nanomaterials

Review
Microfluidics Technology for the Design and Formulation
of Nanomedicines
Eman Jaradat 1 , Edward Weaver 1 , Adam Meziane 2 and Dimitrios A. Lamprou 1, *

1 School of Pharmacy, Queen’s University Belfast, Belfast BT9 7BL, UK; [email protected] (E.J.);
[email protected] (E.W.)
2 Fluigent, 94270 Le Kremlin-Bicêtre, France; [email protected]
* Correspondence: [email protected]

Abstract: In conventional drug administration, drug molecules cross multiple biological barriers,
distribute randomly in the tissues, and can release insufficient concentrations at the desired patholog-
ical site. Controlling the delivery of the molecules can increase the concentration of the drug in the
desired location, leading to improved efficacy, and reducing the unwanted effects of the molecules
under investigation. Nanoparticles (NPs), have shown a distinctive potential in targeting drugs
due to their unique properties, such as large surface area and quantum properties. A variety of
NPs have been used over the years for the encapsulation of different drugs and biologics, acting
as drug carriers, including lipid-based and polymeric NPs. Applying NP platforms in medicines
significantly improves the disease diagnosis and therapy. Several conventional methods have been
used for the manufacturing of drug loaded NPs, with conventional manufacturing methods having
several limitations, leading to multiple drawbacks, including NPs with large particle size and broad
size distribution (high polydispersity index), besides the unreproducible formulation and high batch-





 to-batch variability. Therefore, new methods such as microfluidics (MFs) need to be investigated
Citation: Jaradat, E.; Weaver, E.; more thoroughly. MFs, is a novel manufacturing method that uses microchannels to produce a
Meziane, A.; Lamprou, D.A. size-controlled and monodispersed NP formulation. In this review, different formulation methods of
Microfluidics Technology for the polymeric and lipid-based NPs will be discussed, emphasizing the different manufacturing methods
Design and Formulation of and their advantages and limitations and how microfluidics has the capacity to overcome these
Nanomedicines. Nanomaterials 2021, limitations and improve the role of NPs as an effective drug delivery system.
11, 3440. https://doi.org/10.3390/
nano11123440 Keywords: drug delivery; liposomes; microfluidics; nanoparticles; nanomedicine; PLGA

Academic Editor: Bing Yan

Received: 5 November 2021

1. Introduction
Accepted: 15 December 2021
Published: 18 December 2021
Nanotechnologies are one of the most inspiring technologies in recent centuries that
appear as a novel and promising research filed. It gained awareness and attention when
Publisher’s Note: MDPI stays neutral
the American physics Nobel laureate Richard P Feynman discussed the hypothesis about
with regard to jurisdictional claims in
nanotechnology in his lecture “There’s plenty of room at the bottom” at a conference by
published maps and institutional affil- the American Physical Society in December 1959 [1]. However, in reality, nanoparticles
iations. (NP) structures are not new; these structures have existed on the earth since ancient times;
for example, Romans used NPs in glass manufacturing from the fourth century AD, since
they used Nano-glass particles to fabricate a glass cup, acknowledged as Lycurgus cup,
which was famous and distinctive due to its contrasting colour appearance under the
Copyright: © 2021 by the authors.
different tones of light [2]. These days nanotechnology is a science in itself, with several
Licensee MDPI, Basel, Switzerland.
applications in different fields, including water purification, information technologies,
This article is an open access article
drug development, environmental, food industry, and making more robust and lighter
distributed under the terms and materials [3,4].
conditions of the Creative Commons The nanotechnology topic includes creating and manipulating nanometre-size mate-
Attribution (CC BY) license (https:// rials by reducing bulk materials or scaling up atomic materials. NPs are small particles
creativecommons.org/licenses/by/ that vary in physical dimensions from 10 nm to 1000 nm. The engineered nanoparticles are
4.0/). commercial particles created especially with dimensions less than 100 nm [2].

Nanomaterials 2021, 11, 3440. https://doi.org/10.3390/nano11123440 https://www.mdpi.com/journal/nanomaterials

Nanomaterials 2021, 11, 3440 2 of 30

NP structure is composed of specific molecules produced at the molecular and atomic

level. NPs possess significant chemical, structural, electrical, biological, and mechanical
characteristics. There exist diverse groups of NPs, including fullerenes, ceramic, polymeric,
metal and lipid-based NPs. The value of these materials was recognized when researchers
found that size can influence the physicochemical properties of the materials [5]. For
example, the optical properties of gold and silver NPs depend mainly on the particle size,
which conveys different colours because of the absorption in the visible spectrum [6]. In
addition, toughness, melting point, reactivity, thermal conductivity, quantum effects, and
other properties are related directly to a NP’s unique size and morphology. Moreover,
Nanotechnology’s unique properties show a major improvement in the industrial area,
with a great performance in biotechnology, aerospace engineering, and electronics [2].
Many researchers have realized a significant impact of NPs in improving various
medical fields such as in the biomedical and pharmaceutical applications, as well as tissue
engineering and in vivo imaging. NPs have a special extended medical importance in
enhancing the drug delivery system, including encapsulating and targeting the delivery
of molecules (e.g., chemotherapy) and biologics (e.g., DNA, proteins, and antibodies).
Nanomedicines (NMs) have the potency to surmount the disadvantages and limitations of
conventional drug administration, such as insufficient efficacy, poor biodistribution, lack
of sensitivity, and toxicity. Therefore, nanotechnology represents an active area for research
to improve drug formulations, controlled drug release and targeted delivery.

2. Nano Based Drug Delivery Systems

Applying nanotechnology in the medical field was an encouraging step, especially
in improving the diagnosis and treatment methods. Many nanotechnology applications
have been reported in medicine, including in vivo and in vitro diagnosis, drug delivery, nu-
traceuticals, and producing biocompatible materials [7,8]. Engineered NPs are an essential
tool to achieve some of these applications. In the medical field, not all of the used particle
sizes comply to the stated definition of nanoparticles with a size of ≤100 nm [9]. Hence, the
particle size is not only what affects the functionality of NPs in medical applications; the
main reason for using NPs is their essential characteristics, such as high mass-to-surface-
area ratio, their ability to adsorb and function as a carrier to the other compounds, and their
quantum properties. NPs possess a significantly large “functional” surface that can adsorb,
bind, and carry different compounds, like proteins or other functional moieties. Depending
on their chemical properties and morphology, NPs can be classified into several categories
such as ceramic nanoparticles, carbon-based nanoparticles, semiconductor nanoparticles,
metal nanoparticles, lipid-based nanoparticles, and polymeric nanoparticles [10,11]. The
entire system drives to a particular purpose associated with treating, diagnosing, and
preventing disease, and it’s termed smart-drugs or theranostics [12].
The main objective of nanotechnologies in drug delivery is increasing the specificity
of drug targeting and delivery, improving safety and the biocompatibility, reducing the
toxicity whilst maintaining the therapeutic effects simultaneously. The controlled drug
delivery systems (DDSs) are based on carrying and transporting the active pharmaceutical
ingredients (APIs) to reach the exact site of action. Consequently, it affects the essential
and desired tissue and minimizes any unwanted effects. DDS can also keep the drug
away from the accelerated degradation or clearance by increasing the drug concentration
at the target tissue. Lower doses of the drug are therefore needed, potentially leading to
reduced manufacturing costs [13]. It is essential to apply this modern therapy mode when
the relationship is discrepant between the drug dose or concentration and the therapeutic
effect or toxicity. Attaching the drug to individually created carriers can achieve cell-
specific targeting and the recent advancements in nanotechnology record that NPs with
dimensions less than 100 nm, are excellent as drug carriers [12]. Owing to the nanosize
of these structures, they possess unique biological and physiological properties, such
as the capacity to cross tissue and cell barriers, making them preferable for biomedical
applications [14].
Nanomaterials 2021, 11, 3440 3 of 30

NPs also hold a higher possibility of being taken up by cells more than other particles
due to their larger surface area, which permits in some cases increasing of protein loading
by adsorption [15]. When NPs have any contact, especially with biological substances,
it’s coated with various proteins called a “protein corona”. NP corona has a primary
component named “opsonins”, which are directly responsible for enhancing the NP uptake
by the “Reticuloendothelial system” RES [15,16]. Therefore, it can be used as a functional
carrier for targeted drug delivery to the disease site in order to increase the poorly soluble
drug uptake, target the drugs to a specific location, and enhance drug bioavailability [16].

3. Application of the Nano-Drug Delivery Systems

The application of NPs in nanomedicine, have been investigated in different areas,
from controlled drug delivery to the diagnosis and prevention of diseases. Therefore, this
section discusses NP potential for targeted biological molecules delivery (e.g., siRNA)
for nucleic acid-based treatment and a nanoparticle’s potency in targeting chemotherapy
delivery to different types of cancerous cells. Highlighted below in this section are few
emerging applications for nanoparticles within the medical filed. The authors would like
to note that nanoparticles have a broader scope than those mentioned in this article.

3.1. Nanoparticles for Nucleic Acid-Based Treatment

Nucleic acid-based treatment is one of the promising techniques for drug delivery
targeting, especially to treat genetic disorders and cancer. For example, the plasmid [17] can
be utilized to repair the defective genes, and the small interfering RNA (siRNA) [18] can
be utilized to regulate the therapeutic process[19]. Compared to conventional therapeutic
molecules, Nucleic acid-based treatments need delivery carriers to facilitate their entry
into the cells and protect them from nucleases and other agents[19]. NPs possess many
advantages over molecular medicine, giving them the potential to surmount various
challenges and limitations of nucleic acid-based treatment, particularly the therapeutic
agent’s bio distribution and bioavailability. NPs’ superior retention in vivo is an exceptional
property through the decrease in enzymatic degradation and sequestration by phagocytes
of the RES [20]. That property is related to their immunochemically inert surfaces in
association with the biological environment. Via compromised vasculatures, NPs can
guide the precipitation of the drug to the site of disease in a phenomenon called enhanced
permeability and the retention effect also participates to their enhanced deposition in the
disease areas [21]. siRNAs are used in various applications as a robust way to control gene
expression. The free form of siRNA is unstable in the bloodstream and is unable to cross
cell membranes and tissues. Different NP types have been used for delivering the RNAi,
for example, Quantum dots (QD) have been used to control the delivery of RNAi [22],
NPs based polylactic acid (PLA), and Poly(lactic-co-glycolic) (PLGA), have also applied
for the delivery of RNAi in vitro [23]. Some success in delivering siRNA can be noticed
using several nanomaterials (e.g., chitosan, PLGA, and lipid NP). Tracking their delivery
and observing their transfection efficiency is complicated unless a proper tracking agent
or tag is used. At the same time, there’s significant difficulty to produce an effective and
self-tracking transfection agent for RNA interference.
Latterly, Tan et al. [24], designed a nano-carrier by synthesizing chitosan NPs and
encapsulating them with quantum dot nanomaterials to transport the human epidermal
growth factor receptor-2 (HER2/neu) siRNA [24]. It acts as a new and unique nano-carrier
that helps in monitoring the delivery of siRNA due to the existence of fluorescent QDs
into the encapsulated chitosan NPs. The targeted delivery of HER2 siRNA to reach HER2
receptors (an overexpressed receptor by SKBR3 breast cancer cells) can be more specific
using chitosan—QDs technology. This nano-carrier can be functionalized by grafting the
NP with HE ER2 antibody groups to enhance the target specificity [24].
Marking NPs with a fluorescent label, such as Cy-5, aids in visualizing the process of
nanotube uptake and accumulation by using fluorescent microscopy. Howard et al. [25],
integrated chitosan NPs with siRNA and utilized it mainly to the BCR/ABL-1 junction
Nanomaterials 2021, 11, 3440 4 of 30

sequence. A 90% decrease is detected in the expression of BCR/ABL-1 leukaemia fusion

protein in K562 (Ph (+)) cells. Also, using nasal administration of chitosan/siRNA for-
mulations in vivo drives the RNA molecule effectively to inhibit the gene expression or
translation in the bronchiolar epithelial cells of transgenic EGFP mice (RNA interference).
The same characteristics and features that have been approved in chemotherapeutic
delivery are confirmed to be used in siRNA delivery and packaging [26,27]. The siRNA
delivery and packaging is started by combining siRNA molecules with the NPs in a stable
way and retaining them in circulation. Several techniques, such as complexation and the
encapsulation and the non-covalent union of siRNA within numerous nano-constructs
have been reported in combining siRNAs to different biocompatible and inert molecules,
including long-chain fatty acids and cholesterol [28,29]. Unfortunately, the success is still
restricted until this day, due to the different challenges and limitations faced during the
delivery process. Moreover, recently issued reports discussed the toxicity and instability of
many siRNA-nanoparticle complexes in vivo [30,31].

3.2. Nanoparticles for Cancer Cell Targeting

Today, cancer exhibits as a significant hurdle in the area of pathology. The lack of
chemotherapy selectivity and the random attack on the healthy cells is one of the main
limitations that directly affects the treatment’s safety and efficacy. It also produces severe
side effects, and can generate insufficient drug concentrations within the desired tumour
tissues. Applying NPs for therapeutic purposes, particularly for targeting the delivery as
DDS, makes a significant development in the drug administration systems and a potential
role in overcoming the limitations of chemotherapy.
One of the NP’s recent applications is the use of glyconanoparticles targeting prostate
cancer cells. The glyconanoparticle is a highly branched polymer of glucose, function-
alized with lactose to add the β-galactosidase side to it and named as galacto-glycogen
(GG) [32]. GG, can fully interact with lectins, such as the Galectin-1 (Gal-1) and peanut
agglutinin (PNA) [32]. Gal-1, is a β-galactosidase binding protein highly expressed on
the PC3 prostate cancer cells and plays a vital role in the progression of the disease by
making multiple interactions with endothelial and other cells [33]. Various manufactured
platforms have been employed to enhance the attraction between the glycoconjugates and
their receptors, for example, glycopolymers [33], glycopeptides [34], sugar-functionalized
gold [35], glycodendrimers [36], iron oxide NPs [37], silica, liposomes [38], and carbon
nanotubes [39]. The engineered GG shows a high affinity to the cancer cell membrane
and a strong interaction with Gal-1, which makes them capable of inhibiting the Gal-1
proliferative activity or as being used as a carrier to target the delivery of an anticancer
drug to the PC3 cells [40].
Other applications of using NPs in cancer therapy have also been reported. There’s
serious difficulty in passing chemotherapeutic agents through the blood-brain barrier (BBB)
and towards the brain [41,42]. Researchers have discovered that NPs hold a potency for
carrying therapeutic active ingredients into the brain. Apolipoprotein E is a recommended
receptor for stimulating drug transportation across the BBB [43]. For example, Loperamide
is a drug with an antinociceptive effect but lacks the capacity to pass the BBB; therefore,
when it’s administered directly to the brain by intracerebroventricular injection, it exerts
antinociceptive effects. The drug was loaded into human serum albumin NPs and attached
to Apolipoprotein, then administrated intravenously to mice; the results confirmed an
antinociceptive effect in the tail-flick test. Typically, the efficiency of that complex drug
delivery system relies on lipoprotein receptor recognition.
Doxorubicin (another form of chemotherapy) can access the brain and come across the
intact BBB and release the therapeutic concentrations when it’s bound to polysorbate-coated
NPs [44]. Various anti-cancer drugs, counting doxorubicin-5-fluorouracil, dexamethasone,
and paclitaxel, are formulated conventionally by nanomaterials. For example, PLGA
(polylactic co glycolic acid) and PLA (polylactic acid) based NPs are used to encapsulate
dexamethasone (a glucocorticoid that acts intracellularly at the site of action) [45].
Nanomaterials 2021, 11, 3440 5 of 30

An additional application of a special nanoscale sensor can be used effectively to target

cancer cells. The Probes Encapsulated by Biologically Localized Embedding (PEBBLE), is
a nanoscale sensor that Kopelman designed with potency for carrying diverse kinds of
individual molecules on their surface and displays many functions [42]. Fixation of a target
molecule on the PEBBLE surface permits its distribution directly to the tumour site. Also,
a second agent could be attached to help in visualizing the target in magnetic resonance
imaging (MRI). In contrast, another agent can be joined to PEBBLE to cooperate in the
delivery of fatal dose of drug or toxin adjacent to the cancer cells. These agents’ capacities
can be merged in a small individual polymer sphere to create powerful weaponry against
cancer. Therefore, different platforms of nanomaterials perform as an optimum drug carrier
to target cancer cells.

3.3. Nanoparticles and Angiogenesis

The circulatory system plays a fundamental role in supplying the cells with oxygen and
nutrients to maintain a cell’s growth and development [46]. Cancerous cells, like healthy
cells, need blood circulation for sustained feeding with oxygen and nutrients to speed
their growth and invasiveness. The tumour cells induce vascularization (angiogenesis) to
receive their needs and enhance tumour metastasis [47].
Angiogenesis means the generation of new blood vessels from the existing vessels
after the tumour cells release angiogenic factors such as vascular endothelial growth factor
(VEGF) [47]. Due to the fundamental role of angiogenesis in tumour cell survival, suppress-
ing angiogenesis’s formation is the primary mechanism to repress tumour progression.
Angiogenesis can be suppressed by inhibiting the angiogenesis stimulating factor such as
integrin’s [48]. Integrins are a group of proteins that represent a critical part in tumour
migration, metastasis, and survival; these integrins’ tumour cell expression is linked to dis-
eases in several kinds of tumours such as breast cancer, prostate cancer, pancreatic cancer,
and melanoma [49]. At first, the only integrins targeted to repress tumour angiogenesis
were αvβ3 and αvβ5, but now researchers have targeted a vast collection of integrin’s such
as α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, αvβ5, αvβ6 and αvβ3 to repress tumour angio-
genesis [50]. Recent reported studies notes that αvβ3 integrin and vascular endothelial
growth factors (VEGFs) perform the primary regulatory functions for angiogenesis [51].
Hence, selective targeting of αvβ3 integrin and VEGFs is an innovative method to treat
various solid tumours. One of the approaches is to use NPs coated with peptides that specif-
ically bind to the αvβ3 integrin and the VEGF receptor [51]. The peptide-coated NPs have
an ArgGly-Asp (RGD) sequence, which binds especially to the αvβ3 integrin presented
on endothelial cells in the angiogenic blood vessels and probably inhibits tumour exten-
sion and proliferation. A reported study proves that using αv binding agents to targeted
chemotherapy is a useful technique, which applied an ArgGly-Asp (RGD) peptide joined
to doxorubicin to enhance this chemotherapeutic drug uptake. Doxorubicin effectiveness
has been experimented with in breast cancer xenografts in nude mice [52]. NP-mediated
αvβ3 targeting agents that contain different therapeutics have been examined [53].
Murphy and co-workers performed another study about doxorubicin delivery [54]
by synthesizing a αvβ3 targeting NP, capable of delivering doxorubicin into the tumour
vasculature. These NPs consist of distearoylphosphatidylcholine (DSPC), cholesterol
dioleoylphosphatidylethanolamine (DOPE), and distearoylphosphatidylethanolamine
(DSPE)-mPEG2000 simultaneously, alongside the RGD and was successful in incorpo-
rating doxorubicin. The same authors have also reported in a different study that these
NPs have the ability in controlling pancreatic cancer and renal cell carcinoma metastasis
in orthotopic mouse models [54]. The experiments showed a 15-fold enhancement in the
dose-response of the drug to the tumour vascularization when using the encapsulated
doxorubicin in RGD guided NPs instead of using free doxorubicin [49].
Other studies have been investigating tetraiodothyroacetic acid (tetrac), a thyroid
hormone analogue [55]. The anti-angiogenic qualities of tetrac have been confirmed to
be relevant to the vascular supply of tumours alongside the capability to attack a plasma
Nanomaterials 2021, 11, 3440 6 of 30

membrane receptor on integrin αvβ3 [55,56]. Due to all the powers of tetrac, laboratories
have started working on developing a nanoformulation integrating the biodegradable
polymer poly(lactide-co-glycolide) (PLGA) with tetrac [55–57]. However, the free form
of tetrac can easily penetrate the cell nucleus and exhibit adverse genomic effects (when
administered as an anticancer agent). The nanoformulation strategy in combining tetrac to
PLGA nanoparticles (T-PLGA-NPs) can resolve this complication by stopping NPs from
accessing the nucleus. The NPs larger size is the principal reason for preventing them
from going into the nucleus [58]. Simultaneously, this property of the NPs allows the
utilization of this nanoformulation as a therapeutic means to study the anti-angiogenic and
anti-proliferative effects of tetrac via the integrin’s extracellular domain [58].

3.4. Nano Systems in Inflammation

The power of macrophages in the rapid detection and clearance of foreign substances
provides a rational strategy for controlling inflammation using macrophage-specific tar-
geting with NPs. The ability of Macrophages to secrete several inflammatory mediators
enables them to control inflammation in various conditions; for example, activation signals
comprise cytokines (e.g., interferon-gamma, colony-stimulating factor, tumour necro-
sis factor-alpha, and granulocyte-monocyte), extracellular matrix proteins, and bacterial
lipopolysaccharide. Hence, they have a primary role in muscle repair, wound healing,
and cancer. Macrophages can kill most microbes and many microorganisms such as
Leishmania [59], Listeria monocytogenes [60], and Mycobacterium tuberculosis [61].
NPs that mediate antimicrobial agent(s) delivery inside pathogen-containing intra-
cellular vacuoles in macrophages could help in excreting cellular reservoirs [62]. By this
method, greater than one purpose can be reached. It can help achieve the therapeutic drug
concentrations in the vacuoles of infected macrophages whilst reducing the unwanted
effects correlated to drug administration and free the pro-inflammatory cytokines. Another
example of nanoformulations used as carriers into macrophages is the Polyalkylcyanoacry-
lates (PACA) NPs, which are used for targeting the anti-leishmanial drugs. This nanoma-
terial inhibits the macrophages’ ability to release interleukin-1 [21]. Hence, nanosystems
designed in a similar way could be extremely beneficial in targeting macrophage infections
in chronic diseases.
Amphotericin B (AmB), an antifungal and anti-leishmanial therapy, has been com-
bined with lipid-based nanotubes for investigating the generation a less toxic formulation
of AmB. Gupta and Viyas attempted to formulate AmB in trilaurin-based nanosized lipid
particles (emulsomes), then stabilised the complex with soya phosphatidylcholine (PC) to
produce a novel system for the intravenous (IV) drug delivery for macrophage targeting.
Macrophage’s nanocarrier-mediated delivery for toxins has been confirmed as a potent
strategy to clear out the undesired macrophages in gene therapy and other associated clini-
cal conditions, including rheumatoid arthritis, autoimmune blood diseases, T cell-mediated
autoimmune diabetes, sciatic nerve injury, restenosis after angioplasty, and spinal cord
injury [63]. On the other hand, the lethal properties of NPs with macrophages can be
utilized, and demonstrate a more useful antigen delivery approach and target a particular
nanocarrier [64].
However, the potential success of NPs platforms in the clinical trials as drug carriers
relies on essential parameters such as NP fabrication strategies, their physical properties,
drug loading efficiencies, drug release potential, and most importantly the toxicity of the
carrier itself. Among these platforms, lipid-based nanoparticles have some of the lowest
toxicity in in vivo experiments, having the ability to carry hydrophilic or hydrophobic
molecules and extend the duration of action, which means prolonged half-life and a
controlled drug release. Lipid-based NPs can be subjected to chemical adjustment to
bypass the immune system’s detection, such as PEG, or can be attached to antibodies to
recognize the tumour cells receptors, like folic acid (FoA) [65]. Lipid NPs can be prepared
as sensitive PH formulations to enhance the drug release in acidic conditions [66]. All
Nanomaterials 2021, 11, 3440 7 of 30

of that, promotes the efforts to expand the studies on lipid NPs development, scale-up
production, and manufacturing methods.

4. Manufacturing Methods for Lipid Formulations

Lipid-based NPs formulations, including liposomes and solid lipid nanoparticles
(SLN), have been manufactured using different methods. The several manufacturing
methods can be categorized into conventional methods and novel methods. The varying
use of manufacturing method mainly impacts the final quality of the NPs with their size,
polydispersity index (PDI), and morphology. Different lipid-based NPs manufacturing
methods are discussed in this section.

4.1. Liposomes
Based on years of research, liposomes have achieved importance and interest in
biological, pharmaceutical, and medical research, as the most effective carrier for a variety
of molecules in cell targeting and other various applications. For example, Doxil® is a
form of liposomal chemotherapy used to treat different cancer cells (e.g., Breast cancer,
bladder cancer, and lymphoma). A liposome, is a microsphere lipid constructed from
one or multiple phospholipid bilayers, closely mirroring the cell membranes’ structure.
Liposomes have a broad spectrum of applications extending from employment in basic
research in biophysics to various practical applications like cosmetics, pharmaceuticals,
production of ultrafine particles [67,68].
According to the definition that issued by the New York Academy of Sciences [64], lipo-
somes are classified to three categories: multilamellar large vesicles (MLV; 0.1–6 µm), small
unilamellar vesicles (SUV; 0.02–0.05 µm), and large unilamellar vesicles (LUV; >0.06 µm).
Other shapes (than spherical) and structures have been reported, including oligolamel-
lar [69], giant unilamellar, multivesicular [67], stable pausilamellar [68], helical [69] and
cochleate [70]. The liposome preparation procedure can be generalized and split into three
stages [70]:
1. Preparation of the aqueous and lipid phases;
2. Primary processing involving lipid;
3. Secondary processing steps (essential in some formulations and optional in others);
Practically, all methods require the dissolution of phospholipids in an organic solvent;
after that for most methods, the organic solvent should be removed by the evaporation
during the process. The previous dissolution, followed by removing the organic solvent, is
a critical step in the liposome’s formation process.
Phospholipids and cholesterol are the main building blocks of liposomes. The most
commonly used phospholipids have a critical micelle concentration in the nanomolar scale.
But phospholipids concentrations that are utilized in the liposome formation process should
be higher than the critical micelle concentration. The three-dimensional cylinder shape
of phospholipids stimulates liposome formation, that when phospholipids are exposed
to the aqueous atmosphere, the lipids start to assemble and aggregate. Mixing the lipid
and the aqueous phase can be achieved by in a few ways such as creating a thin lipid layer
before exposing it to the aqueous phase or inserting the lipid phase in the aqueous phase
in a controlled flow rate to form liposomes. Due to this, all of the liposome formation
methods, such as solvent dispersion/antisolvent, solvent evaporation (lipid hydration), or
detergent removal, concentrate mainly on breaking phospholipids into single phospholipid
molecules [71]. Then, exposing it to an aqueous condition to allow forming several classes
of liposomes, viz. MLVs, SUVs, GUVs, OLVs, MVVs [72].
The lately recorded techniques are classified as conventional, which often involves
easy and simple methods for using on the laboratory scale; moreover, the novel techniques
seem more useful for scaling up production due to using exceptional tools controlling the
process [73].
 Nanomaterials 2021, 11, 3440 8 of 30

4.1.1. Conventional Methods

The common strategies to create liposomes followed the identical basic stages are:
• Lipids dissolution in organic solvents;
• Drying the obtained solution;
• Hydration of the dried lipid (applying different aqueous solution);
• Separation of the liposomal vesicles;
• Quality control assays.

Film Hydration
Film hydration resembles the most straightforward and the earliest approach applied
in liposome science; it is named as the Bangham method. Firstly, the lipid is dissolved in
an organic phase (e.g., Chloroform, methanol, dichloromethane or trichloromethane) then
dried using a vacuum rotary evaporator it until a thin lipid layer is created in the bottom of
the flask. After that, the resultant lipid film needs to be hydrated with a suitable aqueous
fluid (e.g., deionized water) to provide dispersed liposomes (Figure 1). Hydration condi-
tions can directly affect the structural organization of the obtained liposomes. Therefore,
the soft and mild hydration of the lipid yields giant unilamellar vesicles (GULV). On the
other hand, confusion hydration can increase the generation of MLV with inadequate size
uniformity. The sizing methods commonly used are probe and bath sonication that allow
the creation of SUV. Probe sonication has higher effectiveness compared to other methods.
Still, at the same time, it is often condemned with the possible contamination (by titanium
from the titanium-based nozzle, which is utilized for mechanical agitation), as well as
the heat generated during the process affecting the stability of the lipid and drug. Even
though both of the sonication strategies provide liposomes with similar properties, using
the bath sonication method is still a more desirable choice because of the simple control of
operational parameters. After the sonication the solution must be left to rest before being
subjected to centrifugation. The main limitations of this method are the application of
organic solvent and mechanical agitation, which produce large and uncontrolled particles
Nanomaterials 2021, 11, x 9 o
sizes (this demands an additional downsizing step like the sonication), heterogeneous
distribution of the particles size, poor encapsulation efficiencies of hydrophilic materials,
scaling up difficulties, time-consuming and sterilization issues [73,74].

Figure 1. Thin film hydration method for empty liposome preparation. The liposomes produced from this method are often
Figure 1. Thin film
polydisperse; hydration
however, there ismethod for between
a correlation empty liposome preparation.
the duration spent during The liposomes
the rotary produced
evaporation from
step and this method are
the quality
oftenofpolydisperse; however, there is a correlation between the duration spent during the rotary evaporation step and the
thin film produced. Factors such as mixing speed and temperature after the hydration will also effect the quality of the
quality of thin film produced. Factors
liposomes so this must be monitored. such as mixing speed and temperature after the hydration will also effect the quality
of the liposomes so this must be monitored.

Solvent Injection
In solvent injection, the lipid solution will be injected at a fast-rate (ethanol or diet
Figure 1. Thin film hydration method for empty liposome preparation. The liposomes produced from this method are
often polydisperse; however, there is a correlation between the duration spent during the rotary evaporation step and the
quality of thin film produced. Factors such as mixing speed and temperature after the hydration will also effect the quality
of the liposomes so this must be monitored.
Nanomaterials 2021, 11, 3440 9 of 30
Solvent Injection
In solvent injection, the lipid solution will be injected at a fast-rate (ethanol or dieth
ether)Solvent
into the aqueous phase (Figure 2). Room temperature or higher, like 60 °C, can
Injection
used to achieve
In solventthis operation;
injection, the lipidthis depends
solution will bespecifically on the(ethanol
injected at a fast-rate level of organic solve
or diethyl
ether) into the aqueous phase (Figure 2). Room temperature or higher, like 60 ◦ C, can be
miscibility with water. Moreover, other parameters may affect the injection pressure a
used to achieve this operation; this depends specifically on the level of organic solvent
the ethanol to lipid ratio. Most of the time, solvent injection liposomes’ hold a relativ
miscibility with water. Moreover, other parameters may affect the injection pressure and
high polydispersity
the ethanol to lipid with distinguished
ratio. organic
Most of the time, solvent solvent contamination,
injection liposomes’ holdparticularly
a relatively ethan
due to creating
high an azeotrope
polydispersity combination
with distinguished with
organic water.
solvent The several
contamination, drawbacks
particularly are the m
ethanol,
due that
problem to creating an azeotrope
the solvent combination
injection methodwith has water.
faced, The several
mainly drawbacks
because are the
the therapeutic su
main problem that the solvent injection method has faced, mainly because the therapeutic
stance is continuously exposed to the organic solvent at high temperatures, both of the co
substance is continuously exposed to the organic solvent at high temperatures, both of
ditions
theaffect the liposomal
conditions products’
affect the liposomal stability
products’ and safety
stability [75,76].
and safety TheThe
[75,76]. high polydispersity
high poly-
the obtained
dispersity liposomes will,liposomes
of the obtained as a consequence, indicate a non-homogenous
will, as a consequence, formulation [7
indicate a non-homogenous
formulation [77].

Figure 2. An illustration for the procedure of the solvent injection method to produce empty liposomes. The major factors
Figure 2. An illustration for the procedure of the solvent injection method to produce empty liposomes. The major factors
to consider during the solvent injection method are the temperature during injection (as this will vary depending upon
to considertheduring the solvent injection method are the temperature during injection (as this will vary depending upon
phase transition temperatures of individual lipids), and the injection rate. These factors will affect the size, shape and
the phase polydispersity
transition temperatures of produced.
of the liposomes individual lipids), and
A following thesuch
method injection rate. Theseisfactors
as micro-extrusion normally will affectafter
required thesolvent
size, shape and
polydispersity of the liposomes produced. A following method
injection to obtain a therapeutically viable formulation. such as micro-extrusion is normally required after solvent
injection to obtain a therapeutically viable formulation.
Detergent Removal
In this method, the primary step is preparing an aqueous solution containing a critical
micelle concentration (CMC) of detergent, then dissolving the phospholipid within the
solution. Through the process, the reaction’s medium causes the release of a single phos-
pholipid, which begins to assemble around each counterpart, building bilayer structures.
This process’s success depends on using polystyrene-based absorber beads or Sephadex
columns (gel permeation chromatography) and a dialysis bag. The obtained compound
should be diluted with an appropriate aqueous liquid; this can help reform the devel-
oped micelles, which contain the liposomes [73,74]. The weakness of this method, are
the exposure to an organic solvent, time-consuming, detergent residual, inadequate yield,
sterilization needed, and low entrapment performance.

4.1.2. Re-Sizing of Lipid Suspension

Liposomes produced by conventional methods may hold large particle sizes or high
polydispersity. The provided lipid suspension can be downsized using sonication. Soni-
cation is a simple technique that breaks the aggregated large MLVs liposomes into small
Nanomaterials 2021, 11, 3440 10 of 30

liposomes with low polydispersity. The sonication technique can be used for liposome
preparation or as a second step of the liposome’s conventional manufacturing methods, as
discussed in the film hydration method.

Sonication
Sonic energy, or in other words sonication, is a powerful energy that has the potential
to alter a LMV suspension to SUV with a size range between 15 and 50 nm [78]. The
sonication process’s accomplishment depends on the sonicators used, bath or probe tip.
Probe tip sonicators, can pass a high amount of energy through the lipid suspension, but
exposure the suspension to high-temperature heating can cause degradation. Also, the
sonication tips can contaminate the suspension via releasing titanium particles, which need
to be cleared by centrifugation. All these limitations of probe sonication were the reason to
consider bath sonication as the most commonly used instrument for SUV preparation. The
LMV dispersion is subjected to the sonication process by moving the container that contains
the lipid suspension to the sonicator bath or to put the sonicator tip in the container itself,
and sonicate the suspension for 5–10 min at a temperature higher than the Tc of the lipid.
The suspension starts to refine into a lightly misty transparent solution. The mistiness of
the solution is produced because of the light scattering of the large remaining particles
in the suspension. The SUV solution becomes clear after removing these particles by
centrifugation. The resultant particles’ diameter and polydispersity are affected by different
factors such as sonicator tuning, the strength and duration of sonication, composition and
concentration, volume, and temperature. The particle size variation in the produced
batches at various times is expected, which is due to the difficulty of reproduction the
sonication conditions. Because of the high-curving degree of the liposome membranes, the
obtained SUV naturally shows a lack of stability, so it assembles impulsively to create more
giant blisters if stored under their phase transition temperature [79].
The complexity of the liposome production offers many barriers such as the variability
and distribution of particle diameter and “shell” deterioration. This is clearly displayed
in the start-up difficulties, long development process, and bioequivalence studies that are
compulsory when any modification occurs to the process. Thus, that makes the production
of liposomes and art more than an area of study. Moreover, liposomes have an essential
advantage of carrying the API in a targeted way to the active site. The targeting accuracy
of the “homing” surface of the phospholipid/liposome can be easily created and designed
for various applications. In general, liposome formulation still has physical and chemical
stability difficulties, which might induce liposome assemblage and drug degradation
during the storage period and compromise the administration of liposome-based drug
carriers [79].

Extrusion
The lipid extrusion method, is driving a specific lipid-containing liquid through a
filter pore size membrane producing particles precisely similar to the filter pore size. The
lipid suspension is subjected to several passes through the polycarbonate filter (Figure 3).
These passes can be forced by a machine with a pump (extruder) or can be manually in
small-scale production. After these passes, the LMV suspension transforms into more
petite and uniform SUVs or large LUVs. Between all the methods for reducing MLV size,
the extrusion method needs to be at a temperature higher than the Tc of the lipid; all
attempts to extrude below the Tc could be failed as the membrane become rigid and lose
the ability to pass through the pores. Tc relates to the transition temperature of a material,
where a material begins to transition between physical states. Various parameters can affect
the diameter and polydispersity of the obtained liposomes using the extrusion method,
especially the pore size. Extrusion through 100 nm pore filters produce LUV with a mean
diameter of 120–140 nm [80]. Also, the applied pressure, number of cycles and the lipid
composition could impact the process. This method has a central role in decreasing the
polydispersity to produce a more homogenous final suspension liposome. On the other
 diameter and polydispersity of the obtained liposomes using the extrusion method, esp
cially the pore size. Extrusion through 100 nm pore filters produce LUV with a mean
ameter of 120–140 nm [80]. Also, the applied pressure, number of cycles and the lip
composition could impact the process. This method has a central role in decreasing t
Nanomaterials 2021, 11, 3440 11 of 30
polydispersity to produce a more homogenous final suspension liposome. On the oth
hand, the membrane extrusion technique has some problems in large-scale productio
such as the clogging of the membrane pores, especially when the suspension is high
hand, the membrane extrusion technique has some problems in large-scale production,
concentrated
such as theorclogging
when the diameter
of the membrane ofpores,
the liposomes is much
especially when bigger than
the suspension the pores.
is highly
addition, the pore
concentrated orsurface
when thearea is only
diameter of 20% of the whole
the liposomes surface
is much biggerarea
than of
thethe membrane
pores. In th
limits the available space for extrusion, consequently limits the extrusion output [79].
addition, the pore surface area is only 20% of the whole surface area of the membrane that
limits the available space for extrusion, consequently limits the extrusion output [79].

Figure 3. The preparation of liposomes by extrusion method using polycarbonate filters. Increasing the number of
Figure 3. The preparation
transitions through of
theliposomes
membrane willby reduce
extrusion method using
the polydispersity polycarbonate
of the formulation. Thefilters.
processIncreasing theperformed
should also be number of transi-
tions through the membrane will reduce the polydispersity of the formulation. The process should also be
at a temperature similar to that of the lipid transition temperature, to prevent lipid cleavage (and subsequent liposomeperformed at a
temperature similar to
breakdown) that
upon of the lipid transition temperature, to prevent lipid cleavage (and subsequent liposome break-
extrusion.
down) upon extrusion.
4.2. Solid Lipid Nanoparticles
Solid nanoparticles (SLN) have gained much attention in the last years as an alternative
4.2. Solid Lipid Nanoparticles
for other lipid formulation NPs such liposomes or polymeric NPs. SLN are colloidal
Solid nanoparticles
particles with a spherical(SLN) have
shape that gained
vary in sizemuch
from 10attention
to 1000 nm.inThe
thedrug
lastmobility
years as
in aan altern
solid lipid is more limited than other lipid formulations, which
tive for other lipid formulation NPs such liposomes or polymeric NPs. SLN are enhances the achievement of colloid
controlled drug release [81]. It has been realized that SLNs could hold the benefits and avoid
particles with a spherical shape that vary in size from 10 to 1000 nm. The drug mobility
the limitations of other colloidal carriers; for example, SLNs offer targeted the drug delivery
a solid
andlipid is controlled
achieve more limited than [82].
drug release other lipid
Also, formulations,
it has whichlarge
an increased stability, enhances
capacity the
for achiev
mentscaled-up
of controlled drugand
production, release [81].the
bypasses It usage
has been realized
of organic that[81].
solvents SLNs SLNcould hold the benef
are composed
of a solid lipid core in an aqueous medium with a surfactant (emulsifier) [83].
and avoid the limitations of other colloidal carriers; for example, SLNs offer targeted t The surfactant
type is changeable due to the different route of administration as it is limited to use with the
parenteral formulation. The solid lipid core consists of lipids such as steroids (cholesterol),
fatty acid (palmitic acid, stearic acid), triglycerides (tripalmitin, trimyristin), complex
fat types (witepsol), glyceryl monostearate (imwitor), and waxes (e.g., cetyl palmitate,
beeswax) that possess a melting point higher than body temperature [84]. Stabilizing
the lipid core-shell inside the aqueous phase requires adding surfactants or emulsifiers
such as nonionic surfactants (pluronic, poloxamer), polyvinyl alcohol, polyethylene glycol
(peg), ionic surfactants (sodium cholate, sodium lauryl sulfate), and phospholipids (soy
lecithin, egg lecithin) [85,86]. The drug should be inserted into the gaps of the solid lipid
matrix since the solid lipid core should be melted through the process then subjected
to recrystallization at room temperature [83]. The drug insertion process relies on drug
Nanomaterials 2021, 11, 3440 12 of 30

hydrophobicity, solid lipid category, and the polymeric alteration in the lipid crystals [87].
There are various techniques to produce and manufacture SLNs, including high-pressure
homogenization, solvent evaporation, ultrasonication, hot homogenization, microemulsion,
solvent emulsification–diffusion, double emulsion, microfluidics and supercritical fluid
technique [88].

4.2.1. High Pressure Homogenization Method

High-pressure homogenization (HPH) is an initial, reliable, and conventional method
used for SLN manufacturing. The use of a homogenizer device is the central concept of
this method; the liquids are propelled with high pressure (100–2000 bar) in a narrow gap
(sub-micron dimension), and the fluid is subjected to a high velocity (above 1000 km/h)
in a small area which generates high-stress to cause a downsizing of the particles to the
submicron diameter [89]. HPH can be completed in two different conditions: hot pressure
homogenization and cold pressure homogenization [90].

Hot Homogenization Method

The hot homogenization approach includes the preparation of SLNs at a temperature
higher than the melting point of the used lipid; considered as emulsion homogenization.
A pre-emulsion should have been prepared by mixing the appropriate lipid and drug
then warming the lipid/drug mixture to a specific temperature above the melting point
until melted, and on the other side, the aqueous phase with the surfactant is heated to
the same temperature [91]. The aqueous phase is added to the drug-loaded lipid and
the mixture is equilibrated to the same temperature. The quality of the pre-emulsion can
highly affect the end product quality as it’s a desire to have droplets within the micrometer
size. The pre-emulsion is subjected to external mixing power to disperse the emulsion
at different speeds graduated from 5000 to 30,000 rpm; the varying rates of mixing is
practiced with different durations times [91]. Finally, the lipid should be cooled to obtain
the SLN; the cooling conditions could be gradually at room temperature or accelerate the
process by using a refrigerator [91]. Also, it may be cooled by using cold water during the
mixing at low speed (5000 rpm) or by adding the lipid to cold water with a considered
ratio such as 1:10 emulsion to water (water temperature between 2 and 25 ◦ C) [91]. The
speed of the mixing, the duration of emulsification, and the cooling states, are all essential
parameters that determine the final particle size and ζ-potential. To achieve high-quality
SLN production, it must be consider that the higher preparation temperature obtains a
smaller particle size due to the lower viscosity of the lipid solution [92]. Also, multiple
passes through the high-pressure homogenization can increase the emulsion temperature
(about 10◦ at 500 bar), generally 3 to 5 passes (at 500–1500 bar) are enough to have the best
results; if the number of passes increases, the particle size will be increased and coalescence
may occur [93]. Like any other methods, this method has also some limitations, such as the
high consumption of mechanical energy, the lack of control on the effective particles size,
long term stability, and the loading capacity, as well as the high cost of production due to the
multiple steps of preparation and the high energy requirement [94]. All these restrictions
boost the researchers to investigate other novel methods to overcome these limitations.

Cold Homogenization Method

The second version of the high-pressure homogenization is the cold homogenization
method, which is regarded as the milling of a suspension. The entire process of this method
needs to be under a well-controlled temperature to keep the material in a solid-state [93].
The cold homogenization method is developed to overcome the limitations of the hot
homogenization method, such as the complication of the nanoemulsion crystallization
process that may cause some modification and supercooled melts, the potential drug
degradation due to the high exposure to heat, and the possible splitting up and hence loss
of the drug into the aqueous phase during the homogenization process [89].
Nanomaterials 2021, 11, 3440 13 of 30

The first step of the cold homogenization method is comparable to the hot homog-
enization, as the desired drug is dispersed into the melted lipid. Then, the drug-loaded
lipid is cooled using liquid nitrogen or dry ice. The cooling process should be completed
quickly to have homogenous drug distribution in the lipid matrix. Then the active solid
lipid is subjected to milling using a ball or mortar miller to produce microparticles in
the range of 50–100 microns. Chilled processing further promoted particle milling by
increasing lipid fragility [89]. Disperse the solid microparticles into a surfactant solution.
The pre-suspension undergoes high-pressure homogenization at or below room tempera-
ture with proper temperature. The high-pressure process usually raises the temperature
spontaneously, so concise monitoring of the temperature change is needed [95].
By comparing the cold homogenization with the hot homogenization samples, the
cold homogenization produces solid NPs with larger dimensions and less homogeneity
(high PDI) [96]. However, the cold homogenization method’s advantages include the
reduction of exposure to heat; drug-loaded lipids are exposed to the heat only in the first
step of the process.

4.2.2. Ultrasonication or High Speed Homogenization

High-speed homogenization or sonication is one of the most straightforward dispers-
ing techniques to produce SLNs [97]. The drug and the suitable Phospholipid are mixed
and added to an aqueous solution to prepare a pre-emulsion. Then, the pre-emulsion
is subjected to high-speed homogenizing for a sufficient period of time [98]. During the
process, it is recommended to keep the temperature 5 ◦ C above the lipid melting point and
ultrasonicate the pre-emulsion for 15 min after the mixing to avoid lipid crystallization [99].
The oil/water (o/w) emulsion should be cooled at room temperature with continuous
stirring to recrystallize the lipid and SLN created [99]. The main limitation of this method
is the high polydispersity of the produced particles, which affects the physical stability of
the particle directly and induces particle growth in storage, alongside the possible metal
contamination [89]. Several research groups have combined high-speed stirring and ultra-
sonication and perform the method at high temperatures in order to increase the formula’s
stability and overcome the limitations [81].

4.2.3. Microemulsion
A lipid-based microemulsion is prepared by the continuous stirring of an optically
transparent solution constituted of emulsifier (e.g., polysorbate 20), low melting temper-
ature lipid (e.g., stearic acid), and water at a temperature close to 65–70◦ . The obtained
microemulsion should be dispersed with continuous stirring in cold water (2–3 ◦ C) [100].
The volume ratio of the water medium and microemulsion are in the range of 25:1 to 50:1,
and the dilution procedure is decided according to the composition of the microemul-
sion [101].
The highlighted advantage of this method is that the microemulsion contains the
droplet structure spontaneously without the need of using external energy to get the
submicron size. According to the literature [102], the particle size of SLNs is determined
mainly by the speed of the distribution process; the hydrophilicity of the used solvent can
impact the size of the SLN. Using a hydrophilic solvent can enhance the distribution velocity
in the aqueous phase compared to the nanoparticles that are distributed in hydrophobic
solvent, which develops larger particles [81]. Considering microemulsions, the construction
of the microemulsion as well as the temperature gradient forms the product quality. High-
temperature gradients promote rapid lipid crystallization and avoid aggregation [81]. The
main limitation of this method is the low concentration of the produced NPs, alongside the
need for a high quantity of water and the high sensitivity to change [103].
Nanomaterials 2021, 11, 3440 14 of 30

5. Manufacturing Methods of Polymeric Formulations

Polymeric nanotechnology has a primary role in current investigations in DDSs, due
to its specified chemical composition, polyvalent surface, and three-dimensional construc-
tion [104]. Both natural and synthetic polymers are developed into special nanocarriers
to enhance drug loading capability, target the action site, and control drug release [105].
Different formulations of NPs can be formulated using polymers, including nanocapsules,
nanospheres, and emulsion droplets. The various types of polymers that are used for the
manufacturing of NP carriers are approved materials by the Food and Drug Administration
(FDA), and includes hydrophobic polymers such as polycaprolactone (PCL), poly-lactic
acid (PLA), poly-(lactic-co-glycolic acid) (PLGA), and hydrophilic polymers such as al-
bumin, chitosan, gelatine, and alginate [104]. Natural polymers are biodegradable and
biocompatible, confirming a complete elimination from the body and making them less
toxic than synthetic polymers [106]. However, the use of natural polymers is still limited
due to poor batch-to-batch reproducibility and the possible immunogenicity [107]. The
sustained release of the incorporated drug over days to weeks is the main advantage for
synthetic hydrophobic polymers over natural hydrophilic polymers. However, the use of
synthetic polymers is still limited due to the high organic solvent usage and the possible
toxicity [105].
Recently, many efforts have been centred on preparing well-characterized NPs by
choosing the most suitable polymer type, preparation method, and other factors that can
highly affect the quality and performance of the polymeric nanoparticles in vivo [107].
Different methods are used to manufacture polymeric NPs, which can be categorized into
two groups: two-step processes that need to prepare the emulsification system as the first
step and then form NPS, and the one-step process that exempts the emulsification step. A
comparison between the different manufacturing method of polymeric NP and lipid NP
are in (Table 1).

Table 1. Comparison of the Advantages and disadvantages of manufacturing methods of lipid NP and polymeric NP.

Nanoparticles Manufacturing
Advantages Disadvantages References
Type Method
1- High consuming of the
organic solvents
1- Established method 2- High PDI
Film hydration 2- Understood method 3- Lack of reproducibility [108]
4- Need for additional
downsizing step
5- Difficulties in scaling-up

1- Exposing to organic
1- Simple and fast solvent
Lipid Solvent injection 2- Scaling- up possibility [108]
2- high PDI
formulation 3- stability problems

1- uniform and homogenous 1- possible clogging of the

Extrusion formulation membrane pores. [109]
2- Difficulties in scaling-up

1- Scaling- up possibility 1- High energy consumption

High pressure
2- Uniform formulation 2- Multiple steps [108]
homogenization
3- Bulky system

3- Difficulty in removing the

1- Small particle size excess water
Microemulsion 2- Homogenous formulation [110]
4- Use high concentration of
surfactants
Nanomaterials 2021, 11, 3440 15 of 30

Table 1. Cont.

Nanoparticles Manufacturing
Advantages Disadvantages References
Type Method

1- Avoids surfactants and 2- Need for purification steps

Emulsification
chlorinated solvents. 3- Encapsulate lipophilic [107,111]
-salting out
drugs only

3- The possible diffusion of

the hydrophilic drug into
1- Scaling- up possibility the aqueous phase
Polymeric Emulsification- 2- Batch-to-batch 4- The need to eliminate high [107,112]
solvent diffusion reproducibility volume of aqueous phase
from the colloidal
dispersion

2- Risk of nanodroplets
Emulsification- 1- Simple and versatile coalescence during the
[111]
evaporation evaporation process
3- Time consuming

1- Effective and simple 1- Time consuming

method 2- Use of high amount of
Dialysis 2- Produce PNP with narrow dialyzing medium, which [111,113]
distribution stimulate the premature
release of NPs content

1- simple and established 1- Restricted for lipophilic

method drugs
Nonparticipation [107,111]
2- use low concentrations of 2- Low polymer
surfactant concentration obtained

5.1. Two-Step Emulsification Procedures

In two-step procedure, the first step is emulsifying the organic polymer solvent
(including the drug) and the aqueous solution. An emulsifier device should be used to
produce the nanodroplets then the NPs [114]. In these methods, the droplet formation
step is the control step that determines the obtained polymeric nanoparticle (PNP) size
and homogeneity [107]. After the nanodroplets are formed, the organic solvent should
be eliminated to condense the polymers into the nanodroplets and form the PNP [107].
Several methods are used for removing the polymer organic solvent, such as fast diffusion
after dilution, solvent evaporation, or salting out. In all the method procedures, the drug is
added to the polymeric organic solvent as the first step to encapsulate it [107].

5.1.1. Emulsification-Salting Out

The salting-out method is a simple method based on emulsification. The emulsion is
composed of a water-miscible solvent (e.g., acetone) containing the drug and an aqueous gel
including a surfactant and salting-out agent. Electrolytes are examples of salting-out agents,
including calcium chloride, magnesium chloride or non-electrolytic salting agent such as
sucrose [107]. The emulsification process is accomplished without using homogenization
energy, as the ouzo effect is the causative of the homogenization process [115]. The
miscibility of water and acetone is reduced, and an o/w emulsion is formed by increasing
the saturation of the aqueous phase. The obtained o/w emulsion should be diluted with a
high amount of water to induce the diffusion of the acetone into the aqueous phase and get
a reverse salting-out effect. The reverse salting-out effect will facilitate the precipitation of
the dissolved polymer in the nanodroplets. The remaining salting-out agent and polymer-
solvent can be removed using cross-flow filtration [116].
This method holds an advantage avoiding the use of chlorinated solvents, which
harm the physiological system and environment. The main disadvantages are the limited
Nanomaterials 2021, 11, 3440 16 of 30

utilization for the lipophilic drugs encapsulating and the need for additional purification
steps due to the using salts [107].

5.1.2. Emulsification-Solvent Diffusion

The solvent diffusion method is an advanced method of the salting out method [107].
The first step of the emulsification-solvent diffusion method is preparing an emulsion that
includes a partially water-miscible solvent with the polymers and drug, and an aqueous
solution with surfactant. To succeed in this process, the polymeric solvent solution and
aqueous solution should be mutually saturated at room temperature to ensure the initial
thermodynamic equilibrium of the liquids [107]. After the droplets are formed, the mixture
is diluted with a high quantity of water and the colloidal particles formed [117]. In general,
this method can obtain nanospheres, but adding a small amount of oil to the organic
solvent is required to get nanocapsules. This method shows clear advantages such as the
scaling up probability, high yields, the needless of high-pressure homogenizer, the high
encapsulation capability, batch-to-batch reproducibility [118]. The disadvantages of this
method include the increased water consumption, the potential loss of the water-soluble
drugs in the external phase during the emulsification process [119], low stability of the
emulsion, and the low solubility of the lipids in organic solvent [103].

5.1.3. Emulsification-Solvent Evaporation

Emulsification solvent preparation is the first applied method to prepare PNP with
the desired properties [107]. This method, as a simple and versatile method, was applied
successfully to produce drug carriers using biocompatible polymers. The hydrophobic
drug and polymer are dissolved in a suitably volatile solvent as the first step. Previously,
chloroform and Dichloromethane were used as a solvent; however, they are now substituted
with ethyl acetate, which has a safer toxicological profile and greater compatibility with
biomedical applications [118]. The resultant organic solution is subjected to emulsification
in an aqueous solution utilizing a surfactant and high-speed homogenization to produce a
nanodroplet dispersion [120]. The evaporation of the polymer-solvent is the critical step to
obtain a NP suspension. The polymer-solvent is enabled to diffuse through the continuous
emulsion phase and can be evaporated by constant magnetic stirring at room temperature
or by being subjected to reduced pressure, which is a slow process [121]. The produced
PNP can be collected by centrifugation then put through freeze-drying for long storage
purposes [122].
The solvent evaporation method is simplistic and versatile. The main limitations
are the suitability for specific drugs only (lipophilic drugs), energy-consuming, time-
consuming, and the possible coalescence of particles through the evaporation process [107].
A limited energy-consuming homogenization method (e.g., phase inversion composition
(PIC) method) is more preferred for scaling up production [123].

5.2. One Step Procedure

In one step procedures the emulsification step is not critical for nanodroplet formation.
The one step procedures include: Nanoprecipitation procedure, Dialysis, and superficial
fluid technology.

5.2.1. Dialysis
The dialysis method is a one-step method that has been used effectively to produce
NPs with narrow size distribution and suitable particle size [124]. The concept of the
dialysis method is the reassembly of the polymers due to the reduction of their solubility.
Generally, the polymer and the drug are dissolved in a suitable organic solvent, moved to
a dialysis membrane, and placed for dialysis in a non-solvent phase [107]. Semipermeable
membranes with conventional molecular weight cut-off (MWCO) or dialysis tubes are
used as a polymer barrier [111]. The aqueous solvent’s displacement into the membrane
changes the polymer-solvent solubility, as the solubility of polymers reduces with the
Nanomaterials 2021, 11, 3440 17 of 30

increase of the interfacial tension, which leads to polymers aggregate and development of
a nanoparticles suspension [107]. The miscibility between the organic polymer solvent and
the dilute dialysis solvent is required. Different parameters can affect the size of particles,
such as the type of solvent, the solvent’s viscosity, the miscibility with water, the dialysis
MWCO, the polymer concentration, and the rate of solvent mixing [125].
The main limitation of this method is the use of high amount of dialyzing medium,
which can stimulate the premature release of the NPs content [107]. In addition, it’s a
time-consuming procedure, and faced difficulty in scaling up the production.

5.2.2. Nanoprecipitation Procedures

The Nanoprecipitation method, or solvent replacement, is a one-step method that
relies mainly on the interfacial participation of the polymer after replacing the organic
solvent from lipophilic solvent to aqueous solvent [107]. The desired drug and polymer
are dissolved in a water-miscible solvent with moderate polarity, and then the solution
is injected as one shot into a stirred aqueous solution [126]. The polymer-solvent will
diffuse quickly and spontaneously to the aqueous solution, and the nanoparticles will be
formed [107]. The Marangoni effect was influential for the sequences of the steps [107].
Reducing the interfacial tension between two different phases will spontaneously increase
the surface area due to the rapid diffusion, and small droplets of the organic solvent will
be created [107].
The diffusing of the solvent out of the nanodroplets stimulates the participation of
polymers as nanospheres or nanocapsules. Acetone is one of the commonly used organic
solvents in this process due to its miscibility with water and the ease of evaporation [107].
Also, solvent mixtures can be used, such as mixing the methanol or ethanol with a small
quantity of water [127]. The protocol of this method is to inject the organic solvent into
the aqueous solvent, but the process can be conversed without affecting the formation of
the NPs.
Compared to the emulsification solvent-evaporation method, the nano-participation
particles are characterized with appropriate particle size and limited size distribution [107].
Regulating the different experimental conditions can impact the final NP properties, such
as the ingredient’s nature, ratios of organic solvent to the aqueous solvent, the mixing
speed, and the injecting rate [126]. For example, a reported study has shown a variance in
the particle size by using different solvents (e.g., acetone, acetonitrile, and tetrahydrofu-
ran), that the most water-miscible solvent (acetone) produces the smallest particles [113].
Chancón et al. [128], investigated the effect of polymer concentration, injection rate and
needle gauge, the lowest polymer concentration, lowest needle gauge, and the highest
injection rate produced smaller particle sizes (~46 nm) [128].
In general, this method is commonly used due to its rapid, simple, and reproducible
results. However, the method is restricted for lipophilic drugs as the loss of the hydrophilic
drugs is expected through the diffusion to the aqueous phase [107]. Controlling the quality
of the drug, polymer, solvent, and non-solvent system is the nanoprecipitation method’s
challenge [126]. Moreover, controlling the mixing process at a constant rate during the
nano-precipitation is the main challenge of this method [107].
The microfluidic method could be a promising approach to overcome the uncontrolled
mixing process by maintaining the hydrodynamic flow of the solvents at a constant rate
and ensuring a well-controlled mixing [129].

6. Microfluidics
Microfluidics is a novel, versatile technology that has been developed recently and
has been utilized in several areas. A microfluidic system can manipulate a small fluid
volume (10−9 to 10−18 L) using micrometre channels, microvalves, and micromixers as
an interconnected system [130]. The principle of microfluidics is based on the rules of a
controlled mixing process [131]. By studying the material nature in a number of previous
studies, it has been realized that the fluid in microscale has different flow dynamics from
Nanomaterials 2021, 11, 3440 18 of 30

those in the macroscale [131]. The flow of the fluid can be categorized into laminar flow and
turbulent flow. Turbulent flow is an irregular fluid distribution in different orientations, and
most macroscale fluids flow is a turbulent flow. The laminar flow is the parallel orderly flow
at constant velocity without scattering of the layers. Laminar flow fluids are controlled by
viscous forces rather than inertial forces, which means a constant and continuous flow [132].
The microfluidic system provides microscale continuous laminar flow; this type of flow
offers a high mixing quality and enhances the performance of microscale devices [133].
The constant continuous flow provides the same quality of production over time and
limiting the batch-to-batch variability [133]. Many studies have described microfluidics as
a mechanism to manage the mixing process on a microscale [134,135].
The microfluidic chips have been manufactured by different materials, with monocrys-
talline silicon and glass borosilicate are the most common due to their suitable proper-
ties [131]. However, the disadvantages of these materials, such as the high cost, the brittle
nature, and the lack of self-sealing, lead to drawbacks and influence the development of
new materials and manufacturing methods [136]. The microfluidic chip’s fabrication mate-
rial should have appropriate properties, such as flexibility and elasticity [137]. Different
polymers (e.g., Polymethyl Methacrylate (PMMA), Polystyrene (PS), and Cyclic Olefin
Copolymer (COC)) hold these properties and have been widely used to fabricating microflu-
idic devices. The polymer structure and manufacturing method can modify the degree of
flexibility, rigidity, elasticity, and brittleness. Both, the polymer type and manufacturing
methods, can be determined depending on the application of microfluidics [137].
The polymers used for the chip manufacturing can be categorized into four groups
of elastomers, thermoplastics, thermosets and, thermoplastics elastomers [137]. The
main thermoplastics and elastomers that are used for microfluidic fabrication, include
Poly(dimethylsiloxane) (PDMS), Poly (ethylene terephthalate) (PET), polymethylmethacry-
late (PMMA), and Cyclo-olefin polymer (COP) (Table 2).

Table 2. Summary of materials used for microfluidic chips, including advantages and disadvantages.

Tg 1 Manufacturing
Material Advantages Disadvantages References
(◦ C) Method
Low Tg, easiness of shaping in
hydrophobic nature, sensitive
the channels, optical soft lithography,
PDMS to organic solvents (e.g.,
−125 transparency, resistance to plasma-enhanced [137–139]
(elastomer) strong acids, hydrocarbon,
water, ability to produce bonding
amines.
microscale features precisely.
solvent imprinting,
low cost, optical transparency, High Tg, Sensitive to alcohol,
hot embossing
attractive mechanical/chemical isopropyl alcohol and acetone,
PMMA 105 thermal bonding, [140,141]
characteristics, and simple high bonding temperature,
injection molding
fabrication processes. Commercial availability
and laser ablation.
Low Tg, low rigidity, low
reduced chemical resistance,
surface energy, easiness of
require surface treatment for hot embossing,
PET 69–78 molding, chemically inertness, [137,142]
bonding due to the low thermal bonding
good gas and moisture barrier
plasma bonding strength.
characteristics, recyclable.
High stability, low interaction
with protein, suitable rigidity, High Tg, brittleness and low hot embossing,
resistance to almost all solvents heat diffusivity, not resistance chemical etching,
COP 70–180 [143]
including ethanol, IPA, and to the non-polar organic thermal bonding
acetone, low water absorbency, solvent (e.g., hexane). methods.
high moisturiser barrier.

Other thermoplastics that have the potential to be used for chip fabrication include
polycarbonate (PC), polystyrene (PS), perylene, and polyvinylchloride (PVC) [144]. In
addition, other types of materials could be used to fabricate microfluidic devices such as
Nanomaterials 2021, 11, 3440 19 of 30

hydrogels, especially for biological applications (cell-related), and paper, particularly for
the open channel microfluidic systems [144]. Several methods have been used to fabricate
the polymer’s microfluidic devices, including hot embossing or imprinting, injection
molding, laser ablation, soft lithography or X-ray photolithography and 3D printing [145].
3D printing is a modern technology that has been used recently to fabricate microfluidic
devices. Compared to other mentioned microfluidic fabrication systems, 3D printing is
low-priced, time-saving, and multiple-purpose [146].
Microfluidics offers several useful applications in different fields, such as electronics,
chemistry, biology, medicine, and material science, among many others. The principal
innovation of microfluidics is the facility to transfer the traditional bulk technique to
100 nm width microchannels. Within these channels, solvents can be mixed by a pump-
ing system and can be exploited for analysis, separation, and synthesis purposes. For
example, microfluidics allows material synthesis, especially nanomaterials and nanostruc-
tures, enhancing the production of micro/nano-sized DDSs [147,148]. For example, Wang
et al. [149], have used the microfluidic system to create a DDS of exosomes. Exosomes
are a novel therapeutic delivery system that suffer from poor targeting ability and lack
an effective isolation technique. The workers used a microfluidic system to isolate the
exosomes by lining the wall with carbon nanotubes by chemical deposition to catch the
exosomes and add the CD63 as a targeting agent to help the exosomes achieve targeted
deliver. The system was investigated on cancer cells, and the results showed that exosomes
show a significant targeting efficiency with high cellular uptake when exposed to the
cells, which enhanced the anticancer effect of the used drug. The results can present that
microfluidics was an effective technique to isolate exosomes and improve the targeting
capacity [149].

6.1. Microfluidics in Nanomedicine

Nanomedicine is one of the most critical topics of present-day investigations. NPs
have been approved for clinical use and utilized for multiple medical purposes, including
drug and gene delivery. Different types of NPs can be used for the encapsulation of
pharmaceutical molecules, including lipid based and polymeric NPs. Several traditional
methods have been used for NP manufacturing as discussed previously in Sections 4 and 5.
The utilization of microfluidic system in NP manufacturing will be discussed in this section.

6.1.1. Microfluidics in the Manufacturing of Liposomes

As a novel and unique technology, the MF system utilizes microscopic channels that
vary in range of dimension (5–500 µm). The power of these channels is in providing the
laminar flow of fluids, which enhances the performance and mixing quality of microfluidic
devices. In the MF process, the lipids dissolve in a suitable organic solvent (ethanol or
isopropanol). The lipid can consist of different types of phospholipids and is usually
coupled with cholesterol; the ratio between the lipid and cholesterol highly affects the
characteristics of the final formulations. The pre-prepared lipid phase is moved to a specific
chamber, and the aqueous phase is transferred to the opposite one (Figure 4). The flow rate
ratio (FFR) and total flow ratio (TFR) are adjusted to control the flow of both phases. Then,
the mixing process of the organic and aqueous phases is started as a continuous controlled
mixing. Mixing the fluids causes a local diffusion of phospholipids in the aqueous phase,
which promotes the lipids’ self-assembly and consequently produces liposomes.
MF technology provides a high control on the flow rate, which allows a continuous
production of monodisperse and homogenous liposomes. Compared to different MF meth-
ods, such as droplets, micro hydrodynamic focusing (MHF), and the pulsed jet flow MF,
the MHF method is the most utilized method to fabricate liposomes; furthermore, it has the
potential to produce SUVs and LUVs with an appropriate size and homogeneity by a one-
step procedure. The success of the MHF method was reported firstly by Jahn et al. [150],
reporting the production of liposomes using the perpendicular flow of lipid dissolved in
an organic solvent mixed with an aqueous buffer. Several parameters can critically impact
Nanomaterials 2021, 11, 3440 20 of 30

the production process, such as the total flow rate (TFR), the flow rate ratio (FRR), the
lipid concentration, the solvents, temperature, and the combination of lipids. For example,
Aghaei et al. [151] used flow-focusing MFs method, to encapsulate methotrexate within
liposomes. This study investigates the impact of different parameters, including TFR,
FFR, and total lipid concentration, on the resultant physicochemical characteristics such
as particle size, PDI, and encapsulation efficacy of the liposomes. The particle size of the
liposomes was within 90–230 nm, and PDI ≤ 0.32 whilst also boasting a high efficiency of
Nanomaterials 2021, 11, x encapsulation (≥60%). The drug release studies display a sustained release of the 21 drug
of 31
and long-acting effect up to 72 h (≤48%). The high encapsulation efficacy and slow drug
release directly relate to the manufacturing process [151].

Schematicpresentation
Figure4.4.Schematic
Figure presentationof ofliposomes
liposomesmanufacturing
manufacturingusing
usingthe
themicrofluidic
microfluidicsystem.
system.Figure
Figure
adapted from Weaver et al. [152].
adapted from Weaver et al. [152].

Guimarães et al. [153] have used dimyristoylphosphatidylcholine (DMPC) and dis-

Guimarães et al. [153] have used dimyristoylphosphatidylcholine (DMPC) and dis-
tearoylphosphatidylcholine (DSPC) to prepare liposomes using two different methods, the
tearoylphosphatidylcholine (DSPC) to prepare liposomes using two different methods,
microfluidics and the thin-film hydration method. The results of the different formulations
the microfluidics and the thin-film hydration method. The results of the different formu-
were compared to assess the positive and negative impacts of microfluidics. Comparing
lations were compared to assess the positive and negative impacts of microfluidics. Com-
the two processes shows that microfluidics is a fast, simple, and single-step technique for
paring the two processes shows that microfluidics is a fast, simple, and single-step tech-
liposome manufacturing. The versatility of the method makes the encapsulation process
nique for liposome manufacturing. The versatility of the method makes the encapsulation
faster with keeping the encapsulation efficiency high. Also, the single-step process of mi-
process faster
crofluidics withthe
makes keeping
scalingtheup encapsulation efficiency
of the production morehigh. Also,
reliable bythe single-step
reducing process
the variables
of
experienced within the process. However, producing large industrial patches usingthe
microfluidics makes the scaling up of the production more reliable by reducing mi-
variables
crofluidicexperienced
technology iswithin
still a the process.
challenge, However,
which producing
is hindering large industrial
its wide-scale patches
implementation
using microfluidic method.
as a formulation technology Thisis still a challenge,
is mainly due towhich is hindering
the high its wide-scalesystems,
cost of microfluidic imple-
mentation as a formulation method. This is mainly due to the high
although once they are established, multiple systems running in parallel could provide cost of microfluidic
systems,
continuousalthough once of
production they are established,
liposomes. Once anmultiple systems
established running
synthesis in parallel
method could
is chosen for
provide
liposomal continuous
productionproduction of liposomes.
using microfluidics, Once
it could an established
attract various sourcessynthesis method
of funding whichis
chosen for liposomal
could lead production
to it eventually being used usingasmicrofluidics, it couldmaterials
a method to provide attract various sources
for clinical use.of
funding which et
Gkionisa could lead to
al. [154], it eventually
reported being used asof
the manufacturing a method
co-loaded to provide
liposomes materials for
using thin-
clinical use.
film hydration and MFs and comparing the performance of both formulations. Using
Gkionisa
different et al. [154],
methods, DSPCreported
is used the for manufacturing
manufacturing of co-loadedand
liposomes liposomes
loadingusing
them thin-
with
film hydrationumbelliprenin
doxorubicin: and MFs and comparing
chemotherapy. the performance of both formulations.
The physicochemical properties and Using dif-
toxicity
ferent
profilemethods,
compared DSPC
for is used
both for manufacturing
formulations. liposomes
The study and loading
investigated them with
the ability doxo-
of MFs to
rubicin: umbelliprenin chemotherapy. The physicochemical properties and toxicity pro-
file compared for both formulations. The study investigated the ability of MFs to provide
cost-effective production of co-loaded liposomes with enhanced stability and advanced
drug loading capacity. The results show a controlled particle size (100–250 nm), uniform
Nanomaterials 2021, 11, 3440 21 of 30

provide cost-effective production of co-loaded liposomes with enhanced stability and

advanced drug loading capacity. The results show a controlled particle size (100–250 nm),
uniform formulation, and doxorubicin drug loading of >80%. Both formulations were
examined in a panel of human breast cancer cells to test their potency. MFs shows a quick,
reliable method to produce size-controlled liposomes with preserved doxorubicin efficacy
and presents low toxicity effect in human breast cells in vitro [154].
The second microfluidic method is the droplet microfluidic process, which requires the
dissolution of the phospholipids in hexane solvent to prepare large liposomes (4–20 µm).
The use of the organic solvent hexane during the manufacturing process of liposomes can
lead to a risk of toxicity. Also, the pulsed jet flow process looks like a modified form of
the film hydration method, that the lipid phase should be dried inside microtubes. That is
followed by hydration of the resulted lipid film by insertion and perfusion process to create
more giant vesicles, 200–534 µm, with an exceptional encapsulation efficiency [155,156].
This method’s primary advantage is given an opportunity to produce vesicles with the
wanted size because of the methods’ versatility and flexibility. The main limitations of this
method involve the difficulty for large-scale production, as well as the commanding use of
organic solvent and extreme agitation [75].

6.1.2. Microfluidics in the Production of SLNs

Solid lipid nanoparticle (SLN) is a promising alternative for the conventional NP-
based DDS (e.g., lipid emulsions, polymeric, liposomes). SLN formulation has shown high
stability, biocompatibility, and well controlling of the drug release [139]. Despite that, the
broad size distribution, the unreproducible formulation and the difficulties in scaling-up
lead to multiple drawbacks of SLN formulations [139]. MF has the potential to overcome
the limitations of the conventional methods, for example, reducing the consumption of
the organic solvents, avoiding the wasting of large quantities of drugs and reagents, and
provides a low-cost automated system. Compared to the other conventional method for
SLN manufacturing, MFs offers high control on the process parameters and conditions [157].
The MF constant continuous flow with well-controlled conditions produces SLN with
controlled particle size, narrow size distribution, high reproducibility, and low batch-to-
batch variations [139].
Based on the literature [139,158], most of the papers that used microfluidics for SLN
manufacturing use the same procedure. The reported procedure is started by dissolving
both lipid and drug in an organic solvent (e.g., ethanol) and inserting thus into the MF
device as the inner fluid beside an aqueous phase with a surfactant inserted as the con-
tinuous outer fluid. Both fluids could flow at a constant rate from separated syringes to
the MF tubes and then to the chip [158]. Regulating the lipid to drug concentration, FRR,
and flow velocity could affect the final SLN results. After the SLN is formed, the organic
solvent should be evaporated, and the particles cooled at 4 ◦ C. An additional purification
step should be performed using a dialysis bag to remove the residual surfactant and the
non-encapsulated drug [157].
Arduino et al. [139] have investigated the effect of utilizing the microfluidic system on
optimizing the SLN characteristics. SLN formulation, was prepared using the bulk method
oil-in-water homogenization process and a MF method to compare the physicochemical
characteristics of both formulations. The SLN was also loaded with paclitaxel (PXT) and
sorafenib (SFN) to test the anti-proliferative efficiency of SLN produced by MFs and those
produced by the bulk method. There was a significant variation in the particle size and PDI
of the different formulated SLN. MFs produced SLNs with a small particle size (121 ± 0 nm)
and well controlled PDI (0.11 ± 0.20). On the other hand, the bulk method SLN was larger
(246 ± 6 nm) and less homogenous (PDI 0.32 ± 080). The encapsulation efficiency of
PXT and SFN was more effective in the MF formulation, and SLN-PXT achieved the best
encapsulation efficiency (54%). The cytotoxic studies of microfluidic SLN were tested in
human alveolar adenocarcinoma (A549) and human glioblastoma cell lines (U87-MG). The
Nanomaterials 2021, 11, 3440 22 of 30

results, show that PTX-SLNs and SFN-SLNs provide a potent anti-proliferative effect and
higher capacity to penetrate the cancer cells than the free drug [139].
However, research using MFs to produce SLN is still limited. Most of the MF applica-
tions are centred on manufacturing liposomes or polymeric NPs. The use of this technology
to make SLNs seems to be a promising area of research, especially since it has overcome
many limitations of the conventional method. One of the main challenges of manufacturing
SLN by MF is concerning the microfluidic chips fabrication material [131].
PDMS based MF chips are sensitive when used with organic solutions, which is a
fundamental component of SLN production as well as the lipophilicity of the PDMS that
may lead to lipid aggregation or adsorption to the microfluidic chip inner surface [131].
Despite that, using a MF glass or silicon chip can resolve these limitations due to their
chemical inertness.

6.1.3. Microfluidics in the Production of Polymeric Formulations

Polymeric NPs gained a high interest in the DDS field, due to their capacity to provide
a controlled drug release profile and effectively encapsulate various drugs and adjust their
release by altering the use of the polymer characteristics [159]. For example, PCL has a
slow degradation rate, making it useful for long-acting formulations [160]. Polymeric
NP systems are commonly used to improve the solubility, reduce toxicity, and control
the retention time of the drug [161]. The effectiveness of the polymeric NPs is based on
the polymer’s nature, the composition of the polymer, and the manufacturing method
(discussed in Section 5) of the polymeric NPs [159]. The main limitations of the polymeric
NP manufacturing methods include time consumption, their capacity for specific type
of drugs (hydrophilic or hydrophobic), requirement for a solvent evaporation step, and
demand for a high quantity of the used materials. MFs has been recognized as an advanced
method that can overcome some of these limitations and improve the manufacturing of PNP
process. MHF is commonly used for the manufacturing of PNP. A suitable polymer can
be dissolved in an organic solvent and mixed with an aqueous stream at specific TFR and
FFR. The technique of MFs is inducing the lipids self-assembly to produce monodispersed
NP without solvent evaporation within seconds [162]. For example, Abstien et al. [163]
compared the particles size and polydispersity of two different PNP formulations, one of
them is prepared by bulk method (nanoprecipitation) and the other by MFs. A copolymer
composed of poly (lactic acid)-poly (ethylene glycol) (PLA-PEG) and poly (lactic-co-glycolic
acid) (PLGA) has been used to manufacture PNP. The different parameters of MFs such as
TFR, FFR, solvent to aqueous ratio are investigated in this study. The TFR was changed from
2 to 17 mL min−1 , and the FRR of the aqueous phase to the organic polymer solution was
either 10:1 or 5:1. The particle size of the bulk preparation method was larger (52–65 nm)
with wide distribution, and the particles prepared by MF produced a smaller particle size
(24–43 nm) with the monodispersed distribution. Also, the MFs’ parameters impact on
particle size has been investigated and the FFR has been identified as the key parameter
to control particle size and improve polydispersity. The results displayed that increasing
the FFR of an equal aqueous to organic phase formulation produces a smaller particle size,
and the PDI was slightly decreased at a low flow rate of 2 mL min−1 and improved at flow
rates of 7–17 mL min−1 (0.064–0.129) [163].
Hanh T.H. Vu et al. [164] have used PLGA to encapsulate rutin (citrus flavonoid) as
a PNP using different methods. One of them is prepared using the bulk method (single
emulsion evaporation method), and the other is by microfluidics. The comparative study
investigates the impact of using microfluidics on particle size, encapsulation efficiency (EE),
and zeta potential. Also, the rutin release was tested in bio relevant media and phosphate
buffer (PBS). The reported results show that the microfluidic formulation has a higher
encapsulation efficiency (34 ± 2%), smaller particle size (123 ± 4 nm), and faster release
than the bulk method (EE 27 ± 1%, size 179 ± 13 nm).
MFs has been used to optimize the loading capacity of PNP. For example, Behnke et al. [165],
have investigated different MF parameters including FRR, TFR, initial polymer concen-
Nanomaterials 2021, 11, 3440 23 of 30

tration, the concentration of the PVA as a surfactant, and their effect on the improvement
of encapsulation efficiency. By using PLGA to encapsulate an anti-inflammatory drug
candidate (dual inhibitor of the 5-lipoxygenase-activating protein (FLAP) and the microso-
mal prostaglandin E2 synthase-1 (mPGES-1)), which are named BRP-18. Clinical studies
reported that the formulation lacks a sufficient loading capacity (LC). To increase the
loading capacity and overcome the limitation the manufacturing method is changed from
bulk to MFs. The impact of several MF parameters have been investigated, such as the
concentration of the PVA as a surfactant, initial polymer concentration, TFR, and FFR. The
resultant particles were in the range from 120 to 260 and PDI values from 0.05 to 0.2. A full-
encapsulation of the drug was reported with 3% BRP-187 (w/w PLGA), 0.3% (w/w) PVA as
a surfactant, and a polymer concentration of 25 mg mL−1 . The highest drug loading (7.3%)
was observed for the formulation of PLGA with c = 15 mg mL and 10% BRP-187 (w/w
PLGA) with 0.3% (w/w) PVA. Changing the manufacturing method from bulk method to
microfluidic increase the (LC) significantly and simplified the whole procedure [165].

6.1.4. Lipid–Polymer Hybrid NPs

Lipid–polymer hybrid nanoparticles (LPHNPs) are a new generation of NPs that
merge the biocompatibility of lipid polymer and the high stability of polymeric NPs [166].
This hybrid system shows a high encapsulation efficiency, cellular targeting properties, and
well-controlled release [166]. Tahira et al. [167], have used MF to encapsulate Sorafenib
(chemotherapy) into LPHNPs. The LPHNPs were composed of PLGA, Lecithin, and DSPE-
PEG 2000. In the study, the LPHNPs formulation were prepared by a bulk method and MFs,
and the encapsulation efficiency results were compared. The formulation produced by MFs
shows a higher encapsulation efficiency (95.0%, 93.8%, and 88.7%) and a well-controlled
release in comparison to the formulation prepared by the bulk methods. The cell-growth
inhibition experiment, safety and stability studies have been performed on breast and
prostate cancer cells. The results show that the LPHNPs formulation was more stable, less
toxic, and more potent in inhibiting cancer cell growth than free Sorafenib [167].

6.1.5. Microfluidics in Synthesis of Inorganic Nanoparticles

Recently, microfluidics has been used to synthesize different types of inorganic NPs,
including gold [168], quantum dots [169], and magnetic NPs [170]. The high controlled
system of Microfluidics can produce inorganic NPs with higher quality than those pro-
duced with conventional methods. For example, Larrea et al. [170], have used gas slug
microfluidics to create a magnetic nanostructure. The results show that microfluidic gas
slugs can produce iron oxide nanoparticles with high purity and provide high control on
the product characteristics (shape, size, and crystalline phase). As well as increasing the re-
producibility of the iron oxide nanoparticles due to the reduction in the total manufacturing
process duration.

7. Conclusions & Future Predictions

The nanoscale DDSs shows significant success in targeting drug delivery and improv-
ing the whole therapeutic process. NPs have been employed in various drug delivery
formulations that are today in markets and utilized for disease therapy or diagnosis. The
development of drug-loaded NPs from the preclinical stage to the industrial scaling up
is challenging due to the difficulty of producing a size-controlled, reproducible, stable
formulation with high encapsulation efficiencies and low batch-to-batch variety. The par-
ticle size, PDI, and encapsulation efficiency are affected mainly by the manufacturing
method. The different NP conventional manufacturing methods are limited in producing
NPs with small particle size and narrow size distribution. Also, the multiple steps of
the traditional manufacturing procedures (e.g., using mechanical energy for mixing or
milling, the requirement for an additional purification step) make them time-consuming,
high cost, and negatively affect the properties of the particles. The development of the
microfluidic system provides a one-step procedure with a constant and continuous process
Nanomaterials 2021, 11, 3440 24 of 30

of NP production. The advanced microfluidic design enables the automatic adjustment of

the process parameters, making the process controllable and enhances the possibility to
produce the optimum particle size and PDI. Many comparative studies are reported in the
literature displaying the difference between the bulk methods and microfluidic method
in the assembled particle size, PDI, and encapsulation efficiency. Moreover, cell culture
studies and cytotoxicity studies, confirm that enhancing the physicochemical properties of
NPs can improve their therapeutic effectiveness, boost potency, and reduce the toxicity of
the drug. It can be concluded that the manufacturing method highly affects the resultant
particles. Among all conventional methods, the NPs produced by microfluidics methods
are more desirable in size, PDI, morphology, therapeutic efficacy, and toxicity.
Future advancements of microfluidic systems can optimize NPs production, extend
their use in therapy, and integrate them into new medical applications. Coupling the
system with complementary devices (e.g., Process analytical technology (PAT)) can control,
analyze, and observe the sample changes during and at different stages of the particle’s
fabrication process. Inserting PAT probes can test the physicochemical properties, including
the particle size, surface area, surface charge, and morphology beside the FTIR analyses
of the manufacturing particle. This significant modification will make the formation,
characterization, and testing of NP a complete, interconnected system. Also, incorporating
molecular imaging technology (e.g., cryo-EM, XFEL) with the organ-on-chip microfluidic
system for addressing the sample delivery shows promising results.
Microfluidics is a high-yielding field for research and development. Improving the
microfluidic system itself or merging it with another device to develop NPs, process
en-bloc is a vital step for the future of NPs. MFs in the near future can expect innova-
tive devices, solutions, and designs to be applied in the NP field and for different new
medical applications.

Author Contributions: E.J.: writing—original draft, writing—review and editing. E.W.: writing—
review and editing. A.M.: writing—review and editing. D.A.L.: project administration, resources,
supervision, writing—review and editing. All authors have read and agreed to the published version
of the manuscript.
Funding: Partial of this research was funded by Fluigent.
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 is
not applicable to this article.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Shirai, Y.; Osgood, A.J.; Zhao, Y.; Yao, Y.; Saudan, L.; Yang, H.; Yu-Hung, C.; Alemany, L.B.; Sasaki, T.; Morin, J.-F. Surface-rolling
molecules. J. Am. Chem. Soc. 2006, 128, 4854–4864. [CrossRef]
2. Bayda, S.; Adeel, M.; Tuccinardi, T.; Cordani, M.; Rizzolio, F. The History of Nanoscience and Nanotechnology: From Chemical-
Physical Applications to Nanomedicine. Molecules 2019, 25, 112. [CrossRef] [PubMed]
3. Grobert, N.; Hutton, D. Nanoscience and nanotechnologies: Opportunities and uncertainties. Lond. R. Soc. R. Acad. Eng. Rep.
2004, 46, 618.
4. Thiruvengadam, M.; Rajakumar, G.; Chung, I.M. Nanotechnology: Current uses and future applications in the food industry.
3 Biotech 2018, 8, 74. [CrossRef] [PubMed]
5. Khan, I.; Saeed, K.; Khan, I. Nanoparticles: Properties, applications and toxicities. Arab. J. Chem. 2019, 12, 908–931. [CrossRef]
6. Zhang, J.Z.; Noguez, C. Plasmonic Optical Properties and Applications of Metal Nanostructures. Plasmonics 2008, 3, 127–150.
[CrossRef]
7. Duncan, R. The dawning era of polymer therapeutics. Nat. Rev. Drug Discov. 2003, 2, 347–360. [CrossRef]
8. De Jong, W.H.; Borm, P.J. Drug delivery and nanoparticles:applications and hazards. Int. J. Nanomed. 2008, 3, 133–149. [CrossRef]
9. Bowman, D.M. More than a decade on: Mapping today’s regulatory and policy landscapes following the publication of
nanoscience and nanotechnologies: Opportunities and uncertainties. NanoEthics 2017, 11, 169–186. [CrossRef]
Nanomaterials 2021, 11, 3440 25 of 30

10. Cascone, M.G.; Lazzeri, L.; Carmignani, C.; Zhu, Z. Gelatin nanoparticles produced by a simple W/O emulsion as delivery
system for methotrexate. J. Mater. Sci. Mater. Med. 2002, 13, 523–526. [CrossRef]
11. Kipp, J.E. The role of solid nanoparticle technology in the parenteral delivery of poorly water-soluble drugs. Int. J. Pharm. 2004,
284, 109–122. [CrossRef] [PubMed]
12. LaVan, D.A.; McGuire, T.; Langer, R. Small-scale systems for in vivo drug delivery. Nat. Biotechnol. 2003, 21, 1184–1191. [CrossRef]
[PubMed]
13. Abdolmaleki, A.; Asadi, A.; Gurushankar, K.; Karimi Shayan, T.; Abedi Sarvestani, F. Importance of Nano Medicine and New
Drug Therapies for Cancer. Adv. Pharm. Bull. 2021, 11, 450–457. [CrossRef]
14. Abdel-Mottaleb, M.M.; Neumann, D.; Lamprecht, A. Lipid nanocapsules for dermal application: A comparative study of
lipid-based versus polymer-based nanocarriers. Eur. J. Pharm. Biopharm. 2011, 79, 36–42. [CrossRef] [PubMed]
15. Nel, A.E.; Mädler, L.; Velegol, D.; Xia, T.; Hoek, E.M.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding
biophysicochemical interactions at the nano-bio interface. Nat. Mater. 2009, 8, 543–557. [CrossRef] [PubMed]
16. Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K.A. Nanoparticle size and surface properties determine the
protein corona with possible implications for biological impacts. Proc. Natl. Acad. Sci. USA 2008, 105, 14265–14270. [CrossRef]
17. Bonadio, J.; Smiley, E.; Patil, P.; Goldstein, S. Localized, direct plasmid gene delivery in vivo: Prolonged therapy results in
reproducible tissue regeneration. Nat. Med. 1999, 5, 753–759. [CrossRef]
18. Hamilton, A.J.; Baulcombe, D.C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 1999,
286, 950–952. [CrossRef] [PubMed]
19. Ding, Y.; Jiang, Z.; Saha, K.; Kim, C.S.; Kim, S.T.; Landis, R.F.; Rotello, V.M. Gold Nanoparticles for Nucleic Acid Delivery. Mol.
Ther. 2014, 22, 1075–1083. [CrossRef]
20. Draz, M.S.; Fang, B.A.; Zhang, P.; Hu, Z.; Gu, S.; Weng, K.C.; Gray, J.W.; Chen, F.F. Nanoparticle-mediated systemic delivery of
siRNA for treatment of cancers and viral infections. Theranostics 2014, 4, 872–892. [CrossRef]
21. DePristo, M.A.; Banks, E.; Poplin, R.; Garimella, K.V.; Maguire, J.R.; Hartl, C.; Philippakis, A.A.; del Angel, G.; Rivas, M.A.;
Hanna, M.; et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet.
2011, 43, 491–498. [CrossRef]
22. Chen, A.A.; Derfus, A.M.; Khetani, S.R.; Bhatia, S.N. Quantum dots to monitor RNAi delivery and improve gene silencing.
Nucleic Acids Res. 2005, 33, e190. [CrossRef]
23. Shinde, R.R.; Bachmann, M.H.; Wang, Q.; Kasper, R.; Contag, C.H. PEG-PLA/PLGA Nanoparticles for In-Vivo RNAi Delivery; NSTI
Nano Tech.: Santa Clara, CA, USA, 2007.
24. Tan, W.B.; Jiang, S.; Zhang, Y. Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference.
Biomaterials 2007, 28, 1565–1571. [CrossRef] [PubMed]
25. Howard, K.A.; Rahbek, U.L.; Liu, X.; Damgaard, C.K.; Glud, S.Z.; Andersen, M.Ø.; Hovgaard, M.B.; Schmitz, A.; Nyengaard, J.R.;
Besenbacher, F.; et al. RNA Interference in Vitro and in Vivo Using a Novel Chitosan/siRNA Nanoparticle System. Mol. Ther.
2006, 14, 476–484. [CrossRef]
26. Gao, W.; Xiao, Z.; Radovic-Moreno, A.; Shi, J.; Langer, R.; Farokhzad, O.C. Progress in siRNA delivery using multifunctional
nanoparticles. Methods Mol. Biol. 2010, 629, 53–67. [CrossRef] [PubMed]
27. Liu, G.; Swierczewska, M.; Lee, S.; Chen, X. Functional nanoparticles for molecular imaging guided gene delivery. Nano Today
2010, 5, 524–539. [CrossRef] [PubMed]
28. Soutschek, J.; Akinc, A.; Bramlage, B.; Charisse, K.; Constien, R.; Donoghue, M.; Elbashir, S.; Geick, A.; Hadwiger, P.; Harborth, J.;
et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 2004, 432, 173–178.
[CrossRef] [PubMed]
29. Wolfrum, C.; Shi, S.; Jayaprakash, K.N.; Jayaraman, M.; Wang, G.; Pandey, R.K.; Rajeev, K.G.; Nakayama, T.; Charrise, K.; Ndungo,
E.M.; et al. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat. Biotechnol. 2007, 25, 1149–1157. [CrossRef]
30. Naito, M.; Ishii, T.; Matsumoto, A.; Miyata, K.; Miyahara, Y.; Kataoka, K. A phenylboronate-functionalized polyion complex
micelle for ATP-triggered release of siRNA. Angew. Chem. Int. Ed. Engl. 2012, 51, 10751–10755. [CrossRef]
31. Lee, S.J.; Huh, M.S.; Lee, S.Y.; Min, S.; Lee, S.; Koo, H.; Chu, J.U.; Lee, K.E.; Jeon, H.; Choi, Y. Tumor-homing poly-siRNA/glycol
chitosan self-cross-linked nanoparticles for systemic siRNA delivery in cancer treatment. Angew. Chem. Int. Ed. 2012, 51,
7203–7207. [CrossRef]
32. Cousin, J.M.; Cloninger, M.J. The role of galectin-1 in cancer progression, and synthetic multivalent systems for the study of
galectin-1. Int. J. Mol. Sci. 2016, 17, 1566. [CrossRef] [PubMed]
33. Ting, S.S.; Chen, G.; Stenzel, M.H. Synthesis of glycopolymers and their multivalent recognitions with lectins. Polym. Chem. 2010,
1, 1392–1412. [CrossRef]
34. Kadam, R.U.; Bergmann, M.; Hurley, M.; Garg, D.; Cacciarini, M.; Swiderska, M.A.; Nativi, C.; Sattler, M.; Smyth, A.R.; Williams,
P. A glycopeptide dendrimer inhibitor of the galactose-specific lectin LecA and of Pseudomonas aeruginosa biofilms. Angew.
Chem. Int. Ed. 2011, 50, 10631–10635. [CrossRef] [PubMed]
35. Marradi, M.; Chiodo, F.; García, I.; Penadés, S. Glyconanoparticles as multifunctional and multimodal carbohydrate systems.
Chem. Soc. Rev. 2013, 42, 4728–4745. [CrossRef]
36. Sharma, R.; Kottari, N.; Chabre, Y.M.; Abbassi, L.; Shiao, T.C.; Roy, R. A highly versatile convergent/divergent “onion peel”
synthetic strategy toward potent multivalent glycodendrimers. Chem. Commun. 2014, 50, 13300–13303. [CrossRef]
Nanomaterials 2021, 11, 3440 26 of 30

37. Liu, L.-H.; Dietsch, H.; Schurtenberger, P.; Yan, M. Photoinitiated coupling of unmodified monosaccharides to iron oxide
nanoparticles for sensing proteins and bacteria. Bioconjugate Chem. 2009, 20, 1349–1355. [CrossRef] [PubMed]
38. Jayaraman, N.; Maiti, K.; Naresh, K. Multivalent glycoliposomes and micelles to study carbohydrate–protein and carbohydrate–
carbohydrate interactions. Chem. Soc. Rev. 2013, 42, 4640–4656. [CrossRef]
39. Vedala, H.; Chen, Y.; Cecioni, S.; Imberty, A.; Vidal, S.; Star, A. Nanoelectronic detection of lectin-carbohydrate interactions using
carbon nanotubes. Nano Lett. 2011, 11, 170–175. [CrossRef]
40. Besford, Q.A.; Wojnilowicz, M.; Suma, T.; Bertleff-Zieschang, N.; Caruso, F.; Cavalieri, F. Lactosylated Glycogen Nanoparticles for
Targeting Prostate Cancer Cells. ACS Appl. Mater. Interfaces 2017, 9, 16869–16879. [CrossRef]
41. Costantino, L.; Gandolfi, F.; Tosi, G.; Rivasi, F.; Vandelli, M.A.; Forni, F. Peptide-derivatized biodegradable nanoparticles able to
cross the blood-brain barrier. J. Control. Release 2005, 108, 84–96. [CrossRef]
42. Sumner, J.P.; Kopelman, R. Alexa Fluor 488 as an iron sensing molecule and its application in PEBBLE nanosensors. Analyst 2005,
130, 528–533. [CrossRef]
43. Michaelis, K.; Hoffmann, M.M.; Dreis, S.; Herbert, E.; Alyautdin, R.N.; Michaelis, M.; Kreuter, J.; Langer, K. Covalent linkage of
apolipoprotein e to albumin nanoparticles strongly enhances drug transport into the brain. J. Pharmacol. Exp. Ther. 2006, 317,
1246–1253. [CrossRef] [PubMed]
44. Steiniger, S.C.; Kreuter, J.; Khalansky, A.S.; Skidan, I.N.; Bobruskin, A.I.; Smirnova, Z.S.; Severin, S.E.; Uhl, R.; Kock, M.; Geiger,
K.D.; et al. Chemotherapy of glioblastoma in rats using doxorubicin-loaded nanoparticles. Int. J. Cancer 2004, 109, 759–767.
[CrossRef] [PubMed]
45. Acharya, S.; Sahoo, S.K. PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect. Adv. Drug
Deliv. Rev. 2011, 63, 170–183. [CrossRef]
46. Rademakers, T.; Horvath, J.M.; van Blitterswijk, C.A.; LaPointe, V.L. Oxygen and nutrient delivery in tissue engineering:
Approaches to graft vascularization. J. Tissue Eng. Regen. Med. 2019, 13, 1815–1829. [CrossRef] [PubMed]
47. Teleanu, R.I.; Chircov, C.; Grumezescu, A.M.; Teleanu, D.M. Tumor Angiogenesis and Anti-Angiogenic Strategies for Cancer
Treatment. J. Clin. Med. 2020, 9, 84. [CrossRef] [PubMed]
48. Fu, S.; Xu, X.; Ma, Y.; Zhang, S.; Zhang, S. RGD peptide-based non-viral gene delivery vectors targeting integrin αvβ3 for cancer
therapy. J. Drug Target. 2019, 27, 1–11. [CrossRef]
49. Majumder, P. Integrin-mediated delivery of drugs and nucleic acids for anti-angiogenic cancer therapy: Current landscape and
remaining challenges. Bioengineering 2018, 5, 76. [CrossRef]
50. Bharali, D.J.; Rajabi, M.; Mousa, S.A. Chapter 11—Application of Nanotechnology to Target Tumor Angiogenesis in Cancer
Therapeutics. In Anti-Angiogenesis Strategies in Cancer Therapeutics; Mousa, S.A., Davis, P.J., Eds.; Academic Press: Boston, MA,
USA, 2017; pp. 165–178. [CrossRef]
51. Li, L.; Wartchow, C.A.; Danthi, S.N.; Shen, Z.; Dechene, N.; Pease, J.; Choi, H.S.; Doede, T.; Chu, P.; Ning, S. A novel antiangiogen-
esis therapy using an integrin antagonist or anti–Flk-1 antibody coated 90Y-labeled nanoparticles. Int. J. Radiat. Oncol. Biol. Phys.
2004, 58, 1215–1227. [CrossRef] [PubMed]
52. Zuo, H. iRGD: A promising peptide for cancer imaging and a potential therapeutic agent for various cancers. J. Oncol. 2019,
2019, 9367845. [CrossRef]
53. Yuan, Y.; Wang, Z.; Cai, P.; Liu, J.; Liao, L.-D.; Hong, M.; Chen, X.; Thakor, N.; Liu, B. Conjugated polymer and drug co-
encapsulated nanoparticles for chemo-and photo-thermal combination therapy with two-photon regulated fast drug release.
Nanoscale 2015, 7, 3067–3076. [CrossRef]
54. Murphy, E.A.; Majeti, B.K.; Barnes, L.A.; Makale, M.; Weis, S.M.; Lutu-Fuga, K.; Wrasidlo, W.; Cheresh, D.A. Nanoparticle-
mediated drug delivery to tumor vasculature suppresses metastasis. Proc. Natl. Acad. Sci. USA 2008, 105, 9343–9348. [CrossRef]
55. Rebbaa, A.; Chu, F.; Davis, F.B.; Davis, P.J.; Mousa, S.A. Novel function of the thyroid hormone analog tetraiodothyroacetic acid:
A cancer chemosensitizing and anti-cancer agent. Angiogenesis 2008, 11, 269–276. [CrossRef] [PubMed]
56. Mousa, S.A.; Bergh, J.J.; Dier, E.; Rebbaa, A.; O’Connor, L.J.; Yalcin, M.; Aljada, A.; Dyskin, E.; Davis, F.B.; Lin, H.-Y. Tetraiodothy-
roacetic acid, a small molecule integrin ligand, blocks angiogenesis induced by vascular endothelial growth factor and basic
fibroblast growth factor. Angiogenesis 2008, 11, 183–190. [CrossRef] [PubMed]
57. Rajabi, M.; Sudha, T.; Darwish, N.H.; Davis, P.J.; Mousa, S.A. Synthesis of MR-49, a deiodinated analog of tetraiodothyroacetic
acid (tetrac), as a novel pro-angiogenesis modulator. Bioorganic Med. Chem. Lett. 2016, 26, 4112–4116. [CrossRef] [PubMed]
58. Bharali, D.J.; Yalcin, M.; Davis, P.J.; Mousa, S.A. Tetraiodothyroacetic acid-conjugated PLGA nanoparticles: A nanomedicine
approach to treat drug-resistant breast cancer. Nanomedicine 2013, 8, 1943–1954. [CrossRef] [PubMed]
59. Kautz-Neu, K.; Kostka, S.L.; Dinges, S.; Iwakura, Y.; Udey, M.C.; von Stebut, E. IL-1 signalling is dispensable for protective
immunity in Leishmania-resistant mice. Exp. Dermatol. 2011, 20, 76–78. [CrossRef] [PubMed]
60. Dewamitta, S.R.; Nomura, T.; Kawamura, I.; Hara, H.; Tsuchiya, K.; Kurenuma, T.; Shen, Y.; Daim, S.; Yamamoto, T.; Qu, H.; et al.
Listeriolysin O-dependent bacterial entry into the cytoplasm is required for calpain activation and interleukin-1 alpha secretion
in macrophages infected with Listeria monocytogenes. Infect. Immun. 2010, 78, 1884–1894. [CrossRef]
61. Robinson, M.W.; O’Brien, R.; Mackintosh, C.G.; Clark, R.G.; Griffin, J.F. Immunoregulatory cytokines are associated with
protection from immunopathology following Mycobacterium avium subspecies paratuberculosis infection in red deer. Infect.
Immun. 2011, 79, 2089–2097. [CrossRef]
Nanomaterials 2021, 11, 3440 27 of 30

62. Zhang, D.; Tan, T.; Gao, L.; Zhao, W.; Wang, P. Preparation of azithromycin nanosuspensions by high pressure homogenization
and its physicochemical characteristics studies. Drug Dev. Ind. Pharm. 2007, 33, 569–575. [CrossRef]
63. Suri, S.S.; Fenniri, H.; Singh, B. Nanotechnology-based drug delivery systems. J. Occup. Med. Toxicol. 2007, 2, 16. [CrossRef]
[PubMed]
64. Chen, X.; Plasencia, C.; Hou, Y.; Neamati, N. Synthesis and biological evaluation of dimeric RGD peptide− paclitaxel conjugate
as a model for integrin-targeted drug delivery. J. Med. Chem. 2005, 48, 1098–1106. [CrossRef] [PubMed]
65. R Rama, A.; Jimenez-Lopez, J.; Cabeza, L.; Jimenez-Luna, C.; C Leiva, M.; Perazzoli, G.; Hernandez, R.; Zafra, I.; Ortiz, R.;
Melguizo, C. Last advances in nanocarriers-based drug delivery systems for colorectal cancer. Curr. Drug Deliv. 2016, 13, 830–838.
[CrossRef] [PubMed]
66. García-Pinel, B.; Porras-Alcalá, C.; Ortega-Rodríguez, A.; Sarabia, F.; Prados, J.; Melguizo, C.; López-Romero, J.M. Lipid-Based
Nanoparticles: Application and Recent Advances in Cancer Treatment. Nanomaterials 2019, 9, 638. [CrossRef] [PubMed]
67. Gregoriadis, G. Fate of injected liposomes: Observations on entrapped solute retention, vesicle clearance and tissue distribution
in vivo. In Liposomes as Drug Carriers: Recent Trends and Progress; Wiley: New York, NY, USA, 1988; pp. 3–18.
68. Lurquin, P.F. Incorporation of genetic material into liposomes and transfer to cells. In Liposome Technology, 2nd ed.; Gregoriadis,
G., Ed.; CRC Press: Boca Raton, FL, USA, 1993; Volume 2, pp. 129–139.
69. Gregoriadis, G. Liposome Technology: Volume II; CRC Press: Boca Raton, FL, USA, 2018.
70. Thompson, A.K. Structure and Properties of Liposomes Prepared from Milk Phospholipids: A Thesis Presented in Partial
Fulfilment of the Requirements for the Degree of Doctor of Philosophy in Food Technology. Ph.D. Thesis, Massey University,
Palmerston North, New Zealand, 2005.
71. Shah, S.; Dhawan, V.; Holm, R.; Nagarsenker, M.S.; Perrie, Y. Liposomes: Advancements and innovation in the manufacturing
process. Adv. Drug Deliv. Rev. 2020, 154–155, 102–122. [CrossRef]
72. Salim, M.; Minamikawa, H.; Sugimura, A.; Hashim, R. Amphiphilic designer nano-carriers for controlled release: From drug
delivery to diagnostics. MedChemComm 2014, 5, 1602–1618. [CrossRef]
73. Alam, S.; Mattern-Schain, S.; Best, M. Targeting and triggered release using lipid-based supramolecular assemblies as medicinal
nanocarriers. In Comprehensive Supramolecular Chemistry II; Elsevier: Oxford, UK, 2017; pp. 329–364.
74. Immordino, M.L.; Dosio, F.; Cattel, L. Stealth liposomes: Review of the basic science, rationale, and clinical applications, existing
and potential. Int. J. Nanomed. 2006, 1, 297.
75. Maherani, B.; Arab-Tehrany, E.; Mozafari, M.R.; Gaiani, C.; Linder, M. Liposomes: A review of manufacturing techniques and
targeting strategies. Curr. Nanosci. 2011, 7, 436–452. [CrossRef]
76. Machado, A.R.; de Assis, L.M.; Machado, M.I.R.; de Souza-Soares, L.A. Importance of lecithin for encapsulation processes. Afr. J.
Food Sci. 2014, 8, 176–183.
77. Sharma, D.; Ali, A.A.E.; Trivedi, L.R. An Updated Review on: Liposomes as drug delivery system. PharmaTutor 2018, 6, 50–62.
[CrossRef]
78. Dua, J.; Rana, A.; Bhandari, A. Liposome: Methods of preparation and applications. Int. J. Pharm. Stud. Res. 2012, 3, 14–20.
79. Couvreur, P.; Dubernet, C.; Puisieux, F. Controlled drug delivery with nanoparticles: Current possibilities and future trends. Eur.
J. Pharm. Biopharm. 1995, 41, 2–13.
80. Burgess, S. Liposome Preparation-Avanti® Polar Lipids; Sigma-Aldrich: St. Louis, MO, USA, 1998.
81. Mehnert, W.; Mäder, K. Solid lipid nanoparticles: Production, characterization and applications. Adv. Drug Deliv. Rev. 2001, 47,
165–196. [CrossRef]
82. Mishra, V.; Bansal, K.K.; Verma, A.; Yadav, N.; Thakur, S.; Sudhakar, K.; Rosenholm, J.M. Solid lipid nanoparticles: Emerging
colloidal nano drug delivery systems. Pharmaceutics 2018, 10, 191. [CrossRef]
83. Lin, C.-H.; Chen, C.-H.; Lin, Z.-C.; Fang, J.-Y. Recent advances in oral delivery of drugs and bioactive natural products using
solid lipid nanoparticles as the carriers. J. Food Drug Anal. 2017, 25, 219–234. [CrossRef] [PubMed]
84. Geszke-Moritz, M.; Moritz, M. Solid lipid nanoparticles as attractive drug vehicles: Composition, properties and therapeutic
strategies. Mater. Sci. Eng. C 2016, 68, 982–994. [CrossRef] [PubMed]
85. Helgason, T.; Awad, T.S.; Kristbergsson, K.; McClements, D.J.; Weiss, J. Effect of surfactant surface coverage on formation of solid
lipid nanoparticles (SLN). J. Colloid Interface Sci. 2009, 334, 75–81. [CrossRef]
86. Botto, C.; Mauro, N.; Amore, E.; Martorana, E.; Giammona, G.; Bondì, M.L. Surfactant effect on the physicochemical characteristics
of cationic solid lipid nanoparticles. Int. J. Pharm. 2017, 516, 334–341. [CrossRef] [PubMed]
87. Uner, M.; Yener, G. Importance of solid lipid nanoparticles (SLN) in various administration routes and future perspectives. Int. J.
Nanomed. 2007, 2, 289–300.
88. Battaglia, L.; Gallarate, M. Lipid nanoparticles: State of the art, new preparation methods and challenges in drug delivery. Expert
Opin. Drug Deliv. 2012, 9, 497–508. [CrossRef]
89. Yadav, N.; Khatak, D.; Sara, U.V. Solid lipid nanoparticles—A review. Int. J. Appl. Pharm. 2013, 5, 8–18.
90. Kasongo, K.W.; Müller, R.H.; Walker, R.B. The use of hot and cold high pressure homogenization to enhance the loading capacity
and encapsulation efficiency of nanostructured lipid carriers for the hydrophilic antiretroviral drug, didanosine for potential
administration to paediatric patients. Pharm. Dev. Technol. 2012, 17, 353–362. [CrossRef] [PubMed]
91. Triplett, M.D.; Rathman, J.F. Optimization of β-carotene loaded solid lipid nanoparticles preparation using a high shear
homogenization technique. J. Nanopart. Res. 2009, 11, 601–614. [CrossRef]
Nanomaterials 2021, 11, 3440 28 of 30

92. Lander, R.; Manger, W.; Scouloudis, M.; Ku, A.; Davis, C.; Lee, A. Gaulin homogenization: A mechanistic study. Biotechnol. Prog.
2000, 16, 80–85. [CrossRef] [PubMed]
93. Müller, R.H.; Peters, K. Nanosuspensions for the formulation of poorly soluble drugs: I. Preparation by a size-reduction technique.
Int. J. Pharm. 1998, 160, 229–237. [CrossRef]
94. Ahlin, P.; Kristl, J.; Smid-Korbar, J. Optimization of procedure parameters and physical stability of solid lipid nanoparticles in
dispersions. Acta Pharm. (Zagreb) 1998, 48, 259–267.
95. Mukherjee, S.; Ray, S.; Thakur, R.S. Solid lipid nanoparticles: A modern formulation approach in drug delivery system. Indian J.
Pharm. Sci. 2009, 71, 349–358. [CrossRef]
96. Zur Mühlen, A. Feste Lipid Nanopartikel mit Prolongierter Wirkstoffliberation: Herstellung, Langzeitstabilität, Charakterisierung,
Freisetzungsverhalten und-Mechanismen (Solid Lipid Nanoparticles with Prolonged Drug Rrelease: Production, Long-Term
Stability, Characterisation, Release Pproperties and Mechanisms). Ph.D. Thesis, Free University of Berlin, Berlin, Germany, 1996.
97. Eldem, T.; Speiser, P.; Hincal, A. Optimization of spray-dried and -congealed lipid micropellets and characterization of their
surface morphology by scanning electron microscopy. Pharm. Res. 1991, 8, 47–54. [CrossRef]
98. Nikam, S.; Chavan, M.; Sharma, P.H. Solid lipid nanoparticles: A lipid based drug delivery. Nanotechnology 2014, 1, 5.
99. Magenheim, B.; Levy, M.Y.; Benita, S. A new in vitro technique for the evaluation of drug release profile from colloidal carriers—
Ultrafiltration technique at low pressure. Int. J. Pharm. 1993, 94, 115–123. [CrossRef]
100. del Pozo-Rodríguez, A.; Solinís, M.A.; Gascón, A.R.; Pedraz, J.L. Short- and long-term stability study of lyophilized solid lipid
nanoparticles for gene therapy. Eur. J. Pharm. Biopharm. 2008, 71, 181–189. [CrossRef] [PubMed]
101. Mehnert, W.; Mäder, K. Solid lipid nanoparticles: Production, characterization and applications. Adv. Drug Deliv. Rev. 2012, 64,
83–101. [CrossRef]
102. Gasco, M. Solid Lipid Nanospheres from Warm Micro-Emulsions: Improvements in SLN production for more efficient drug
delivery. Pharm. Technol. Eur. 1997, 9, 52–58.
103. Ganesan, P.; Narayanasamy, D. Lipid nanoparticles: Different preparation techniques, characterization, hurdles, and strategies
for the production of solid lipid nanoparticles and nanostructured lipid carriers for oral drug delivery. Sustain. Chem. Pharm.
2017, 6, 37–56. [CrossRef]
104. Ahlin Grabnar, P.; Kristl, J. The manufacturing techniques of drug-loaded polymeric nanoparticles from preformed polymers.
J. Microencapsul. 2011, 28, 323–335. [CrossRef] [PubMed]
105. Zhong, Y.; Meng, F.; Deng, C.; Zhong, Z. Ligand-directed active tumor-targeting polymeric nanoparticles for cancer chemotherapy.
Biomacromolecules 2014, 15, 1955–1969. [CrossRef] [PubMed]
106. Marin, E.; Briceño, M.I.; Caballero-George, C. Critical evaluation of biodegradable polymers used in nanodrugs. Int. J. Nanomed.
2013, 8, 3071.
107. Crucho, C.I.C.; Barros, M.T. Polymeric nanoparticles: A study on the preparation variables and characterization methods. Mater.
Sci. Eng. C 2017, 80, 771–784. [CrossRef]
108. Sala, M.; Miladi, K.; Agusti, G.; Elaissari, A.; Fessi, H. Preparation of liposomes: A comparative study between the double solvent
displacement and the conventional ethanol injection—From laboratory scale to large scale. Colloids Surf. A Physicochem. Eng. Asp.
2017, 524, 71–78. [CrossRef]
109. Ong, S.G.; Chitneni, M.; Lee, K.S.; Ming, L.C.; Yuen, K.H. Evaluation of Extrusion Technique for Nanosizing Liposomes.
Pharmaceutics 2016, 8, 36. [CrossRef]
110. Rodríguez-Rodríguez, A.A.; Moreno-Trejo, M.B.; Meléndez-Zaragoza, M.J.; Collins-Martínez, V.; López-Ortiz, A.; Martínez-
Guerra, E.; Sánchez-Domínguez, M. Spinel-type ferrite nanoparticles: Synthesis by the oil-in-water microemulsion reaction
method and photocatalytic water-splitting evaluation. Int. J. Hydrog. Energy 2019, 44, 12421–12429. [CrossRef]
111. Rao, J.P.; Geckeler, K.E. Polymer nanoparticles: Preparation techniques and size-control parameters. Prog. Polym. Sci. 2011, 36,
887–913. [CrossRef]
112. Kozlowski, M. Polymeric Nanomaterials for Nanotherapeutics. Ed. C. Vasile, Elsevier, 1st Edition. 2018. Series Volume Editors:
Cornelia Vasile, eBook ISBN: 9780128139332; Paperback ISBN: 9780128139325: Elsevier, Published Date: 2nd November 2018;
558 pages. In Micro and Nano Technologies Series. Polymers 2019, 11, 1452. [CrossRef]
113. Crucho, C.I.; Barros, M.T. Formulation of functionalized PLGA polymeric nanoparticles for targeted drug delivery. Polymer 2015,
68, 41–46. [CrossRef]
114. Allouche, J. Synthesis of organic and bioorganic nanoparticles: An overview of the preparation methods. In Nanomaterials:
A Danger or a Promise? Springer: London, UK, 2013; pp. 27–74.
115. Ganachaud, F.; Katz, J.L. Nanoparticles and nanocapsules created using the Ouzo effect: Spontaneous emulsification as an
alternative to ultrasonic and high-shear devices. ChemPhysChem 2005, 6, 209–216. [CrossRef]
116. Konan, Y.N.; Gurny, R.; Allémann, E. Preparation and characterization of sterile and freeze-dried sub-200 nm nanoparticles. Int. J.
Pharm. 2002, 233, 239–252. [CrossRef]
117. Petros, R.A.; DeSimone, J.M. Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discov. 2010, 9,
615–627. [CrossRef] [PubMed]
118. Chaudhary, S.A.; Patel, D.M.; Patel, J.K.; Patel, D.H. Solvent Emulsification Evaporation and Solvent Emulsification Diffusion
Techniques for Nanoparticles. In Emerging Technologies for Nanoparticle Manufacturing; Patel, J.K., Pathak, Y.V., Eds.; Springer
International Publishing: Cham, Switzerland, 2021; pp. 287–300. [CrossRef]
Nanomaterials 2021, 11, 3440 29 of 30

119. Kudr, J.; Haddad, Y.; Richtera, L.; Heger, Z.; Cernak, M.; Adam, V.; Zitka, O. Magnetic nanoparticles: From design and synthesis
to real world applications. Nanomaterials 2017, 7, 243. [CrossRef] [PubMed]
120. Moinard-Chécot, D.; Chevalier, Y.; Briançon, S.; Beney, L.; Fessi, H. Mechanism of nanocapsules formation by the emulsion-
diffusion process. J. Colloid Interface Sci. 2008, 317, 458–468. [CrossRef] [PubMed]
121. Vauthier, C.; Bouchemal, K. Methods for the preparation and manufacture of polymeric nanoparticles. Pharm. Res. 2009, 26,
1025–1058. [CrossRef] [PubMed]
122. Liu, Z.; Jiao, Y.; Wang, Y.; Zhou, C.; Zhang, Z. Polysaccharides-based nanoparticles as drug delivery systems. Adv. Drug Deliv.
Rev. 2008, 60, 1650–1662. [CrossRef] [PubMed]
123. Fornaguera, C.; Feiner-Gracia, N.; Calderó, G.; García-Celma, M.; Solans, C. Galantamine-loaded PLGA nanoparticles, from
nano-emulsion templating, as novel advanced drug delivery systems to treat neurodegenerative diseases. Nanoscale 2015, 7,
12076–12084. [CrossRef] [PubMed]
124. Jeon, H.-J.; Jeong, Y.-I.; Jang, M.-K.; Park, Y.-H.; Nah, J.-W. Effect of solvent on the preparation of surfactant-free poly (DL-lactide-
co-glycolide) nanoparticles and norfloxacin release characteristics. Int. J. Pharm. 2000, 207, 99–108. [CrossRef]
125. Chronopoulou, L.; Fratoddi, I.; Palocci, C.; Venditti, I.; Russo, M.V. Osmosis based method drives the self-assembly of polymeric
chains into micro-and nanostructures. Langmuir 2009, 25, 11940–11946. [CrossRef] [PubMed]
126. Mora-Huertas, C.; Fessi, H.; Elaissari, A. Influence of process and formulation parameters on the formation of submicron particles
by solvent displacement and emulsification–diffusion methods: Critical comparison. Adv. Colloid Interface Sci. 2011, 163, 90–122.
[CrossRef] [PubMed]
127. Chang, J.; Jallouli, Y.; Kroubi, M.; Yuan, X.-B.; Feng, W.; Kang, C.-S.; Pu, P.-Y.; Betbeder, D. Characterization of endocytosis of
transferrin-coated PLGA nanoparticles by the blood–brain barrier. Int. J. Pharm. 2009, 379, 285–292. [CrossRef]
128. Chacon, M.; Berges, L.; Molpeceres, J.; Aberturas, M.; Guzman, M. Optimized preparation of poly D, L (lactic-glycolic) micro-
spheres and nanoparticles for oral administration. Int. J. Pharm. 1996, 141, 81–91. [CrossRef]
129. Xie, H.; Smith, J.W. Fabrication of PLGA nanoparticles with a fluidic nanoprecipitation system. J. Nanobiotechnology 2010, 8, 18.
[CrossRef]
130. Cai, G.; Xue, L.; Zhang, H.; Lin, J. A Review on Micromixers. Micromachines 2017, 8, 274. [CrossRef] [PubMed]
131. Chiesa, E.; Dorati, R.; Pisani, S.; Conti, B.; Bergamini, G.; Modena, T.; Genta, I. The Microfluidic Technique and the Manufacturing
of Polysaccharide Nanoparticles. Pharmaceutics 2018, 10, 267. [CrossRef] [PubMed]
132. Wang, J.; Song, Y. Microfluidic synthesis of nanohybrids. Small 2017, 13, 1604084. [CrossRef] [PubMed]
133. Liu, D.; Zhang, H.; Fontana, F.; Hirvonen, J.T.; Santos, H.A. Current developments and applications of microfluidic technology
toward clinical translation of nanomedicines. Adv. Drug Deliv. Rev. 2018, 128, 54–83. [CrossRef]
134. Lu, M.; Yang, S.; Ho, Y.P.; Grigsby, C.L.; Leong, K.W.; Huang, T.J. Shape-controlled synthesis of hybrid nanomaterials via
three-dimensional hydrodynamic focusing. ACS Nano 2014, 8, 10026–10034. [CrossRef] [PubMed]
135. Rhee, M.; Valencia, P.M.; Rodriguez, M.I.; Langer, R.; Farokhzad, O.C.; Karnik, R. Synthesis of size-tunable polymeric nanoparticles
enabled by 3D hydrodynamic flow focusing in single-layer microchannels. Adv. Mater. 2011, 23, H79–H83. [CrossRef] [PubMed]
136. Greener, J.; Li, W.; Ren, J.; Voicu, D.; Pakharenko, V.; Tang, T.; Kumacheva, E. Rapid, cost-efficient fabrication of microfluidic
reactors in thermoplastic polymers by combining photolithography and hot embossing. Lab Chip 2010, 10, 522–524. [CrossRef]
[PubMed]
137. Fallahi, H.; Zhang, J.; Phan, H.P.; Nguyen, N.T. Flexible Microfluidics: Fundamentals, Recent Developments, and Applications.
Micromachines 2019, 10, 830. [CrossRef] [PubMed]
138. Martin, S.; Bhushan, B. Transparent, wear-resistant, superhydrophobic and superoleophobic poly(dimethylsiloxane) (PDMS)
surfaces. J. Colloid Interface Sci. 2017, 488, 118–126. [CrossRef]
139. Arduino, I.; Liu, Z.; Rahikkala, A.; Figueiredo, P.; Correia, A.; Cutrignelli, A.; Denora, N.; Santos, H.A. Preparation of cetyl
palmitate-based PEGylated solid lipid nanoparticles by microfluidic technique. Acta Biomater. 2021, 121, 566–578. [CrossRef]
[PubMed]
140. Zhang, W.; Lin, S.; Wang, C.; Hu, J.; Li, C.; Zhuang, Z.; Zhou, Y.; Mathies, R.A.; Yang, C.J. PMMA/PDMS valves and pumps for
disposable microfluidics. Lab Chip 2009, 9, 3088–3094. [CrossRef] [PubMed]
141. Chen, Y.; Zhang, L.; Chen, G. Fabrication, modification, and application of poly(methyl methacrylate) microfluidic chips.
Electrophoresis 2008, 29, 1801–1814. [CrossRef]
142. Li, J.; Liu, C.; Qiao, H.; Zhu, L.; Chen, G.; Dai, X. Hot embossing/bonding of a poly (ethylene terephthalate)(PET) microfluidic
chip. J. Micromechanics Microengineering 2007, 18, 015008. [CrossRef]
143. Gencturk, E.; Mutlu, S.; Ulgen, K.O. Advances in microfluidic devices made from thermoplastics used in cell biology and analyses.
Biomicrofluidics 2017, 11, 051502. [CrossRef]
144. Ren, K.; Zhou, J.; Wu, H. Materials for Microfluidic Chip Fabrication. Acc. Chem. Res. 2013, 46, 2396–2406. [CrossRef]
145. Becker, H.; Locascio, L.E. Polymer microfluidic devices. Talanta 2002, 56, 267–287. [CrossRef]
146. Ballacchino, G.; Weaver, E.; Mathew, E.; Dorati, R.; Genta, I.; Conti, B.; Lamprou, D.A. Manufacturing of 3D-Printed Microfluidic
Devices for the Synthesis of Drug-Loaded Liposomal Formulations. Int. J. Mol. Sci. 2021, 22, 8064. [CrossRef]
147. Sengupta, P.; Khanra, K.; Chowdhury, A.R.; Datta, P. Lab-on-a-chip sensing devices for biomedical applications. In Bioelectronics
and Medical Devices: From Materials to Devices-Fabrication, Applications and Reliability; Elsevier: Amsterdam, The Netherlands, 2019;
pp. 47–95.
Nanomaterials 2021, 11, 3440 30 of 30

148. Niculescu, A.G.; Chircov, C.; Bîrcă, A.C.; Grumezescu, A.M. Fabrication and Applications of Microfluidic Devices: A Review. Int.
J. Mol. Sci. 2021, 22, 2011. [CrossRef] [PubMed]
149. Wang, J.; Li, W.; Zhang, L.; Ban, L.; Chen, P.; Du, W.; Feng, X.; Liu, B.-F. Chemically Edited Exosomes with Dual Ligand Purified
by Microfluidic Device for Active Targeted Drug Delivery to Tumor Cells. ACS Appl. Mater. Interfaces 2017, 9, 27441–27452.
[CrossRef] [PubMed]
150. Jahn, A.; Vreeland, W.N.; Gaitan, M.; Locascio, L.E. Controlled vesicle self-assembly in microfluidic channels with hydrodynamic
focusing. J. Am. Chem. Soc. 2004, 126, 2674–2675. [CrossRef] [PubMed]
151. Aghaei, H.; Solaimany Nazar, A.R.; Varshosaz, J. Double flow focusing microfluidic-assisted based preparation of methotrexate–
loaded liposomal nanoparticles: Encapsulation efficacy, drug release and stability. Colloids Surf. A Physicochem. Eng. Asp. 2021,
614, 126166. [CrossRef]
152. Weaver, E.; Uddin, S.; Cole, D.K.; Hooker, A.; Lamprou, D.A. The Present and Future Role of Microfluidics for Protein and
Peptide-Based Therapeutics and Diagnostics. Appl. Sci. 2021, 11, 4109. [CrossRef]
153. Guimarães Sá Correia, M.; Briuglia, M.L.; Niosi, F.; Lamprou, D.A. Microfluidic manufacturing of phospholipid nanoparticles:
Stability, encapsulation efficacy, and drug release. Int. J. Pharm. 2017, 516, 91–99. [CrossRef]
154. Gkionis, L.; Campbell, R.A.; Aojula, H.; Harris, L.K.; Tirella, A. Manufacturing drug co-loaded liposomal formulations targeting
breast cancer: Influence of preparative method on liposomes characteristics and in vitro toxicity. Int. J. Pharm. 2020, 590, 119926.
[CrossRef] [PubMed]
155. Pattni, B.S.; Chupin, V.V.; Torchilin, V.P. New developments in liposomal drug delivery. Chem. Rev. 2015, 115, 10938–10966.
[CrossRef]
156. Akbarzadeh, A.; Rezaei-Sadabady, R.; Davaran, S.; Joo, S.W.; Zarghami, N.; Hanifehpour, Y.; Samiei, M.; Kouhi, M.; Nejati-Koshki,
K. Liposome: Classification, preparation, and applications. Nanoscale Res. Lett. 2013, 8, 102. [CrossRef]
157. Capretto, L.; Mazzitelli, S.; Nastruzzi, C. Design, production and optimization of solid lipid microparticles (SLM) by a coaxial
microfluidic device. J. Control. Release 2012, 160, 409–417. [CrossRef] [PubMed]
158. Anderluzzi, G.; Perrie, Y. Microfluidic manufacture of solid lipid nanoparticles: A case study on tristearin-based systems. Drug
Deliv. Lett. 2020, 10, 197–208. [CrossRef]
159. Shepherd, S.J.; Issadore, D.; Mitchell, M.J. Microfluidic formulation of nanoparticles for biomedical applications. Biomaterials
2021, 274, 120826. [CrossRef] [PubMed]
160. Kumari, A.; Yadav, S.K.; Yadav, S.C. Biodegradable polymeric nanoparticles based drug delivery systems. Colloids Surf. B
Biointerfaces 2010, 75, 1–18. [CrossRef]
161. Zhang, R.; Billingsley, M.M.; Mitchell, M.J. Biomaterials for vaccine-based cancer immunotherapy. J. Control. Release 2018, 292,
256–276. [CrossRef] [PubMed]
162. Karnik, R.; Gu, F.; Basto, P.; Cannizzaro, C.; Dean, L.; Kyei-Manu, W.; Langer, R.; Farokhzad, O.C. Microfluidic platform for
controlled synthesis of polymeric nanoparticles. Nano Lett. 2008, 8, 2906–2912. [CrossRef]
163. Abstiens, K.; Goepferich, A.M. Microfluidic manufacturing improves polydispersity of multicomponent polymeric nanoparticles.
J. Drug Deliv. Sci. Technol. 2019, 49, 433–439. [CrossRef]
164. Vu, H.T.; Streck, S.; Hook, S.M.; McDowell, A. Utilization of Microfluidics for the Preparation of Polymeric Nanoparticles for the
Antioxidant Rutin: A Comparison with Bulk Production. Pharm. Nanotechnol. 2019, 7, 469–483. [CrossRef]
165. Behnke, M.; Vollrath, A.; Klepsch, L.; Beringer-Siemers, B.; Stumpf, S.; Czaplewska, J.A.; Hoeppener, S.; Werz, O.; Schubert, U.S.
Optimized Encapsulation of the FLAP/PGES-1 Inhibitor BRP-187 in PVA-Stabilized PLGA Nanoparticles Using Microfluidics.
Polymers 2020, 12, 2751. [CrossRef]
166. Mukherjee, A.; Waters, A.K.; Kalyan, P.; Achrol, A.S.; Kesari, S.; Yenugonda, V.M. Lipid-polymer hybrid nanoparticles as a
next-generation drug delivery platform: State of the art, emerging technologies, and perspectives. Int. J. Nanomed. 2019, 14,
1937–1952. [CrossRef] [PubMed]
167. Tahir, N.; Madni, A.; Li, W.; Correia, A.; Khan, M.M.; Rahim, M.A.; Santos, H.A. Microfluidic fabrication and characterization of
Sorafenib-loaded lipid-polymer hybrid nanoparticles for controlled drug delivery. Int. J. Pharm. 2020, 581, 119275. [CrossRef]
168. Abalde-Cela, S.; Taladriz-Blanco, P.; de Oliveira, M.G.; Abell, C. Droplet microfluidics for the highly controlled synthesis of
branched gold nanoparticles. Sci. Rep. 2018, 8, 2440. [CrossRef] [PubMed]
169. Kubendhiran, S.; Bao, Z.; Dave, K.; Liu, R.-S. Microfluidic synthesis of semiconducting colloidal quantum dots and their
applications. ACS Appl. Nano Mater. 2019, 2, 1773–1790. [CrossRef]
170. Larrea, A.; Sebastian, V.; Ibarra, A.; Arruebo, M.; Santamaria, J. Gas Slug Microfluidics: A Unique Tool for Ultrafast, Highly
Controlled Growth of Iron Oxide Nanostructures. Chem. Mater. 2015, 27, 4254–4260. [CrossRef] [PubMed]