-1-NH-Py, respectively) at a high concentration (50 µg/mL) [134].
Novel MOF-based Fe3 O4 @UiO-66 core-shell composites were used successfully to
incorporate DOX [135]. Different concentrations of DOX dissolved in PBS at pH 8.0 were
stirred with the magnetic composites for 24 h, and the drug payload was evaluated to be 2.5
and 66.3 wt.% when the amount of DOX increased from 0.15 to 15 mg, respectively. DOX
release kinetics were studied in PBS at pH 4.0, 5.0, 6.0, and 7.4 under shaking. The release
percentages at pH 4.0 and 5.0 were measured to be about 36.1% and 21.6%, respectively,
whereas only 17.1% and 13.8% of encapsulated DOX were released at the higher pH values
(6.0 and 7.4) for 41 days. In vitro experiments were conducted for Fe3 O4 @UiO-66, free and
loaded DOX on HeLa cells. The empty MOFs did not show any cytotoxicity after 24 h of
incubation, even at a high concentration (500 mg/L). On the other hand, both free and
loaded DOX killed about 60% of the cells at a low concentration (20 mg/L) [135]. Table 5
presents a few DDSs based on DOX-loaded MOFs to treat different cancers.
Even with the current research advances in MOF-based chemotherapeutic platforms,
the move from bench to bedside is hampered by several potent obstacles [136,137]. These
challenges include (1) difficulties in scaling up the production, (2) incompatibility with
biosafety measures, (3) the need for careful identification of targeting biomarkers and
effective conjugation, and (4) FDA regulations compliance.
Pharmaceutics 2022, 14, 254 21 of 29

Table 5. Recent studies on MOF-based DDSs incorporating DOX for cancer treatment.

Composition Target Cancer Functionalization DOX Loading Study Model Triggering Modality Findings Ref.
MOF nanocarriers are biocompatible and safe (cell
Physical loading at dark viability of h 77 ± 0.71% was observed at the highest
conditions for 72 h Biocompatibility testing MOFs concentration of 1000 µg/mL).
nanoscale Zr where 100 mg of each by SRB assay on human
hepatocellular folic acid (FA), Drug release from dual-ligand LA-GA formulation was
(IV)-based MOFs formulation was fibroblast skin cells.
carcinoma lactobionic acid (LA), added to 35 mg of DOX pH-responsiveness sensitive to pH, releasing 60% and 100% of the [138]
nanoMOFs (HCC) glycyrrhetinic acid (GA) In vitro flow cytometry
(NH2-UiO-66) solution, followed by and MTT assay on encapsulated DOX at pH 7.4 and 4.0, respectively.
pelleting and vacuum HepG2 cells. Dual-targeting was the most efficient approach as these
drying at 40 ◦ C. MOFs exhibited the best anti-tumor activity,
approaching that of free DOX.
DOX loading was
carried out by pelleting
the MOFs and DOX encapsulation efficiency was a function of the
dispersing them in
Solid-state NMR weight ratio of DOX to MOFs during the loading
water before mixing (ssNMR) spectroscopy. process and the time of impregnation. A higher DOX
hepatocellular γ-cyclodextrincitrate
MIL-100(Al) carcinoma 1 mL of aqueous MOFs DOX release in N/A payload was observed with the increase in the weight [139]
nanoMOFs oligomers (CD-CO)
(HCC) coatings (2 mg/mL) with 1 mL phosphate buffer saline ratio and the impregnation time. DOX encapsulation
of DOX solution). The (PBS). had no significant effects on the MOFs’ morphologies
mixture was mixed for
1 to 6 days. The loaded or colloidal stability.
MOFs were centrifuged
and collected.
2 mL of DOX solution
(6.8 mg in 50 mL In vitro Cck-8 assay and DOX entrapment into both types of MOFs resulted in a
methanol) was mixed flow cytometry analysis loaded capacity of 0.65 µg/mg. Release from both types
with 100 mg of MOFs or of mouse breast cancer was sustained for 12 h period, while enhanced kinetics
Alendronate (Aln) Aln-MOFs powder. The
modified ZIF-8 Bone metastasis N/A 4T1 cells. pH-responsiveness were observed at a lower pH (~5.5) than neutral [140]
mixture was gently In vivo model: Balb/c conditions. The modified MOFs (Aln-MOF-DOX)
based MOFs
mixed for two days, mice inoculated with showed superior anti-tumor activity compared to the
followed by 4T1 cells to establish a unmodified MOFs. However, the tumor growth was
centrifugation, washing, bone metastasis model. arrested for 12 days only after which it regrows again.
and freeze-drying.

DOX was loaded into The in vitro dialysis bag

Fe-MOFs by mixing 10 diffusion technique to Both functionalized and unfunctionalized MOFs
mg of DOX with 20 mg study pH-dependent showed stability and long circulation capabilities. The
cationic polymer of Fe-MOFs overnight, release kinetics, MTT release at pH 5.0 after 12-h incubation reached 72% in
Fe-MOFs Different MV-PAH multilayers followed by assay to evaluate pH-responsiveness the functionalized MOFs, while unfunctionalized [141]
cancers toxicity to A549 and MOFs at pH 7.4 released <4% after the same incubation
(PEM) centrifugation. Loaded
Fe-MOFs were then MCF-7 cells. period. Coating with PEM increased the sensitivity of
coated with PEM using In vitro Annexin the DDS towards pH changes.
V-FITC apoptosis
the LBL technique. detection assay.
Pharmaceutics 2022, 14, 254 22 of 29

6. Concluding Remarks and Future Directions

Nanocarriers such as liposomes, micelles, and MOFs present diverse and interesting
designs of promising drug delivery systems that have been rapidly evolving over the years
and continue to be improved/upgraded to achieve their potentials. Liposomes are one of
the most explored and successful nanocarriers with many liposomal formulations at the
clinical pharmaceutical preparation stages. To date, there are more than eighteen liposo-
mal formulations already approved by the FDA to treat cancer and other diseases [142].
Compared to the liposomes, micelles and MOFs are synthesized from artificial materials
and, therefore, are more susceptible to elimination by the RES, affecting the amount of
these nanocarriers accumulating in the tumor. The main advantage of liposomes is their
high versatility, which allows them to successfully carry out different functions. This is due
to the ability to manipulate their designs by controlling the type of natural or synthetic
phospholipids used in their preparation using different headgroups (different charge) and
diverse fatty acids chains with different lengths and saturation levels (different transition
temperatures). In addition, a wide range of compounds can be added to the phospholipid
bilayer, such as PEG, cholesterol and targeting ligands such as carbohydrates, peptides,
proteins, and antibodies.
However, some issues still need to be addressed to improve the stability of liposo-
mal formulations, such as lipid oxidation and aggregation. In addition, the large-scale
production of liposomes is a complex and challenging process. It requires a high level
of precision due to the need to constantly test each produced batch to ensure the purity
and reproducibility of the used technique. Enhancing the liposomes with PEG (pegylated
or stealth liposomes) improves their circulation time and their accumulation inside solid
tumors while considerably reducing the cardiotoxicity of DOX. However, clinical trials
have shown that pegylated liposomes are associated with new side effects such as skin
toxicity and mucositis. Although those side effects are less severe than those caused by
doxorubicin treatment, it is important to improve the design of the pegylated liposomes to
eliminate their toxicity.
Micelles have a smaller size than liposomes and MOFs, making it easier for them to
benefit from the EPR effect when delivering DOX to tumors. In addition, micelles’ ability
to self-assemble and protect their encapsulated drug is a unique feature that allowed these
macromolecules to be investigated as nanocarriers capable of delivering DOX as well as
small particles like proteins and genes. Their simple assembly makes them highly feasible
for large-scale production compared to liposomes and MOFs. However, their low stability
is a major disadvantage that needs to be addressed. When micelles are injected into the
bloodstream and dilute, the concentration decreases below the critical micelle concentration.
As a result, these nanovehicles may disassociate, releasing their encapsulated drug before
reaching the targeted site. More understanding of the in vivo behavior of micelles should
be the focus of future studies. So far, limited studies have investigated the correlation
between micelle stability/in vitro drug release and drug pharmacokinetics. Generally, the
main two obstacles to the clinical success of micelles are their stability issues and the lack
of specific characterization tools. While several structural designs and improved prepa-
ration methods are being developed, these improvements mustn’t complicate the simple
structure of the micelles, which may make their large-scale production a complex process
and technically challenging. The progress to clinical translation depends on securing a
robust manufacturing process that is cost-effective and meets the regulatory requirement,
especially with complex micelles.
MOFs have interesting advantages over liposomes and micelles as they have the
largest surface area/volume ratios and have a highly porous structure. Thus, MOFs can
load more DOX and deliver higher local concentrations of DOX compared to micelles
and liposomes. However, MOFs are still behind the two other nanocarriers clinically.
Understanding the in vivo toxicity of MOFs, drug kinetics while loading and releasing,
the mechanism of MOFs degradation, and pharmacokinetics are still the main focus of
Pharmaceutics 2022, 14, 254 23 of 29

current research. Overcoming these limitations is essential to fully realizing the promising
potential of those unique nanocarriers as DDS in clinical applications. MOFs’ success in
clinical trials will also depend on understanding the mechanism of their cellular uptake by
the cancer cells.
Liposomes, micelles, and MOFs are injected into the blood and expected to reach the
tumor site while still intact, and DOX is encapsulated inside them. Therefore, all three types
of nanocarriers are faced with similar challenges; the physical force applied on them by the
circulating blood and their elimination by the reticuloendothelial system (RES) as well as
the renal system. Another major challenge is the heterogeneity of the EPR effect between
the different solid tumors and within the same tumors. The tumor microenvironment
is complex and comes with unique conditions and different extracellular matrix (ECM)
compared to the normal cells, which directly affect DOX delivery to the cells. Adding a
targeting molecule will surely enhance the active targeting ability of these nanocarriers
and their binding/uptake by the cancer cells once they reach their target. However, it
will not improve their chances of reaching the tumor while facing the many challenges
associated with their blood circulation. It is impossible to determine how much of the
nanocarriers, targeted or non-targeted, will reach the tumor and at what concentration.
Scientific efforts should be directed not only to enhance the properties of the nanocarriers
but also to understanding tumor vasculature. Efforts aiming to regulate vessel permeability
and the ability to physically disturb the vessels surrounding tumors, e.g., a pre-treatment
using photo-immunotherapy with antibody-photosensitizer conjugate, which has shown a
24-fold increase in nanocarriers’ accumulation at the tumor site [143], are all essential to
allow these nanocarriers to benefit fully from the EPR effect.
The use of nanocarriers, including those discussed in this review, to deliver DOX to
solid tumors is an excellent and efficient method to deliver this important and prevalent
anti-neoplastic agent to the targeted sites while reducing its side effects. There is no ideal
drug delivery nanocarrier. Each DDS showed advantages as well as disadvantages. In
addition, some of these systems were successful and made it to clinical trials, while others
were commercialized. Yet, research efforts continue to improve and enhance the current
DDS to unlock the potentials of nanocarriers. Future clinical trials must increase the number
of participants to produce robust results regarding the relationship between the produced
side effects, the effect of the patient’s age, and the treatment regime. One of the possible
future turning points in their application is the use of specially designed nanocarriers for
the personalized treatment of cancer patients depending on the tumor characteristics and
patients’ conditions.

Author Contributions: Writing—original draft preparation, M.I., W.H.A. and N.S.A.; writing—
review and editing, R.S. and G.A.H. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Dana Gas Endowed Chair for Chemical Engineering,
American University of Sharjah Faculty Research Grants (FRG20-L-E48, eFRG18-BBRCEN-03), and
Sheikh Hamdan Award for Medical Sciences MRG/18/2020.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing does
not apply to this article.
Acknowledgments: The authors would like to acknowledge the financial support of the American
University of Sharjah Faculty Research Grants, the Al-Jalila Foundation (AJF 2015555), the Al Qasimi
Foundation, the Patient’s Friends Committee-Sharjah, the Biosciences and Bioengineering Research
Institute (BBRI18-CEN-11), GCC Co-Fund Program (IRF17-003) the Takamul program (POC-00028-
18), the Technology Innovation Pioneer (TIP) Healthcare Awards, Sheikh Hamdan Award for Medical
Sciences MRG/18/2020, the Dana Gas Endowed Chair for Chemical Engineering. We also would
like to acknowledge student funding from the Material Science and Engineering Ph.D. program at
Pharmaceutics 2022, 14, 254 24 of 29

AUS. The work in this paper was supported, in part, by the Open Access Program from the American
University of Sharjah (grant number: OAPCEN-1410-E00014). This paper represents the opinions of
the author(s) and does not mean to represent the position or opinions of the American University
of Sharjah.
Conflicts of Interest: The authors declare no conflict of interest.

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