Trends in Analytical Chemistry 80 (2016) 30–40

Contents lists available at ScienceDirect

Trends in Analytical Chemistry

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a c

PLGA-based nanoparticles: A new paradigm in biomedical

applications
Shweta Sharma *, Ankush Parmar, Shivpoojan Kori, Rajat Sandhir
Institute of Forensic Science & Criminology, Panjab University, Chandigarh 160 014, India

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

Keywords:
Polymers were first introduced three decades ago as bioresorbable surgical devices. Since then, polymer-
Biodegradable
based nanoparticles have been extensively studied in a variety of fields. Nanocarriers formulated with
Poly(lactic-co-glycolic acid)
Nanoparticles
biocompatible and biodegradable polymers approved by the US FDA (Food and Drug Administration) and
Targeting EMA (European Medicines Agency) are being studied for the controlled delivery of various therapeutic
Sustained release agents. Amidst the various polymers synthesized for formulating polymeric nanoparticles, poly(lactic-
co-glycolic acid) (PLGA) is the most popular. PLGA has several interesting properties such as controlled
and sustained release, low cytotoxicity, long-standing biomedical applications, biocompatibility with tissues
and cells, prolonged residence time and targeted delivery. The main aim of this review was to compre-
hensively address the issues related to PLGA-based nanoparticles focusing on the methods of preparation,
characterization techniques, surface modification, mechanism of drug release and the drawbacks. The
review also critically addresses the developmental aspects of PLGA-based nanocarriers in terms of tar-
geted drug delivery, as well as exploring their efficacy in vitro and in vivo.
© 2016 Elsevier B.V. All rights reserved.

Contents

1. Introduction ........................................................................................................................................................................................................................................................... 31
2. Poly(lactic-co-glycolic acid) ............................................................................................................................................................................................................................. 31
2.1. Properties of PLGA ................................................................................................................................................................................................................................. 31
3. Methods of preparing PLGA nanoparticles ................................................................................................................................................................................................. 32
3.1. Emulsification–solvent evaporation method ................................................................................................................................................................................ 32
3.1.1. Single emulsion method ..................................................................................................................................................................................................... 32
3.1.2. Double emulsion method ................................................................................................................................................................................................... 32
3.2. Phase separation (coacervation) ....................................................................................................................................................................................................... 32
3.3. Emulsification–solvent diffusion method ...................................................................................................................................................................................... 32
3.4. Emulsification–reverse salting-out method ................................................................................................................................................................................. 32
3.5. Nanoprecipitation method (solvent displacement) ................................................................................................................................................................... 33
3.6. Dialysis ...................................................................................................................................................................................................................................................... 33
3.7. Spray drying ............................................................................................................................................................................................................................................ 33
3.8. Supercritical fluid technology ........................................................................................................................................................................................................... 33
3.8.1. Rapid expansion of supercritical solutions .................................................................................................................................................................. 33
3.8.2. Rapid expansion of supercritical solutions in liquid solvents .............................................................................................................................. 33
4. Characterization parameters for nanoparticles ......................................................................................................................................................................................... 33
5. Surface modification of PLGA nanoparticles .............................................................................................................................................................................................. 33

Abbreviations: DCC, N,N-Dicyclohexylcarbodiimide; DDS, Drug delivery system; DNA, Deoxyribose nucleic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl carbodiimide);
EGFR, Epidermal growth factor receptor; EMA, European Medicines Agency; EPR, Enhanced permeability and retention; FDA, Food and Drug Administration; FTIR, Fourier
transform infrared; GALT, Gut-associated lymphoid tissue; MDR, Multidrug resistance; MMPs, Matrix metalloproteinases; MPS, Mononuclear phagocyte system; MRI, Mag-
netic resonance imaging; NHS, N-hydroxysuccinimide; NMR, Nuclear magnetic resonance; O/W, Oil in water; PEG, Polyethylene glycol; PGA, Polyglycolic acid; P-gP, P-glycoprotein;
PLA, Polylactic acid; PLGA, Poly(lactic-co-glycolic acid); PNP, Polymeric nanoparticle; RES, Reticuloendothelial system; RNA, Ribose nucleic acid; SiRNA, Short interfering
ribose nucleic acid; 99mTC, Technetium 99m; Tg, Glass transition temperature; TPGS, d-α-tocopherol polyethylene glycol succinate; VCAM, Vascular cell adhesion mole-
cule; VEGFR, Vascular endothelial growth factor receptors; W/O/W, Water in oil–water; XPS, X-ray photoelectron spectroscopy.
* Corresponding author. Tel.: +91 172 2534121(O) +91 9872688577 (M).
E-mail address: 25shweta@pu.ac.in (S. Sharma).

http://dx.doi.org/10.1016/j.trac.2015.06.014
0165-9936/© 2016 Elsevier B.V. All rights reserved.
 S. Sharma et al./Trends in Analytical Chemistry 80 (2016) 30–40 31

5.1. Polyethylene glycol ................................................................................................................................................................................................................................ 33

5.1.1. PEGylation strategies ............................................................................................................................................................................................................ 34
5.1.2. Polysorbate .............................................................................................................................................................................................................................. 34
5.1.3. Vitamin E TPGS ...................................................................................................................................................................................................................... 34
6. Mechanism of drug release from PLGA-based DDSs ............................................................................................................................................................................... 34
7. Physiochemical changes occurring in PLGA-based DDSs ...................................................................................................................................................................... 35
8. Drug release behaviour ..................................................................................................................................................................................................................................... 36
8.1. Phase I ........................................................................................................................................................................................................................................................ 36
8.2. Phase II ...................................................................................................................................................................................................................................................... 36
9. Factors affecting degradation .......................................................................................................................................................................................................................... 36
9.1. Polymer composition and molecular weight ............................................................................................................................................................................... 36
9.2. Drug type .................................................................................................................................................................................................................................................. 36
9.3. Size and shape of the matrix ............................................................................................................................................................................................................. 36
9.4. pH ................................................................................................................................................................................................................................................................ 36
9.5. Drug load .................................................................................................................................................................................................................................................. 37
10. PLGA-mediated drug delivery for cancer treatment ............................................................................................................................................................................... 37
11. Targeting strategies for efficient drug delivery ......................................................................................................................................................................................... 37
11.1. Passive targeting .................................................................................................................................................................................................................................. 37
11.2. Active targeting .................................................................................................................................................................................................................................... 37
11.2.1. Targeting of cancer cells ................................................................................................................................................................................................... 37
11.2.2. Targeting of tumour endothelium ................................................................................................................................................................................ 37
12. Ligand-anchored PLGA nanoparticles for cancer therapy ..................................................................................................................................................................... 38
13. PLGA nanoparticles as thriving mediators .................................................................................................................................................................................................. 38
13.1. Gene delivery for cancer treatment .............................................................................................................................................................................................. 38
13.2. Diagnosis and imaging of cancer ................................................................................................................................................................................................... 38
13.3. Theranostics of cancer ....................................................................................................................................................................................................................... 39
14. Drawbacks of using PLGA nanoparticle-based DDSs .............................................................................................................................................................................. 39
15. Conclusion .............................................................................................................................................................................................................................................................. 39
Acknowledgement ............................................................................................................................................................................................................................................... 39
References .............................................................................................................................................................................................................................................................. 39

1. Introduction nanoparticles [7]. The study by Joo et al. was comprehensive, de-
scribing various features of PLGA including surface modification,
In recent years, nanoparticles have been increasingly studied in targeting aspect and the intrinsic capacity with uptake of the cancer
the field of biomedical sciences. Depending upon the nature of the drug carrier [8]. The present review describes in detail the charac-
polymer used in their formulation, these particles may be catego- teristics of PLGA and its associated potentials in terms of the
rized into natural or synthetic. Delivery of various substances such structure–property relationship. The efficacy of PLGA nanoparticles
as vaccines, macromolecules and hydrophobic drugs to cells and was improved through ligand anchoring, which were applied as me-
organs such as the brain, liver and lungs can be achieved via these diators for gene delivery, in imaging of cancer and in the field of
nanoparticles, thus making them a multifaceted platform for tar- theranostics.
geted delivery [1]. However, for use as a vector for drug delivery
systems (DDSs), a nanoparticle must possess vital properties such 2. Poly(lactic-co-glycolic acid)
as biocompatibility, drug compatibility and proper biodegrada-
tion kinetics. A site-specific action of the drug at a therapeutically Polyester PLGA is a copolymer of polylactic acid (PLA) and
optimal rate and dose regimen can be achieved by controlling for polyglycolic acid (PGA). In terms of design and performance, PLGA
parameters such as particle size, surface properties and release rate is the best candidate as biomaterials for drug delivery. Here, PLGA
during the synthesis and design of the nanoparticles. Polymer- denotes poly-D, L-lactic-co-glycolic acid, where D- and L-lactic acid
based nanoparticles have been used for site-specific delivery, for forms are present in a fixed ratio [9]. The need for efficient and su-
cancer therapy, for clinical bioanalytical diagnostics, to design tissue- perior DDSs has led to the development of assorted block copolymers
engineered scaffolds and in devices [2]. For the synthesis of of polyesters with PEG. PLGA/PEG block copolymers are available
nanoparticles, a variety of polymers have been used, but the co- in two varieties: diblock (PLGA–PEG) or triblock with both ABA
polymer poly(lactic-co-glycolic acid) (PLGA) has been widely used (PLGA–PEG–PLGA) and BAB (PEG–PLGA–PEG).
in this context. PLGA is a biocompatible, biodegradable and safely
administrable polymer approved by the US FDA (Food and Drug Ad- 2.1. Properties of PLGA
ministration) and EMA (European Medicines Agency) [3]. Langer and
Folkman [4] were the first to report the controlled release of mac- PLGA is synthesized via random ring-opening copolymeriza-
romolecules via polymers. Thus, their pioneering work led to further tion of two different monomers, glycolic acid and lactic acid, in the
discoveries in the field of novel DDSs. This work led to the evolu- presence of tin (II) 2-ethylhexanote, tin (II) alkoxides or alu-
tion of anti-angiogenic DDSs to treat serious diseases such as cancer. minium isopropoxide as the catalyst. Glycolic and lactic acid units
Some reports have proved PLGA nanoparticles as an excellent vector are consecutively linked via ester linkages during polymerization,
for the transmission of biomolecules such as RNA, DNA, peptides, resulting in the formation of PLGA.
vitamins, proteins and drugs both in vivo and in vitro. Stevanovic Different forms of PLGA can be obtained by varying the ratio of
and Uskokovic highlighted the efficient delivery of vitamins using lactide to glycolide during the polymerization reaction, for example,
PLGA-based nano- and microparticles [5]. . In their review article, PLGA 50:50 (refers to a copolymer composed of 50% lactic acid and
Choi et al concisely described PLGA-aided tumour targeting [6]. In 50% glycolic acid), PLGA 75:25, PLGA 80:20, etc. Depending on the
another report, Locatelli and Franchini explained the synthesis and molecular weight and the lactide to glycolide copolymer ratio used,
applications of PLGA-b-polyethylene glycol (PEG) polymeric the deterioration time of the polymer may vary from several months
32 S. Sharma et al./Trends in Analytical Chemistry 80 (2016) 30–40
to several years [1,10]. Low molecular weight polymers with higher
glycolide content are more hydrophilic and amorphous, and thus
have a shorter deterioration time. This is because glycolic acid is
more hydrophilic and thus tends to absorb a large amount of water.
Conversely, polymers with higher lactic acid content are more hy-
drophobic, absorb less amount of water and degrade in a more
gradual manner [11]. This phenomenon has been proven useful for
controlled and sustained drug release varying from weeks to months.
The hydrolysis of PLGA yields two metabolic monomers: lactic acid
and glycolic acid. Via the Krebs cycle, these monomers are endog-
enously metabolized into the simpler by-products such as CO2 and
H2O [12]. PLGA can be processed into almost any kind of configu-
ration and can be used to encapsulate a variety of molecules. PLGA
polymers are miscible in various volatile organic solvents such as
tetrahydrofuran, acetone, dichloromethane, chloroform and ethyl
acetate. Physical properties such as molecular weight, polydisper-
sity index, Tg and degree of crystallinity are known to affect the
swelling behaviour, biodegradation rate and mechanical strength
of the polymer [13]. The type and molar ratio of the individual
monomer components in the polymer chain chiefly determine the
degree of crystallinity.
Makadia and Siegel [9] showed that a decrease in the degree of
crystallinity along with an increase in the hydration rate (hydro-
lysis) is observed during copolymerization of a crystalline PGA with
PLA. PLGA copolymers are generally glassy in nature and exhibit a
fairly rigid chain structure. This may be attributed to the Tg of PLGA
exceeding its physiological temperature [14]. It has been demon-
strated that the dose and composition of PLGA nanocarriers has a
demarcating effect on blood clearance and uptake of these
nanocarriers by the mononuclear phagocyte system (MPS) [15].
3. Methods of preparing PLGA nanoparticles
Depending on the process of preparation, the structural orga-
nization of the nanoparticle may vary. Biodegradable PLGA
nanoparticles are generally prepared by dispersing the polymer. The
nanoparticles are actually formed in the initial step, which is
common for all techniques, wherein an emulsification system is pre-
pared [8]. The different methods of preparation are discussed in the
following sections:
3.1. Emulsification–solvent evaporation method
3.1.1. Single emulsion method
This is the most commonly used method for preparing PLGA
nanoparticles. O/W emulsification is generally used when the en-
capsulant is hydrophobic or poorly soluble in water [16]. To prepare
an organic phase, an appropriate amount of the polymer is initial-
ly dissolved in a volatile organic solvent such as dichloromethane,
chloroform or ethyl acetate. Then, the drug or encapsulant is added
to this solution, resulting in dispersion. The dispersion containing
the polymer and the drug is added to a continuously stirring aqueous
solution with surfactants such as polyvinyl alcohol, polysorbate 80,
poloxamer 188 and vitamin E TPGS (d-α-tocopherol polyethylene
glycol succinate), thus generating a stable emulsion. The organic
solvent is then allowed to evaporate either by magnetic stirring or
by maintaining a reduced pressure [9].
3.1.2. Double emulsion method
The double emulsion method is also known as the W/O/W
method. The encapsulation efficiency and particle size are pre-
dominantly affected by the type of solvent and stirring rate. An
appropriate amount of the drug is dissolved in an aqueous phase
(prepared in deionized water) followed by simultaneous addition
of the drug solution to a vigorously stirring organic phase (com-
posed of PLGA dissolved in volatile organic solvents such as ethyl
acetate, chloroform or dichloromethane). Thus, a water-in-oil primary
emulsion is formed. Further emulsification is achieved by adding
the primary emulsion to an aqueous solution, followed by simul-
taneous stirring and thereafter allowing the organic solvent to
evaporate [17]. Both of these techniques are optimal for laborato-
ry synthesis; on a large scale, however, they are applicable only to
liposoluble drugs and require excess energy during homogeniza-
tion. However, it has been reported that any alteration in the process
parameters, for example, stirring speed and temperature, helps over-
come the drawbacks of these techniques [8].
3.2. Phase separation (coacervation)
This process mainly leads to the formation of micrometre-
sized polymeric nanoparticles via liquid–liquid phase separation.
In this method, two liquid phases comprising a coacervate phase
and a supernatant phase depleted in the polymer are formed. The
coacervation process includes three major steps [9]:
(i) Phase separation of the coating polymer solution,
(ii) Adsorption of the coacervate around the drug particles and
(iii) Quenching of the microspheres.
Polymers and solvent are mixed in an appropriate ratio, fol-
lowed by the addition of the drug (i.e., hydrophilic drug in the
W/O/W emulsion and hydrophobic drug in the O/W emulsion). A
soft coacervate of the drug encapsulated in a droplet is extracted
as a result of phase separation of the polymer. The microdroplets
are quenched by dipping the coacervate quickly in an insoluble
medium. However, the morphology and size of the microspheres
can be controlled by altering the following process parameters [18]:
(a) Polymer concentration
(b) Quenching temperature
(c) Quenching time
(d) Solvent composition
(e) Stirring rate
3.3. Emulsification–solvent diffusion method
This technique is a modification of the salting-out process. An
initial thermodynamic equilibrium is attained by mutually satu-
rating the solvent and water at room temperature before use.
Emulsification of the organic phase containing the polymer and drug
in an aqueous surfactant solution is achieved via high-speed ho-
mogenization. In order to attain a phase transformation reaction and
outward diffusion of the solvent from the internal phase, water is
added while regularly stirring. Colloidal nanoparticles are formed
as a result of nanoprecipitation. Solvent evaporation is then facili-
tated by either evaporation or vacuum distillation [19]. This method
offers several advantages such as enhanced encapsulation efficien-
cy, high batch-to-batch reproducibility, ease of scale-up, narrow size
distribution and simplicity. However, on the contrary, this tech-
nique has limitations such as leakage of water-soluble drugs and
removal of high volume of water from the suspension.
3.4. Emulsification–reverse salting-out method
The organic phase containing the polymer and drug is firstly
added to a water-miscible solvent. An O/W emulsion is formed by
forcefully magnetically stirring the preformed organic solvent with
an aqueous solution containing the salting-out agent (e.g., magne-
sium chloride or calcium chloride) and a colloidal stabilizer (e.g.,
polyvinyl pyrrolidone). An abrupt increase in the continuous phase
of the emulsion is facilitated by further diluting the O/W with a large
volume of water. The volatile organic solvent diffuses into the
 S. Sharma et al./Trends in Analytical Chemistry 80 (2016) 30–40
aqueous phase starts, leading to the formation of nanoparticles [20].
The residual solvent and salting-out agents are removed by filtra-
tion, leaving the nanoparticles behind. This technique offers the
advantage of encapsulating heat-sensitive agents such as pro-
teins, DNA and RNA.
3.5. Nanoprecipitation method (solvent displacement)
It is a one-step process, generally used to entrap hydrophobic
drugs in the polymer matrix. The organic phase is formed by dis-
solving the polymer and drug in a polar solvent (e.g., acetone, ethanol,
methanol and acetonitrile). This solution is added drop-wise to an
aqueous solution containing an emulsifier or a surfactant. The rapid
diffusion of the solvent takes place, resulting in the formation of
nanoparticles [21–23].
3.6. Dialysis
Dialysis is generally used to prepare small-sized nanoparticles
with a narrow distribution. The polymer is dissolved in a volatile
organic solvent and placed in a dialysis tube of appropriate pore
size. Inside the dialysis bag, displacement of the solvent takes place,
along with a loss in the solubility, ultimately leading to the pro-
gressive aggregation of polymer and formation of a homogeneous
suspension of nanoparticles [21,24].
3.7. Spray drying
Spray drying is an alternative to the conventional methods of
preparing polymer nanoparticles. This technique offers several ad-
vantages such as rapidity, convenience and implementation of fewer
processing parameters. In this process, a W/O dispersion is sprayed
in a hot stream of air, leading to the formation of nanoparticles.
However, the adhesion of the nanoparticles to the inner walls of
the spray dryer hinders the effective collection of the formed par-
ticles [25].
3.8. Supercritical fluid technology
Supercritical fluid technology has been proven to be a more
environment-friendly approach to produce polymeric nanoparticles
(PNPs). It can produce PNPs with high purity and fewer traces of
organic solvent [26]. Two principal methods are used to produce
nanoparticles [27]:
3.8.1. Rapid expansion of supercritical solutions
A solution is formed by dissolving the solute in a supercritical
fluid, accompanied by the rapid expansion of the solution in the
ambient air. Homogeneous nucleation is achieved by reducing the
pressure rapidly. Due to the rapid reduction in pressure, a high degree
of supersaturation is achieved, leading to expansion and ultimate-
ly formation of nanoparticles [27].
3.8.2. Rapid expansion of supercritical solutions in liquid solvents
It is a modified form of rapid expansion of supercritical solu-
tions, with expansion of the supercritical solution taking place in
a liquid solvent instead of ambient air. Apparently, particle growth
is suppressed in the expansion jet, by the action of the liquid solvent,
leading to the formation of nanoparticles [27,28].
4. Characterization parameters for nanoparticles
Characterization becomes a prerequisite for understanding the
properties of the nanoparticles. Several parameters can provide
insight into the properties of the nanoparticles. First and fore-
most, size helps determine the efficacy of the nanoparticles, release
33
profile and degradation pattern. Dynamic light scattering, scan-
ning electron microscopy, transmission electron microscopy and
atomic force microscopy can be used to determine parameters such
as size, distribution and morphology of the nanoparticle. It has also
been revealed that the molecular weight of the polymer has an
adverse effect on the particle size, encapsulation efficiency and deg-
radation rate [23]. The chain length of the polymer represents the
molecular weight of the polymer, thus reflecting the chemical nature
of the polymer, i.e. its hydrophobic or hydrophilic nature. It is well
known that polymers with shorter chain length are hydrophobic
and have a faster degradation rate. However, polymers with a longer
chain length are generally hydrophilic in nature and have a shorter
degradation rate. Thus, the molecular weight of the polymer plays
a crucial role in deciding the release kinetics of the drug [29]. Size
exclusion chromatography is useful in determining the molecular
weight of the polymer [30]. It has also been observed that the in
vitro and in vivo release characteristics of the drug are affected by
the physical state of both the drug and polymer. The muco-adhesion,
nanoparticle constancy and intracellular trafficking are greatly de-
pendent on the zeta potential [31]. The biodistribution of the
nanoparticles is greatly dependent upon their hydrophobic nature.
Various studies have corroborated that the retention time of hy-
drophilic particles is more that of hydrophobic particles [32].
Techniques such as water contact angle measurement and hydro-
phobic interaction chromatography may be used to determine the
hydrophobicity and hydrophilicity of the nanoparticles, respective-
ly [33]. The surface chemistry can be analysed with a variety of
techniques such as X-ray photoelectron spectroscopy (XPS), Fourier
transform infrared (FTIR) spectroscopy and nuclear magnetic res-
onance (NMR) spectroscopy [23,34].
5. Surface modification of PLGA nanoparticles
To serve as a targeted drug delivery vehicle, the nanoparticle must
be capable of persisting in the human systematic circulation. A pro-
longed circulation time will help the nanoparticles reach the target
organ. On the contrary, these particles are removed from the blood-
stream by the reticuloendothelial system (RES). This phenomenon
has proven to be one of the foremost challenges in developing
nanoparticle-based DDS [1,35]. This may be attributed to the binding
of nanoparticles with the opsonin proteins present in the blood
serum when administered intravenously. Further, the opsonized par-
ticles become attached to the macrophages, where they are
ultimately internalized by phagocytosis. The final fate of such par-
ticles is their clearance from the body via the renal system. Therefore,
despite their favourable biocompatibility and biodegradability, PLGA
nanoparticles are likely to be cleared rapidly from circulation by
macrophages of the MPS immediately after their intravenous ad-
ministration [36]. To overcome these limitations, the bare
nanoparticles are subjected to surface modification. Surface modi-
fication plays a crucial role in escaping the natural defence system
of the body. Hydrophilic particles with size of about ≤100 nm have
the greatest survival rate in escaping the phagocytic system [23].
The retention time for hydrophilic nanoparticles is comparatively
longer than that for hydrophobic nanoparticles. This is because hy-
drophobic nanoparticles are preferably taken up by the RES and are
thus eliminated from the body [8]. To construct a hydrophilic core
around PLGA nanoparticles, the nanoparticles are coated with surface
derivatizers. The following chemical species act as good surface
modifiers:
5.1. Polyethylene glycol
PEG is a nonionic, hydrophilic polymer with excellent biocom-
patibility. Coating of the nanoparticle surface with PEG, that is,
PEGylation, is the most commonly used technique for surface
34 S. Sharma et al./Trends in Analytical Chemistry 80 (2016) 30–40
Table 1
PLGA–PEG nanoparticles for anticancer drug delivery
Nanoparticles Drug loaded Target Inference
PLGA–PEG Paclitaxel HeLa Entrapment of paclitaxel within a PEGylated PLGA nanoparticle resulted in higher tumour growth
nanoparticles inhibition, followed by a significant augmentation in the tumour-specific localization of the drug
PLGA–mPEG Cisplatin BALB/c miceaa Higher survival rate and delayed tumour growth was observed for PLGA–mPEG nanoparticle-treated
nanoparticles mice in comparison with animals treated with the free drug
PLGA–PEG Docetaxel Solid tumors Biological half-life of the drug is significantly augmented, while imparting considerable solid
nanoparticles tumour accumulation
modification of the nanoparticles. The process allows the
nanoparticles to evade the MPS attack, thus concomitantly increas-
ing their plasma half-life [37]. However, the mechanism underlying
this increase in the plasma half-life remains to be elucidated. It is
assumed that stearic repulsion as well as van der Waals forces
created by the hydrated barriers on the nanoparticle surface pre-
vents the nanoparticles from being opsonized. The high flexibility
of the polymer chain allows the free rotation of the polymer units,
creating a highly hydrophilic stealth corona around the nanoparticles.
Thus, the nanoparticles are prevented from interacting with the mac-
romolecules present in the body. It has also been observed that PEG
molecules with high molecular weight, high surface density and
longer chain length are absorbed at a comparatively lower rate, thus
increasing the residence time of the nanoparticle [38]. Moreover,
PEGylation of PLGA nanoparticles has also been shown to enhance
the drug payload, solubility and kinetic stability, thereby improv-
ing the targeting index, therapeutic index as well as the accessibility
of the nanoparticle to the target site. With the help of PEGylation,
the aqueous solubility and stability can be enhanced, intermolecu-
lar aggregation can be reduced and immunogenicity can be
decreased. In their study, Danhier et al. showed that the entrap-
ment of paclitaxel within a PEGylated-PLGA-based nanoparticle
altered its pharmacokinetics and biodistribution such that the
tumour-specific localization of the drug was significantly aug-
mented, leading to higher tumour growth inhibition efficiency than
the free drug [39]. In another experiment, Gref et al. showed that
the PEGylated PLGA nanoparticles effect an increase in the circu-
lation time and decreased uptake by the liver; that is, the uptake
of nanoparticles was reduced from 66% within the first 5 min of ad-
ministration, but the uptake was <30% (within 2 h) for non-coated
nanoparticles [40]. Although long-circulating nanoparticles formu-
lated with PEGylated PLGA possess many significant advantages, this
strategy is also not free from limitations. Capping of PLGA with PEG
prevent the interaction not only between the nanoparticles and the
opsonin but also between nanoparticles and the cell surface [36].
Table 1 shows the effect of PEGylation on the uptake of PLGA
nanoparticles in cancerous cells [40–42].
5.1.1. PEGylation strategies
5.1.1.1. Direct conjugation. Betancourt et al. reported three differ-
ent methods for the covalent attachment of PEG to PLGA
nanoparticles [43]. Based on their study, varying the reaction con-
ditions can help control the efficacy of conjugation as well as the
final copolymer composition. A non-significant incorporation of PEG
occurred when the PEG molecule was directly conjugated to the car-
boxylic groups present on the surface of PLGA nanoparticles, because
of the inaccessibility of these groups. Moderate efficiency is ob-
served, when the conjugation reactions are carried out in solution.
Direct conjugation of PLGA nanoparticles with PEG would also fa-
cilitate the encapsulation of a desired agent with the nanoparticle,
as PEGylation would occur after the desired agent is encapsulated
within the solid polymer matrix core (Fig. 1a). However, the tech-
nique has certain limitations such as low yield because of the
purification steps. The nanoparticles must be exposed to an aqueous
Reference
[40]
[41]
[42]
environment during the conjugation process for achieving maximum
efficiency.
5.1.1.2. Activated conjugation. It is a two-step process where acti-
vation is succeeded by conjugation. Minimal hydrolysis of the active
intermediate occurs, and the undesirable formation of PEG–PEG con-
jugates is avoided [43]. Fig. 1b shows the synthetic route used for
the conjugation of heterofunctional PEG to PLGA nanoparticles.
5.1.1.3. Ring-opening polymerization. Among all the strategies used
for the production of PEGylated PLGA nanoparticles, ring-opening
polymerization is the most commonly and widely used technique.
The reaction is initiated by protic agents such as the hydroxyl group
of OH–PEG–COOH, leading to the formation of PLGA with hy-
droxyl end groups, whereas the carboxyl end groups of PEG remain
free. Fig. 1c shows the ring-opening polymerization of PEG to PLGA
nanoparticles.
5.1.2. Polysorbate
Polysorbate 20, 40, 60 and 80 are the most commonly used non-
ionic surfactants and emulsifiers in food and cosmetic products. Due
to the surface coating of polymer nanoparticles with polysorbate,
their ability to cross the blood–brain barrier is enhanced. Because
of the binding of the nanoparticles to the inner linings of the brain
capillaries, a large change in the concentration gradient is ob-
served along with an increase in passive diffusion, resulting in
facilitated delivery of the drug to the brain.
5.1.3. Vitamin E TPGS
Vitamin E TPGS is a synthetic water-soluble form of vitamin E.
TPGS is a PEG derivative of α-tocopherol that is water soluble. It is
commonly used as an emulsifier, a solubilizer and a vehicle in drug
delivery formulations. Vitamin E TPGS is also widely used as an emul-
sifier for enhanced encapsulation efficiency, drug loading and release
kinetics of hydrophobic drugs such as paclitaxel, doxorubicin and
5-fluorouracil. The molecule has shown to improve the adhesion
of nanoparticles to the cells and the haemodynamic properties of
the nanoparticles [33].
6. Mechanism of drug release from PLGA-based DDSs
The term ‘release mechanism’ may be defined as the way in which
drug molecules are transported or released and as an event of the
process determining the release rate. Drug molecules are released
via three possible mechanisms [44]:
(i) Transport through water-filled pores
(ii) Transport through the polymer
(iii) Dissolution of the encapsulating polymer
Large and highly hydrophilic molecules such as protein or DNA
are encapsulated agents that are generally transported through the
polymer phase and are released by transport through water-filled
pores. Transport through water-filled pores is mainly achieved via
 S. Sharma et al./Trends in Analytical Chemistry 80 (2016) 30–40 35

Fig. 1. (a). Direct conjugation of PEG to the surface of premade PLGA nanoparticles. (b) Activated conjugation of PLGA to heterofunctional PEG. (c) Ring-opening polymer-
ization of lactide and glycolide.

a process known as diffusion, where the molecules are driven by a
chemical potential gradient. Another phenomenon of transport is
convection, where osmotic pressure is the driving force and hence
the name osmotic pumping. This pressure may generate an influx
of water into a non-swelling system. The phenomenon is most com-
monly confronted in the case of cellulose acetate-derived DDSs [45].
PLGA possesses mobile polymer chains and can absorb a large
amount of water, thus leading to a prominent swelling. Due to re-
arrangement of the polymer chain and swelling, the increase in the
volume of water is compensated. Sometimes, the encapsulated drug
may be released as a result of dissolution of the polymer or erosion.
Pores are created and an increase in the rate of diffusion is ob-
served. The release rate is controlled by diffusion at the initial stages,
whereas degradation or erosion plays a very vital role in the final
stage of the release period. Thus, it can be concluded that drug
release is controlled by more than one mechanism at once [44].
7. Physiochemical changes occurring in PLGA-based DDSs
When the DDS is administered in vitro or immersed in water,
the polymer quickly absorbs water. The water molecules occupy
some volume in the polymer matrix, which is often regarded as pores
and the process is said to be the pore-forming process. Due to the
small size of the pores, a minimal amount of drug is transported
during the initial release phase. However, over time, the water-
filled pores start to grow in size and number, thus creating a porous
connected network. This network facilitates the drug release during
the later phase. However, heterogeneous degradation via an auto-
catalytic phenomenon takes place when PLGA matrix comes in
contact with water. This phenomenon is termed as hydrolysis. During
hydrolysis, the ester bonds are broken, with a decrease in the mo-
lecular weight of the polymer and generation of acids [46]. With
an increase in the dimension of the DDS, degradation is faster at
36 S. Sharma et al./Trends in Analytical Chemistry 80 (2016) 30–40
the centre of the PLGA matrix than at the surface [47]. Further-
more, loss of polymer occurs when the dissolved polymer
degradation products diffuse into the release medium. As it can be
hydrated rapidly, the PLGA polymer undergoes bulk erosion in spite
of surface erosion. Pores are created by the dissolution of polymer
degradation and erosion processes. The generated small pores grow
in size by coalescing with the neighbouring pores, ultimately leading
to the formation of larger pores. The PLGA polymer can be rear-
ranged by mobilizing its polymeric chains, leading to the formation
of larger pores. The effects of dissolved degradation products on DDS
are as follows:
(i) Due to their acidic nature, they catalyse hydrolysis
(ii) An increase in the rate of water absorption along with a de-
crease in the transport resistance of the polymer is observed
due to plasticization of the polymer by the dissolved degra-
dation product
(iii) The osmolality inside the polymer matrix is increased, thereby
enhancing the force of water absorption
(iv) Due to the presence of many repeating monomeric units in
a row, crystallization takes place, thus inhibiting the absorp-
tion of water
The effect of dissolved degradation products on the release of
the polymer ceases with the onset of rapid erosion. The transport
resistance controls the release of the encapsulated moiety and
polymer degradation kinetics. Transport resistance is found to be
affected by various processes such as pore formation, pore closure,
drug dissolution, polymer–drug interaction and drug–drug inter-
action [44]. It has also been found that an increase in the density
of the particle and reduction in the porosity (structural relax-
ation) might result in a decreased burst release of the drug molecule
from the PLGA nanoparticles. The magnitude of drug release in a
particular formulation is directly affected by the rate and extent of
structural relaxation present in the formulated nanoparticle. Struc-
tural relaxation in PLGA nanoparticles is dependent on various
properties such as molecular weight, fabrication methods, drug–
polymer interactions, residual solvents and storage conditions.
Together, these factors contribute to structural relaxation. They may
also lead to a variability in burst release, limiting the develop-
ment of products using this type of drug delivery technology.
8. Drug release behaviour
For mechanistic evaluation, the release profile can be used as a
basic parameter. However, the most commonly preferred release
profile is zero order and the monophasic release is rarely ob-
served. As a result of heterogeneous degradation, a biphasic or
triphasic release profile may be observed [48]. In the case of surface-
coated PLGA nanoparticles, a biphasic release profile with a relatively
rapid second phase is observed. The following patterns are ob-
served in the case of biphasic drug release from PLGA nanoparticles
[49]:
8.1. Phase I
Drug type, drug concentration and polymer hydrophobicity are
some of the parameters associated with an initial burst of drug
release. As soon as the nanoparticle comes in contact with the dis-
solution medium, the water penetrates the polymer matrix rapidly.
The drug present on the surface of the nanoparticle is released as
a function of solubility. Random scission of the PLGA molecule occurs,
significantly decreasing the molecular weight of the polymer.
However, there is no appreciable weight loss and formation of a
soluble monomer product during this phase.
8.2. Phase II
In the second phase, the thicker drug layer is depleted, leading
to progressive release of the drug. Soluble oligomeric and mono-
meric products are formed due to the hydrolysis of the polymer
matrix. Hydrolysis creates a passage drug to be released by diffu-
sion and erosion until complete polymer solubilization takes place.
However, in the case of a triphasic release profile, phase I is usually
referred to as burst release. This kind of behaviour is attributed mainly
to the hydration of non-encapsulated drug particles present on the
surface or close to the surface of the DDS. As degradation and hy-
dration proceed, the polymer begins to grow dense, and consequently
a slow release or diffusion of the drug is observed in phase II. Burst
release is sometimes followed by erosion, which is a comparative-
ly fast release phase and sometimes called the second burst.
9. Factors affecting degradation
The following factors affect the degradation of PLGA
nanoparticles:
9.1. Polymer composition and molecular weight
The composition of the polymer significantly determines the hy-
drophilicity and rate of degradation of any delivery matrix [50]. A
significant loss in the weight of the polymer has been observed with
a substantial increase in the polymer’s glycolic content. The deg-
radation rate of the polymer is directly affected by the amount of
glycolic acid present, and the degradation rate increases with in-
crease in the glycolic acid proportion. Due to higher hydrophilicity,
preferential degradation of glycolic acid proportion is seen, leading
to faster degradation of PLGA 50:50 (PLA:PGA) than PLGA 65:35.
Subsequently, PLGA 65:35 shows faster degradation than PLGA 75:25
and PLGA 85:15. Generally, lower degradation rates are exhibited
by high molecular weight polymers. Polymers of high molecular
weight possess longer polymeric chains and require longer dura-
tions for degradation, whereas polymers of low molecular weight
possess smaller polymeric chains and are degraded in a shorter
duration.
9.2. Drug type
Drug type also determines the mechanistic fate of polymer–
drug matrix degradation and drug release rate. The mechanism of
degradation, as well as the rate of matrix degradation, may be
changed from bulk erosion to surface degradation depending on the
type of encapsulated drug.
9.3. Size and shape of the matrix
The degradation profile of large devices is shown to be signifi-
cantly affected by the ratio of the surface area to volume. Faster and
greater degradation of the matrix takes place with a higher surface
area ratio, but the opposite effect is seen in matrices or devices with
a smaller surface area to volume ratio. Bulk degradation takes place
faster than pure surface degradation, leading to faster release of the
drug from devices with higher surface area to volume ratios.
9.4. pH
In vitro studies have shown that both strongly alkaline and acidic
media tend to accelerate polymer degradation. However, due to the
autocatalysis of the polymer by the carboxylic end groups, the dif-
ference between slightly acidic and neutral media becomes less
pronounced [51].
 S. Sharma et al./Trends in Analytical Chemistry 80 (2016) 30–40
9.5. Drug load
The rate and duration of drug release is significantly affected by
the amount of drug loading in the drug delivery matrix. A larger
initial burst release is seen in matrices with higher drug content,
whereas a smaller burst release is observed in the case of matri-
ces with lower drug content because of their smaller polymer to
drug ratio. Depending on the type of drug, the effect is attenuated
when a certain drug concentration is reached.
10. PLGA-mediated drug delivery for cancer treatment
Cancer treatment via the oral route is preferred because of its
non-invasive nature and better patient compliance. However, due
to their poor oral availability, most anticancer drugs cannot be de-
livered via the oral route. When administered orally, only a small
fraction of the drug becomes available to the systematic circula-
tion. For example, the oral bioavailability of paclitaxel, docetaxel and
doxorubicin has been found to be 1%, < 10% and < 5%, respectively
[52]. The underlying reason for this poor availability is the exten-
sive first-pass metabolic effect by cytochrome P-450 (liver microsomal
enzyme) [44], as well as their efflux by an overexpressed plasma
membrane transporter P-gp efflux pump [53]. The P-gp is encoded
via the gene MDR-1, which acts as an efflux pump. After a pro-
longed chemotherapeutic session, the efflux pump exports a wide
range of chemo drugs and thus tends to decrease the accumula-
tion of functional drugs in MDR cells. Ultimately, the body gradually
stops responding, decreasing the drugs’ therapeutic efficacy simul-
taneously followed by treatment failure [54]. The administration of
P-gp inhibitors based on the drug of interest can be an alternative
for overcoming this limitation. On the contrary, P-gP administra-
tion is generally associated with aberrant toxicity, blocking of
physiological anticancer drug efflux from the normal cells and in-
terrupted efflux of toxins from the body via the P-gP efflux pump.
The small intestine contains two types of cells: enterocytes and M
cells. Any liquid or soluble material is absorbed in the small intes-
tine via the enterocytes directly from the systematic circulation,
whereas particulate matters are absorbed by the M cells via the lym-
phatic system. In the case of orally administered PLGA nanoparticles,
the absorption predominantly occurs via the M cells present on
Peyer’s patches and via the isolated follicles of the gut-associated
lymphoid tissue (GALT). The efflux of drug by the P-gp efflux trans-
porter can be effectively overcome by entrapping the desired
molecules within the voids of the PLGA matrices. The drug ab-
sorbed from the enterocytes in the systematic circulation tends to
undergo first-pass metabolism, whereas the drug absorbed into the
lymphatic system tends to bypass this [36]. Thus, the incorpora-
tion of active agents within the polymer matrix of the PLGA prevents
depletion of the drug in the hostile environment of the gastroin-
testinal lumen. The small size and unique surface chemistry of PLGA
nanoparticles afforded them improved adhesion, absorption and
transport of the drug. The absorption pathway of a particulate de-
livery vehicle occurs via M cells in Peyer’s patches. Encapsulation
with PLGA nanoparticles enhanced the solubility, stability and phar-
macokinetics of chemo drugs [55]. The drug concentration can be
controlled, thereby reducing the risk of unwanted side effects and
maintaining useful treatment cycles, without damaging the healthy
cells.
11. Targeting strategies for efficient drug delivery
Chemo drugs have many advantages, but when administered
inside the biological milieu they pose many potential hazards such
as systemic toxicity, bone marrow suppression, cardiomyopathy and
neurotoxicity. This is because chemo drugs cannot differentiate
between normal and cancerous cells, and the healthy cells or tissues
37
are damaged, limiting the maximal permissible dose of the drug.
A large amount of drug must be administered, as some of the drug
is distributed into the non-targeted organs and tissues, ultimately
leading to non-specific toxicity. This is followed by the rapid removal
of the drug, making the treatment process costly and uneconomi-
cal. With the implementation of nanoparticle-mediated DDSs, these
limitations can be overcome [1]. In general, nanoparticle-mediated
targeting can be achieved via two targeting strategies: passive and
active targeting;
11.1. Passive targeting
The size of nanoparticles offers an additional advantage. They
have the ability to extravasate and accumulate inside the intersti-
tial spaces, thus contributing to enhanced permeability. Enhanced
retention is observed because of the ineffective lymphatic vessels,
leading to inefficient drainage of the tumour tissue [56]. Altogeth-
er, these two phenomena constitute the enhanced permeability and
retention (EPR) effect, which is considered to be a gold standard in
designing effective anticancer DDSs.
11.2. Active targeting
In active targeting, the ligands are grafted at the surface of the
nanoparticle. The tumour cells are found to possess overexpressed
receptors, to which these ligands bind specifically. Improved cel-
lular internalization rather than an increased tumour accumulation
has been implicated in the enhanced antitumoural efficacy of ac-
tively targeted nanoparticles. In the case of active targeting, the
following two cellular targeting strategies are used:
11.2.1. Targeting of cancer cells
Internalization-prone cell surface receptors such as transferrin,
folate, integrins or epidermal growth factor receptor (EGFR) are
mainly overexpressed in cancer cells. Thus, active targeting acts as
an alternative pathway to improve cellular uptake. The ligand-
mediated approach allows the cancer cells to be killed directly,
followed by generating a cytotoxic effect on cells present at the pe-
riphery of the tumour [57].
11.2.2. Targeting of tumour endothelium
Recognition of specific receptors such as vascular endothelial
growth factor receptor (VEGFR)-1 and VEGFR-2, integrins (αvβ3,
α5β), vascular cell adhesion molecule (VCAM)-1 or matrix
metalloproteinases (MMPs) by targeting ligands facilitates effec-
tive targeting of the tumour endothelium [1]. Folkman et al. were
the first to propose the rationale behind this targeting. It has been
suggested that tumour growth can be inhibited by preventing an-
giogenesis [58]. Due to lack of oxygen, the endothelium in tumour
cells is destroyed, ultimately leading to the death of tumour cells.
However, the size and metastatic capabilities of tumours can be con-
trolled by stopping the surge of blood supply. The tumour core is
composed of angiogenic blood vessels, which in turn support the
tumour cells. The nanoparticle binds with and kills these angio-
genic blood vessels, thus indirectly killing tumour cells. Nevertheless,
the nanoparticle-mediated approach has many advantages [8]:
(i) In order to direct these nanoparticles to the target site, no
extravagation of the nanocarrier is required.
(ii) When administered intravenously, these particles tend to bind
quickly to their receptor sites.
(iii) The possible risk of emerging resistance is prevented, as the
endothelial cells are genetically more stable than the tumour
cells.
(iv) The majority of endothelial cells are expressed in almost all
types of tumour, hence making this approach ubiquitous.
38 S. Sharma et al./Trends in Analytical Chemistry 80 (2016) 30–40
12. Ligand-anchored PLGA nanoparticles for cancer therapy
Cancer cells, unlike normal cells, have an innate tendency to pro-
liferate rapidly, supported by some overexposed receptors present
on the surface of the tumour cell. These receptors allow the uptake
of growth factor via receptor-mediated endocytosis. This mecha-
nism can be used as a Trojan horse for site-specific delivery of
anticancer agents. The surface of the nanoparticles is decorated with
ligands such as antibodies, which tend to bind specifically with these
receptors [36]. The desired ligands can be attached to the surface
of the nanoparticles via simple physical associations or conjuga-
tion reactions.
Carbodiimide chemistry is most commonly used for conjuga-
tion reactions. Nanoparticles can be surface-derivatized via
conjugation reactions. A water-soluble carbodiimide reagent such
as 1-ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDC)/N,N-
dicyclohexylcarbodiimide (DCC) is allowed to react with a carboxyl
group present in the PLGA, leading to the formation of an amine-
reactive O-acylisourea intermediate. This intermediate reacts with
the amine group present in the ligand, resulting in the formation
of a PLGA–ligand conjugate. However, this reaction tends to lead
to other unwanted secondary reactions. To avoid any further reac-
tion, N-hydroxysuccinimide (NHS) is added to the reaction mixture,
and the amine-reactive intermediate thus formed early in the re-
action is transformed into an NHS-ester derivative. The ester
derivative immediately reacts with any primary amine group present
in the reaction mixture, along with the liberation of NHS, ultimate-
ly leaving behind the PLGA–ligand conjugate. Importantly, the
conjugation reaction should proceed in an aqueous environment,
as some portions of the free carboxylic end group remain embed-
ded in the PLGA nanoparticle, which can limit availability for direct
conjugation. To overcome this situation, PLGA is dissolved in an
organic solvent such as dimethyl formamide, prior to the conjuga-
tion reaction [59]. The strong conjugation reactions can be avoided
by using other ligands, for example, non-covalent binding of biotin–
PEG–NH2 with an avidin-functionalized PLGA nanoparticle. Ligands
such as antibodies and Fab fragments can be attached to PLGA
nanoparticles.
Substantial research has been conducted on ligand-mediated
PLGA nanoparticles, but certain drawbacks have limited the use of
these engineered nanoparticles in practical situations. In vivo studies
have revealed that a significant amount of the injected dose is ac-
cumulated in various organs of the RES such as the liver and spleen.
This is undesirable as the anticancer drugs may damage the MPS
organs. The clearance time is increased as these targeted
nanoparticles are recognized by the MPS. Despite the limitations
of the ligand-targeted approach, ligand-targeted nanoparticles have
Table 2
Ligand-targeted PLGA nanoparticles for cancer therapeutics
Nanoparticle Drug loaded Ligand Target site
PLGA Paclitaxel ScFv antibody Hepatic cancer
PLGA–PEG Cisplatin PSMA targeting aptamer Prostate
PLGA–PEG Paclitaxel RGDp Human umbilical vein
endothelial cells
Table 3
PLGA nanoparticles for gene delivery
Nanoparticles Gene Delivered
PLGA nanoparticles DNA
Chitosan–PLGA nanoparticles Antisense oligonucleotides DNA/RNA
DEPA–PVA–PLGA nanoparticles Anti-luciferase siRNA
been proven to enhance the anticancer effect of the entrapped drug
by facilitating cellular uptake and intracellular retention of the
nanodrug carriers, in turn augmenting their antitumour efficacy [36].
A comprehensive overview of different ligands anchored or conju-
gated to PLGA nanoparticles is provided in Table 2 [60–62].
13. PLGA nanoparticles as thriving mediators
13.1. Gene delivery for cancer treatment
Currently, gene therapy is considered one of the most promis-
ing tool for targeted drug delivery in cancer treatment. This technique
can be used to treat a variety of infectious diseases such as mono-
genic diseases and cancer [63]. When travelling from outside of the
cells to the cellular milieu, a gene or macromolecule faces many chal-
lenges such as poor permeability, membrane non-selectivity and
degradation in the endo-lysosomal environment. To circumvent these
obstacles, non-viral novel vectors have been proposed.
PLGA nanoparticles have been explored as a delivery vehicle for
DNA, so that the target gene is effortlessly transfected with the cancer
cells. For this purpose, the surface properties of PLGA nanoparticles
such as surface charge or coating must be tailored as efficient car-
riers for DNA transfer into cancer cells.36 Gene silencing via siRNA
is currently the fastest growing sector of the antigene field for target
validation and therapeutic applications. However, the systematic
deliverability of siRNA into target cells is often limited by their rapid
degradation and poor cell penetration ability. To overcome these
limitations, a broad spectrum of viral and non-viral vectors has been
successful in improving the targeted delivery of siRNA to cancer cells
and protecting them from premature degradation in the biologi-
cal milieu. PLGA-based systems have also been investigated for the
targeted delivery of siRNA to cancer cells and for induction of gene
silencing. Studies on the delivery of DNA or a specific gene via PLGA
nanoparticles are listed in Table 3 [64–66].
13.2. Diagnosis and imaging of cancer
In the field of clinical oncology, tumour imaging plays a key role,
as it helps determine the recurrence of solid tumours along with
monitoring the therapeutic responses. The currently available clin-
ical diagnostic methods are unable to detect cancer in the early stages
[1]. However, developing a non-invasive molecular imaging system
might allow the detection of tumours at an early stage. Recent ad-
vancements in nanoparticles have facilitated the use of contrast
agents for imaging [23]. Wang et al. prepared supramagnetic iron
oxide-loaded PLGA nanoparticles for magnetic resonance imaging
(MRI) [67]. The imaging effects are enhanced along with an increase
Inference Reference
Improved cellular cytotoxicity against Chepp-3 cells [60]
The NPs were readily taken up by receptor mediated endocytosis [61]
Enhanced uptake by integrin expressing malignant cells [62]
Target Inference Reference
th
COS-7 and Cf2 cells Localization into the endo-lysosomal compartment, [64]
250-fold protein expression in cells
Lung cancer cells Efficient delivery of antisense oligonucleotides [65]
H1299 cell line 80–90% knockdown of the luciferase reporter gene [66]
 S. Sharma et al./Trends in Analytical Chemistry 80 (2016) 30–40
in the half-life of nanoparticles in the bloodstream, with reduced
severity of side effects. In another experiment, Acharya et al. de-
tected sentinel lymph node of Wistar rats by encapsulating a
radiotracer, 99mTC, in PLGA nanoparticles [23].
13.3. Theranostics of cancer
Theranostics collectively describes the therapeutic and diag-
nostic agents. It may combine passive and active targeting, envi-
ronmentally responsive drug release, molecular imaging and other
therapeutic functions under a single platform [68]. For example, mag-
netic nanoparticles containing doxorubicin were further encapsulated
in PLGA nanoparticles via hydrophobic interactions. In another study,
PLGA-containing magnetic nanoparticles were designed with the
dual function of drug delivery and imaging [69]. MRI scans have
shown that these nanoparticles offer a better contrast than the com-
mercially available contrast agents.
14. Drawbacks of using PLGA nanoparticle-based DDSs
Apart from developmental and nanoethical aspects, PLGA
nanoparticle-based DDSs have certain drawbacks:
(i) One of the major drawbacks is that the EPR is often misun-
derstood. It is a heterogeneous phenomenon that varies
substantially from model to model, and from patient to
patient. Moreover, the potential efficacy of active drug tar-
geting is overestimated sometimes. In theory, targeted
nanoparticles are retained more efficiently and rapidly than
non-targeted nanoparticles. However, an increase in immu-
nogenicity and protein adsorption is often noted with the
introduction of targeting moieties [56].
(ii) Although PLGA-based nanoparticles offer advantages such as
high encapsulation efficiency in comparison to other
nanoparticle-based DDS, they exhibit poor drug loading, which
might prove problematic for some drugs [8].
(iii) High burst release rate of the drug from nanoparticles is often
accompanied by the DDS. Thus, the efficacy of the DDS is lost,
followed by a non-targeted delivery of the payload or drug.
(iv) The degradation of the PLGA polymer leads to the forma-
tion of acids that limit the drug’s therapeutic efficacy. Drug
delivery via PLGA nanoparticles as the encapsulant is not fea-
sible if they are pH sensitive.
(v) The local tissue reactions occur at the site of application, which
is another cause for concern. Studies have also suggested that
specific biodistribution and toxicological profiles emerge when
nanoparticles of any material are used for therapeutic ap-
plication [70].
(vi) Relatively poor drug loading, higher cost of production and
difficulty in scaling up are some of the other limitations to
the use of PLGA nanoparticles in clinical trials.
(vii) Current knowledge on the effect of various process param-
eters on the preparation of nanoparticles is still limited [27].
(viii) The particle size and morphology of the nanoparticles are af-
fected by two major factors: homogenization conditions and
droplet size distribution. A thorough understanding of these
parameters is still lacking.
(ix) During in vivo application, heterogeneous expression levels
of the targeted receptor in cancer cells also pose a chal-
lenge. Various physiological barriers prevent these
nanoparticles from reaching the target tissues.
(x) The in vivo behaviour of these nanoparticles has not been
completely characterized [59].
(xi) Due to the complexity of drug release from PLGA-based
DDSs, the results obtained with a specific DDS cannot be
generalized [44].
39
15. Conclusion
Despite the several advantages of PLGA nanoparticle-based DDSs,
these systems have their limitations as well. A relatively low drug
loading efficiency significantly limits the use of drug-loaded PLGA
nanoparticles in clinical trials. The preparation of polymeric
nanoparticles is a state-of-the-art technology requiring a suitable
protocol, a thorough homogenization process, appropriate surfac-
tants and creditable co-surfactant to obtain the desired polymeric
nanoparticles with optimal properties. Future research should be
directed towards developing techniques that can precisely control
the particle size and morphology, which are considered factors de-
termining the applications of these nanoparticles. Further
investigation is needed to better control nanoparticle size, polydis-
persity and charge surface, and to render these features easily
reproducible during synthesis steps. The limitations of using PLGA
nanoparticles can be overcome through an extensive and thor-
ough evaluation of the pharmacokinetics, biodistribution and toxicity.
PLGA degradation and drug release rate can be increased with greater
hydrophilicity, increase in chemical interactions among the hydro-
lytic groups, less crystallinity and larger volume to surface ratio of
the device. All of these factors should be considered to tailor the
degradation and drug release mechanism as needed. Proper selec-
tion of the preparation method, formulation/reaction variables and
appropriate scale-up techniques/methods, combined with coordi-
nated efforts from the industrial and academic sectors, can lead to
successful commercialization of engineered PLGA nanoparticles. Nev-
ertheless, the prospects of putting drug-loaded PLGA NP technology
into practice are exciting as the next generation of DDSs.
Acknowledgement
The authors are grateful to DST, Government of India, for pro-
viding financial support to IFSC through the PURSE grant.
References
[1] F. Danhier, E. Ansorena, J.M. Silva, R. Coco, A. Le, V. Breton, et al., based
nanoparticles: an overview of biomedical applications, J. Control. Release 161
(2012) 505–522.
[2] Y. Liu, H. Miyoshi, M. Nakamura, Nanomedicine for drug delivery and imaging:
a promising avenue for cancer therapy and diagnosis using targeted functional
nanoparticles, Int. J. Cancer 120 (2007) 2527–2537.
[3] S.A. Wickline, A.M. Neubauer, P.M. Winter, S.D. Caruthers, G.M. Lanza, Molecular
imaging and therapy of atherosclerosis with targeted nanoparticles, J. Magn.
Reson. Imaging 25 (2007) 667–680.
[4] R. Langer, J. Folkman, Polymers for the sustained release of proteins and other
macromolecules, Nature 263 (1976) 797–800.
[5] D. Dragan, S. Magdalena, Poly(lactide-co-glycolide)-based micro and
nanoparticles for the controlled drug delivery of vitamins, Curr. Nanosci. 5
(2009) 1–14.
[6] J.-S. Choi, K. Seo, J.-W. Yoo, Recent advances in PLGA particulate systems for
drug delivery, J. Pharm. Investig. 42 (2012) 155–163.
[7] E. Locatelli, M.C. Franchini, Biodegradable PLGA-b-PEG polymeric nanoparticles:
synthesis, properties, and nanomedical applications as drug delivery systems,
J. Nanoparticle Res. 14 (2012).
[8] F.S.T. Mirakabad, K. Nejati-Koshki, A. Akbarzadeh, M.R. Yamchi, M. Milani, N.
Zarghami, et al., PLGA-based nanoparticles as cancer drug delivery systems,
Asian Pac. J. Cancer Prev. 15 (2014) 517–535.
[9] H.K. Makadia, S.J. Siegel, Poly Lactic-co-Glycolic acid (PLGA) as biodegradable
controlled drug delivery carrier, Polymers (Basel) 3 (2011) 1377–1397.
[10] A. Prokop, J.M. Davidson, Nanovehicular intracellular delivery systems, J. Pharm.
Sci. 97 (2008) 3518–3590.
[11] G. Schliecker, C. Schmidt, S. Fuchs, T. Kissel, Characterization of a homologous
series of D,L-lactic acid oligomers; a mechanistic study on the degradation
kinetics in vitro, Biomaterials 24 (2003) 3835–3844.
[12] S. Acharya, S.K. Sahoo, PLGA nanoparticles containing various anticancer agents
and tumour delivery by EPR effect, Adv. Drug Deliv. Rev. 63 (2011) 170–183.
[13] S.J. Siegel, J.B. Kahn, K. Metzger, K.I. Winey, K. Werner, N. Dan, Effect of drug
type on the degradation rate of PLGA matrices, Eur. J. Pharm. Biopharm. 64
(2006) 287–293.
[14] N. Passerini, D.Q. Craig, An investigation into the effects of residual water on
the glass transition temperature of polylactide microspheres using modulated
temperature DSC, J. Control. Release 73 (2001) 111–115.
40 S. Sharma et al./Trends in Analytical Chemistry 80 (2016) 30–40
[15] Z. Panagi, A. Beletsi, G. Evangelatos, E. Livaniou, D.S. Ithakissios, K. Avgoustakis,
Effect of dose on the biodistribution and pharmacokinetics of PLGA and
PLGA-mPEG nanoparticles, Int. J. Pharm. 221 (2001) 143–152.
[16] R.A. Jain, The manufacturing techniques of various drug loaded biodegradable
poly(lactide-co-glycolide) (PLGA) devices, Biomaterials 21 (2000) 2475–2490.
[17] S. Mao, J. Xu, C. Cai, O. Germershaus, A. Schaper, T. Kissel, Effect of WOW process
parameters on morphology and burst release of FITC-dextran loaded PLGA
microspheres, Int. J. Pharm. 334 (2007) 137–148.
[18] F.J. Hua, T.G. Park, D.S. Lee, facile preparation of highly interconnected
macroporous PLGA scaffolds by liquid-liquid phase separation of a PLGA-
dioxane-water ternary system, Polymer (Guildf.) 44 (2003) 1911–1920.
[19] S.R. D’Mello, S.K. Das, N.G. Das, Polymeric nanoparticles for small-molecule
drugs: biodegradation of polymers and fabrication of nanoparticles. Drug
Delivery Nanoparticles Formulation and Characterization, 2006.
[20] G. Lambert, E. Fattal, P. Couvreur, Nanoparticulate systems for the delivery of
antisense oligonucleotides, Adv. Drug Deliv. Rev. 47 (2001) 99–112.
[21] H. Fessi, F. Puisieux, J.P. Devissaguet, N. Ammoury, S. Benita, Nanocapsule
formation by interfacial polymer deposition following solvent displacement,
Int. J. Pharm. 55 (1989) R1–R4.
[22] T. Govender, S. Stolnik, M.C. Garnett, L. Illum, S.S. Davis, PLGA nanoparticles
prepared by nanoprecipitation: drug loading and release studies of a water
soluble drug, J. Control. Release 57 (1999) 171–185.
[23] Y.-I. Jeong, C.-S. Cho, S.-H. Kim, K.-S. Ko, S.-I. Kim, Y.-H. Shim, et al., Preparation
of poly(dl-lactide-co-glycolide) nanoparticles without surfactant, J. Appl. Polym.
Sci. 80 (2001) 2228–2236.
[24] M. Kostog, S. Kohler, T. Liebert, T. Heinze, Pure cellulose nanoparticles from
trimethylsilyl cellulose, Macromol. Symp. 294 (2) (2010) 96–106.
[25] H. Nie, L.Y. Lee, H. Tong, C.H. Wang, PLGA/chitosan composites from a
combination of spray drying and supercritical fluid foaming techniques: new
carriers for DNA delivery, J. Control. Release 129 (2008) 207–214.
[26] K. Mishima, Biodegradable particle formation for drug and gene delivery using
supercritical fluid and dense gas, Adv. Drug Deliv. Rev. 60 (2008) 411–432.
[27] J.P. Rao, K.E. Geckeler, Polymer nanoparticles: Preparation techniques and
size-control parameters, Elsevier 36 (2011) 887–913.
[28] Y.P. Sun, H.W. Rolling, J. Bandara, J.M. Meziani, C.E. Bunker, Preparation and
processing of nanoscale materials by supercritical fluid technology, in: Y.P. Sun
(Editor), Supercritical Fluid Technology in Materials Science and Engineering:
Synthesis, Properties, and Applications, Marcel Dekker, New York, 2002, pp.
491–576.
[29] G. Mittal, D.K. Sahana, V. Bhardwaj, M.N. Ravi Kumar, Estradiol loaded PLGA
nanoparticles for oral administration: effect of polymer molecular weight and
copolymer composition on release behavior in vitro and in vivo, J. Control.
Release 119 (2007) 77–85.
[30] M. Garinot, V. Fievez, V. Pourcelle, F. Stoffelbach, A. des Rieux, L. Plapied, et al.,
PEGylated PLGA-based nanoparticles targeting M cells for oral vaccination,
J. Control. Release 120 (2007) 195–204.
[31] F. Esmaeili, M.H. Ghahremani, B. Esmaeili, M.R. Khoshayand, F. Atyabi, R.
Dinarvand, PLGA nanoparticles of different surface properties: preparation and
evaluation of their body distribution, Int. J. Pharm. 349 (2008) 249–255.
[32] I. Bala, S. Hariharan, M.N. Kumar, PLGA nanoparticles in drug delivery: the state
of the art, Crit. Rev. Ther. Drug Carrier Syst. 21 (2004) 387–422.
[33] Z. Zhang, S.S. Feng, The drug encapsulation efficiency, in vitro drug release,
cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl
polyethylene glycol succinate nanoparticles, Biomaterials 27 (2006) 4025–4033.
[34] A. Yang, L. Yang, W. Liu, Z. Li, H. Xu, X. Yang, Tumor necrosis factor alpha blocking
peptide loaded PEG-PLGA nanoparticles: preparation and in vitro evaluation,
Int. J. Pharm. 331 (2007) 123–132.
[35] A. Kumari, S.K. Yadav, S.C. Yadav, Biodegradable polymeric nanoparticles based
drug delivery systems, Colloids Surf. B. Biointerfaces 75 (2010) 1–18.
[36] A.K. Jain, M. Das, N.K. Swarnakar, S. Jain, Engineered PLGA nanoparticles:
an emerging delivery tool in cancer therapeutics, Crit. Rev. Ther. Drug Carrier
Syst. 28 (2011) 1–45.
[37] W. Lu, Y.Z. Tan, K.L. Hu, X.G. Jiang, Cationic albumin conjugated pegylated
nanoparticle with its transcytosis ability and little toxicity against blood-brain
barrier, Int. J. Pharm. 295 (2005) 247–260.
[38] R. Gref, M. Luck, P. Quellec, M. Marchand, E. Dellacherie, S. Harnisch, et al.,
‘Stealth’ corona-core nanoparticles surface modified by polyethylene glycol
(PEG): influences of the corona (PEG chain length and surface density) and of
the core composition on phagocytic uptake and plasma protein adsorption,
Colloids Surf. B. Biointerfaces 18 (2000) 301–313.
[39] F. Danhier, N. Lecouturier, B. Vroman, C. Jerome, J. Marchand-Brynaert, O. Feron,
et al., Paclitaxel-loaded PEGylated PLGA-based nanoparticles: in vitro and in
vivo evaluation, J. Control. Release 133 (2009) 11–17.
[40] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, V. Torchilin, R. Langer,
Biodegradable long-circulating polymeric nanospheres, Science 263 (1994)
1600–1603.
[41] E.C. Gryparis, M. Hatziapostolou, E. Papadimitriou, K. Avgoustakis, Anticancer
activity of cisplatin-loaded PLGA-mPEG nanoparticles on LNCaP prostate cancer
cells, Eur. J. Pharm. Biopharm. 67 (2007) 1–8.
[42] M. Senthilkumar, P. Mishra, N.K. Jain, Long circulating PEGylated poly(D,L-
lactide-co-glycolide) nanoparticulate delivery of Docetaxel to solid tumors,
J. Drug Target. 16 (2008) 424–435.
[43] T. Betancourt, J.D. Byrne, N. Sunaryo, S.W. Crowder, M. Kadapakkam, S. Patel,
et al., PEGylation strategies for active targeting of PLA/PLGA nanoparticles,
J. Biomed. Mater. Res. A 91 (2009) 263–276.
[44] S. Fredenberg, M. Wahlgren, M. Reslow, A. Axelsson, The mechanisms of drug
release in poly(lactic-co-glycolic acid)-based drug delivery systems–a review,
Int. J. Pharm. 415 (2011) 34–52.
[45] M. Marucci, Characterization of the mechanisms of drug release from polymer-
coated formulations using experiments and modelling., Doctoral Dissertation.
Lund University, Lund, Sweden, 2009.
[46] A. Shenderova, T.G. Burke, S.P. Schwendeman, The acidic microclimate in
poly(lactide-co-glycolide) microspheres stabilizes camptothecins, Pharm. Res.
16 (1999) 241–248.
[47] M. Dunne, I. Corrigan, Z. Ramtoola, Influence of particle size and dissolution
conditions on the degradation properties of polylactide-co-glycolide particles,
Biomaterials 21 (2000) 1659–1668.
[48] N.S. Berchane, K.H. Carson, A.C. Rice-Ficht, M.J. Andrews, Effect of mean diameter
and polydispersity of PLG microspheres on drug release: experiment and theory,
Int. J. Pharm. 337 (2007) 118–126.
[49] L.C. Amann, M.J. Gandal, R. Lin, Y. Liang, S.J. Siegel, In vitro-in vivo correlations
of scalable PLGA-risperidone implants for the treatment of schizophrenia,
Pharm. Res. 27 (2010) 1730–1737.
[50] L. Lu, S.J. Peter, M.D. Lyman, H.L. Lai, S.M. Leite, J.A. Tamada, et al., In vitro and
in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams,
Biomaterials 21 (2000) 1837–1845.
[51] B.S. Zolnik, D.J. Burgess, Effect of acidic pH on PLGA microsphere degradation
and release, J. Control. Release 122 (2007) 338–344.
[52] I.E. Kuppens, T.M. Bosch, M.J. van Maanen, H. Rosing, A. Fitzpatrick, J.H. Beijnen,
et al., Oral bioavailability of docetaxel in combination with OC144-093
(ONT-093), Cancer Chemother. Pharmacol. 55 (2005) 72–78.
[53] M. Hennessy, J.P. Spiers, A primer on the mechanics of P-glycoprotein the
multidrug transporter, Pharmacol. Res. 55 (2007) 1–15.
[54] B. Li, H. Xu, Z. Li, M. Yao, M. Xie, H. Shen, et al., Bypassing multidrug resistance
in human breast cancer cells with lipid/polymer particle assemblies, Int. J.
Nanomedicine 7 (2012) 187–197.
[55] R. Langer, Drug delivery and targeting, Nature 392 (1998) 5–10.
[56] F. Danhier, O. Feron, V. Preat, To exploit the tumor microenvironment: Passive
and active tumor targeting of nanocarriers for anti-cancer drug delivery,
J. Control. Release 148 (2010) 135–146.
[57] F. Pastorino, C. Brignole, D. Di Paolo, B. Nico, A. Pezzolo, D. Marimpietri, et al.,
Targeting liposomal chemotherapy via both tumor cell-specific and tumor
vasculature-specific ligands potentiates therapeutic efficacy, Cancer Res. 66
(2006) 10073–10082.
[58] J. Folkman, Transplacental carcinogenesis by stilbestrol, N. Engl. J. Med. 285
(1971) 404–405.
[59] H. Sah, L.A. Thoma, H.R. Desu, E. Sah, G.C. Wood, Concepts and practices used
to develop functional PLGA-based nanoparticulate systems, Int. J. Nanomedicine
8 (2013) 747–765.
[60] G. Kou, J. Gao, H. Wang, H. Chen, B. Li, D. Zhang, et al., Preparation and
characterization of paclitaxel-loaded PLGA nanoparticles coated with cationic
SM5-1 single-chain antibody, J. Biochem. Mol. Biol. 40 (2007) 731–739.
[61] S. Dhar, F.X. Gu, R. Langer, O.C. Farokhzad, S.J. Lippard, Targeted delivery
of cisplatin to prostate cancer cells by aptamer functionalized Pt(IV)
prodrug-PLGA-PEG nanoparticles, Proc. Natl. Acad. Sci. U.S.A. 105 (2008)
17356–17361.
[62] F. Danhier, B. Vroman, N. Lecouturier, N. Crokart, V. Pourcelle, H. Freichels, et al.,
Targeting of tumor endothelium by RGD-grafted PLGA-nanoparticles loaded
with paclitaxel, J. Control. Release 140 (2009) 166–173.
[63] S. Diez, G. Navarro, C.T. de ILarduya, In vivo targeted gene delivery by cationic
nanoparticles for treatment of hepatocellular carcinoma, J. Gene Med. 11 (2009)
38–45.
[64] K. Gvili, O. Benny, D. Danino, M. Machluf, Poly(D,L-lactide-co-glycolide acid)
nanoparticles for DNA delivery: waiving preparation complexity and increasing
efficiency, Biopolymers 85 (2007) 379–391.
[65] N. Nafee, S. Taetz, M. Schneider, U.F. Schaefer, C.M. Lehr, Chitosan-coated PLGA
nanoparticles for DNA/RNA delivery: effect of the formulation parameters on
complexation and transfection of antisense oligonucleotides, Nanomedicine 3
(2007) 173–183.
[66] J. Nguyen, T.W. Steele, O. Merkel, R. Reul, T. Kissel, Fast degrading polyesters
as siRNA nano-carriers- for pulmonary gene therapy, J. Control Rel. 132 (2008)
243–251.
[67] Y.G.N.Y. Wang, Y. Chen, B. Shuter, J. Yi, J. Ding, S. Wang, et al., Formulation of
superparamagnetic iron oxides by nanoparticles of biodegradable polymers for
magnetic resonance imaging, Adv. Funct. Mater. 18 (2008) 308–318.
[68] S.M. Janib, A.S. Moses, J.A. MacKay, Imaging and drug delivery using theranostic
nanoparticles, Adv. Drug Deliv. Rev. 62 (2010) 1052–1063.
[69] A. Singh, F. Dilnawaz, S. Mewar, U. Sharma, N.R. Jagannathan, S.K. Sahoo,
Composite polymeric magnetic nanoparticles for co-delivery of hydrophobic
and hydrophilic anticancer drugs and MRI imaging for cancer therapy, ACS Appl.
Mater. Interfaces 3 (2011) 842–856.
[70] L.A. Dailey, N. Jekel, L. Fink, T. Gessler, T. Schmehl, M. Wittmar, et al.,
Investigation of the proinflammatory potential of biodegradable nanoparticle
drug delivery systems in the lung, Toxicol. Appl. Pharmacol. 215 (2006) 100–108.