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European Journal of Applied Sciences – Vol. 10, No. 1
Publication Date: February 25, 2022
DOI:10.14738/aivp.101.11724. Vidawati, S., Barbosa, S., Taboada, P., & Nadhifa, S. F. (2022). Stability of Human Serum Albumin Loaded PLGA Nanoparticles for
Protein Delivery Applications. European Journal of Applied Sciences, 10(1). 366-370.
Services for Science and Education – United Kingdom
Stability of Human Serum Albumin Loaded PLGA Nanoparticles
for Protein Delivery Applications
Sri Vidawati
Faculty of Post Graduate Engineering
National Institute of Science and Technology, Jakarta, Indonesia
Silvia Barbosa
Grupo de F’ısica de Coloides y Pol’ımeros
Departamento de F ́ısica de la Materia Condensada
Universidad de Santiago de Compostela, Santiago de Compostela, Spain
Pablo Taboada
Grupo de F’ısica de Coloides y Pol’ımeros
Departamento de F ́ısica de la Materia Condensada
Universidad de Santiago de Compostela, Santiago de Compostela, Spain
Nadhifa Safira P.
Metallurgy and Materials Engineering University of Indonesia
Depok, Indonesia
ABSTRACT
Poly (lactic-co-glycolic acid) (PLGA) is one of the most effectively developed
biodegradable polymers. Biodegradable polymers based on PLGA are the most
common materials used to encapsulate therapeutic agents. Protein-loaded PLGA
nanoparticles are generally formulated by the double emulsion method. However,
the main problems associated with this method are low encapsulation efficiency
proteins and instability proteins. This study report the stability of encapsulate of
Human Serum Albumin (HSA)-loaded PLGA nanoparticles for protein delivery
applications.
Keywords: Nanoparticles, PLGA, HSA.
INTRODUCTION
In the last decade, nanotechnology has evolved to such an extent that it has become possible to
fabricate, characterize and specifically adapt the functional properties of nanoparticles for
biomedical applications [1-3]. Nanotechnology has been applied to improve drug delivery and
to develop nano-scaled drug delivery devices. Nanoparticulate drug delivery systems appear to
be a viable and promising strategy for the biopharmaceutical industry. They have advantages
over conventional drug delivery systems. They can improve the bioavailability, solubility and
permeability of many potent drugs that are otherwise difficult to deliver orally. The
nanoparticulate drug delivery systems will also reduce the drug dosage frequency and will
improve patient compliance. In the near future nanoparticulate drug delivery systems can be
used to exploit many biological drugs that have poor aqueous solubility, permeability and lack
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Vidawati, S., Barbosa, S., Taboada, P., & Nadhifa, S. F. (2022). Stability of Human Serum Albumin Loaded PLGA Nanoparticles for Protein Delivery
Applications. European Journal of Applied Sciences, 10(1). 366-370.
URL: http://dx.doi.org/10.14738/aivp.101.11724
of bioavailability. Nanoparticles have been developed as a crucial strategy for delivering low
molecular weight drugs, as well as biomacromolecules such as proteins or DNA [4].
Nanoparticles or colloidal particles have been widely used for targeted drug delivery and other
biomedical applications [5,6]. Many studies have shown that the biological distribution of
drugs, proteins or DNA can be modified, both at the cellular and organ levels, using
micro/nanoparticles delivery systems [7]. The nanometer size ranges of these delivery systems
offer different advantages for drug delivery.
Poly(lactic-co-glycolic acid) (PLGA) is one of the most effectively developed biodegradable
polymers. Among the variant polymers developed to formulate polymeric nanoparticles, PLGA
has interested considerable attention due to its interesting properties: (i) biodegradability and
biocompatibility, (ii) FDA and European Medicine Agency approval in drug delivery systems for
parenteral administration, (iii) well explained formulations and production methods adjusted
to different variety of drugs e.g. hydrophilic or hydrophobic small molecules or
macromolecules, (iv) protection of drug from degradation, (v) the potential of sustained
release, (vi) the potential to improve surface behaviour to prepare stealthness and/or better
interaction with biological materials and (vii) the potential to target nanoparticles to certain
organs or cells.
Biodegradable polymers based on poly(lactic-co-glycolic acid) (PLGA) are completely
biocompatible and therefore among the most common materials used to encapsulate
therapeutic agents. PLGA micro/nanoparticles have been used for the delivery of antigens and
the stimulation of immune responses [8,9]. Antigen-loaded PLGA nanoparticles are generally
formulated by the emulsion solvent evaporation method. However, the primary problems
associated with this method are low encapsulation efficiency of highly water-soluble proteins
and instability arising during the formulation, storage, and lyophilization of the nanoparticles.
Proteins instability also occurs during polymer degradation because of the accumulation of
acidic monomers and the consequent generation of a low pH inside the biodegradable
nanoparticles [10,11].
Multiple emulsion methods are very often used to encapsulate hydrophilic drugs, such as
peptides, proteins and nucleic acids. One of the most commonly used technique for the
encapsulating proteins into PLGA nanoparticles is the double emulsion solvent evaporation
procedure, as proteins tend to be hydrophilic macromolecules. The encapsulation of proteins
into PLGA nanoparticles presents some challenges as instability problems [10]. For example, in
the first step of the formulation procedure the protein dissolved in the aqueous phase can be
aggregate or be denatured at the water/organic solvent interface, adsorb to the hydrophobic
polymer or unfold because of the shear stress used for the formation of the primary emulsion
[12]. Denatured or aggregated protein species will not only be therapeutically inactive, but also
can cause induce unpredictable side effects, such as toxicity or immunogenicity [13]. To address
these problems, many studies have focused on optimizing the formulation process to improve
protein stability during classical procedure.
In this study we reported encapsulation of Human Serum Albumin (HSA) loaded PLGA
nanoparticles. HSA encapsulated PLGA nanoparticles are prepared using the double emulsion
method. Further studies are needed to determine the size and surface zeta potential of HSA
loaded PLGA nanoparticles for protein delivery applications.
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European Journal of Applied Sciences (EJAS) Vol. 10, Issue 1, February-2022
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MATERIALS AND METHODS
Materials
PLGA of 38 - 54 kDa with 50:50 lactide-glycolide ratio, Pluronic F127, and Human Serum
Albumin (HSA), are get from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and
solvents are get from Sigma-Aldrich. Pure water of Milli-Q quality is used in all preparations.
Synthesis of HSA Loaded PLGA Nanoparticles
The preparation polymeric encapsulated PLGA nanoparticles, containing of HSA were prepared
using the multiple emulsion solvent evaporation methods. In a typical preparation, PLGA (25
mg) was dis-solved in a sealed vial containing Dichloromethane (1 ml), HSA was dissolved in
pure water (100 μL) by ultrasonic 10 min, by sonication with a probe-type sonicator (20 kHz,
Bandelin Sonopuls, Bandelin GmbH, Berlin, Germany) at some parameters of time and power
in an ice bath. Then, this organic solution was added drop wise with a syringe pump (0.166
mL/min) to an aqueous solution (50 mL) containing Pluronic F127 (typically 1 wt% if not
otherwise stated) while stirring at 10 ̊C. After sonication with power 100 W for 15 minutes of
this experiment to homogenize the resulting dispersion, the organic solvent was completely
evaporated under mechanical stirring over-night, the dispersion subsequently centrifuged
twice at 9000 rpm for 20 min and 20 ̊C. Subsequently, the supernatant was removed and the
final precipitate was kept in the freezer.
Characterization of Nanoparticles
Transmission electron microscopy (TEM) is the most beneficial technique for explained the
particle size and morphology of nanoparticles. Samples were prepared for analysis by
evaporating a drop of the nanoparticles dispersion on a carbon-coated copper grid without
staining (TEM). TEM images of nanoparticles were obtained with a Philips CM-12 (Philips,
Netherlands) micro-scope operating at 120 kV. HR-TEM images and selected area electron
diffraction (SAED) patterns were obtained with a transmission electron microscope (Carl-Zeiss
Libra 200 FE-EFTEM, Germany) operating at 200 kV.
The zeta potentials of nanoparticles are get by triplicate with a Zetasizernano ZS (Malvern, UK),
using disposable folded capillary cells. Each experiment is repeated at least three times.
RESULT AND DISCUSSION
Figure 1. TEM images of the synthesis of HSA loaded PLGA nanoparticles
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Vidawati, S., Barbosa, S., Taboada, P., & Nadhifa, S. F. (2022). Stability of Human Serum Albumin Loaded PLGA Nanoparticles for Protein Delivery
Applications. European Journal of Applied Sciences, 10(1). 366-370.
URL: http://dx.doi.org/10.14738/aivp.101.11724
Given the tremendous advances in biotechnology, biological agents such as proteins, peptides,
aptamers and oligonucleotides have attracted a lot of interest [14, 15]. However, their speed to
reach the clinical phase has faced difficulties. This may be due to certain obstacles including
protein fragility and its short half-life in the body. To overcome these obstacles, Novel's
controlled delivery system has been proposed [16]. Among the many operators applied to this
purpose, biodegradable polymers have gained increasing interest due to biocompatibility, non- toxicity and diversity in physical-chemical properties [17, 18].
One of the most commonly used methods for encapsulating proteins into PLGA nanoparticles
is the double solvent evaporation procedure, since proteins tend to be hydrophilic
macromolecules. Encapsulation of proteins into PLGA nanoparticles presents several
challenges as instability problems [10]. Some investigations have already successfully shown
stabilisation nanoparticles using protein-super paramagnetik iron oxide nanoparticle (SPIONs)
loaded PLGA nanoparticle [19, 20].
The encapsulation of these therapeutic proteins in PLGA nanoparticles has emerged as a
promising alternative to overcome all of these problems as well as to contributing to certain
with additional advantages. The incorporation of the proteins into the polymer matrix provide
protection against enzymatic and hydrolytic degradation in vivo, maintaining their integrity
and activity, can improve their bioavailability and in some cases may target therapeutic protein
to the target area.
In this experiment, we synthesized HSA loaded PLGA nanoparticles using the multiple emulsion
solvent evaporation methods. The size and morphology of HSA loaded PLGA nanoparticle is
characterized by TEM. TEM image are used to obtain key information about the main size and
morphology of nanoparticles. TEM is an importtant technique that has the unique ability to
investigate the internal structure of individual nanoparticles. Figure 1 shows an TEM image of
HSA loaded PLGA nanoparticles with sonication parameter of power 20 W for 12 minutes. The
TEM images in Figure 1 shows the nanoparticles look perfect and stabilize without aggregration
in the sphere nanocapsule around 200 - 300 nm in size. HSA loaded PLGA nanoparticles have a
zeta potential of about −40.15 mV which suggest HSA load PLGA nanoparticles are stabilizing.
Nanoparticles are dense and spherical structures range from 100 nm - 300 nm in size and are
made of natural or synthetic polymers. Various medications can be delivered using
nanoparticles, such as hydrophilic small drug, hydrophobic small drug, vaccines, and biological
macromolecules. Nanoparticles also make it possible administration of certain organs or cells
or controlled drug delivery.
CONCLUSIONS
Nanoparticulate drug/antigen delivery systems show to be a viable and promising strategy for
the biopharmaceutical industry. They have benefits over conventional drug/antigen delivery
systems. They can increase the bioavailability, solubility and permeability of many potent
drugs/antigen which are difficult to deliver orally. Nanoparticles can minimize some of these
drugs/antigen unique problems by maintaining stability and maintaining their structure.
PLGA-based nanoparticles present many advantages for drug/antigen delivery. They can
protect drugs/antigen from degradation and increase its stability. In summarizing our data, we
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argued that HSA loaded PLGA nanoparticles are stability which is one of an important indication
of such as protein delivery applications.
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