International Journal of Pharmaceutics 554 (2019) 224–234

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International Journal of Pharmaceutics

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

Review

Modified gelatin nanoparticles for gene delivery T

a,b,1 a,c,1 a,d,⁎,1
Osama Madkhali , George Mekhail , Shawn D. Wettig
a
School of Pharmacy, University of Waterloo, Waterloo ON, N2L 3G1, Canada
b
Department of Pharmaceutics, Faculty of Pharmacy, Jazan University, Jizan, Saudi Arabia
c
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt
d
Waterloo Institute of Nanotechnology, University of Waterloo, Waterloo ON, N2L 3G1, Canada

A R T I C LE I N FO A B S T R A C T

Keywords: Gelatin nanoparticles (GNPs) are one of the most extensively used natural polymers for gene therapy. With
Gelatin advantages of being biodegradable, biocompatible, low cost and easily modified, gelatin holds great promise as a
Gene delivery non-viral system for gene delivery. This review examines various methods of preparation of modified gelatin
Modification nanoparticles and considers how these modifications apply to gene delivery. The article discusses cationic ge-
Method
latin, PEGylated gelatin, thiolated gelatin, alendronate gelatin, and EGFR gelatin nanoparticles. This article also
considers several advantages of these modifications and their contribution to the improvement in the efficiency
of these systems, resulting in superior transfection and enhanced gene delivery in general.

1. Introduction require a delivery vehicle in order to efficiently travel through extra-

Gene therapy (GT) has received much attention due to its great
potential for the treatment of both acquired and inherited diseases such
as cancer, cystic fibrosis (CF), acquired immunodeficiency syndrome
(AIDS), X-linked combined immune deficiency (X-linked SCID), em-
physema, retinitis pigmentosa, sickle cell anemia, hemophilia,
Duchenne Muscular Dystrophy (DMD), some autosomal dominant dis-
orders, vascular disease, neurodegenerative disorders, polygenic dis-
orders, inflammatory conditions, and other infectious diseases (Keeler
et al., 2017; Nayerossadat et al., 2012; Stone, 2010). A gene therapeutic
should fulfil two characteristics: (a) it should contain an active sub-
stance containing or consisting of a recombinant nucleic acid that is
delivered to the nucleus in order to regulate, repair, replace, add, or
delete a defective gene; (b) its therapeutic, diagnostic, or prophylactic
effect relates to the recombinant nucleic acid it contains (Wirth et al.,
2013). When gene therapy is used in the treatment of genetic diseases,
it restricts these diseases through introducing genes coding for func-
tional proteins to cells; thus, it normalizes the cells and even organs in
question (Jin et al., 2014). Since the first attempt of gene therapy in
1928 done by Fredrick Griffith (Griffith, 1928), enormous and sig-
nificant changes have been seen in the development and improvement
of gene therapy. The most important change was the development of
delivery systems. Nucleic acids (such as plasmid DNA, messenger RNA
(mRNA), small interfering RNA (siRNA), and micro RNA (miRNA))
and intra-cellular barriers. Gene delivery vectors offer the nucleic acids
the necessary protection against various nucleases encountered during
its travel to the target site. Moreover, delivery vectors might enhance
the ability of the nanocomplexes to penetrate the cell membrane via
various penetrating mechanisms. Gene carriers also play a crucial role
in endosomal escape via different mechanisms (Cardoso et al., 2014;
Karimi et al., 2015; Lacerda et al., 2012; Trabulo et al., 2010). Finally,
the delivery vector enters the nucleus to express the required protein to
correct or moderate specific diseases. Gene therapy is based on two
types of delivery systems: viral and non-viral vectors. Viral vectors are
more effective and generally have longer gene expression depending on
the nucleic acid type, virus type and its ability to combine the trans-
gene with the host cell genome (Lundstrom and Boulikas, 2003). For
example, single stranded RNA viruses, such as alphaviruses exhibit a
rapid but transient gene expression. Some of the DNA viruses, like
adenovirus, possess short term gene expression. However, adeno-asso-
ciated viruses predictably engender long-term expression owing to their
ability to integrate into the host genome. Finally, retroviruses, double-
stranded RNA viruses, could similarly induce long-term expression
through chromosomal integration (Lundstrom and Boulikas, 2003;
Nayerossadat et al., 2012). Yet viral vectors have several concerns re-
garding safety. As a result, an alternative method is the use of non-viral
vectors. They are safe, non-toxic, cheap, have low immunogenicity and
can be produced in scalable batches. Although they suffer from short-

⁎
Corresponding author at: School of Pharmacy, University of Waterloo, 200 University Ave. W., Waterloo, ON N2L 3G1, Canada.
E-mail address: wettig@uwaterloo.ca (S.D. Wettig).
1
All authors contributed equally to this work.

https://doi.org/10.1016/j.ijpharm.2018.11.001
Received 30 July 2018; Received in revised form 31 October 2018; Accepted 1 November 2018
Available online 05 November 2018
0378-5173/ © 2018 Elsevier B.V. All rights reserved.
O. Madkhali et al.
term gene expression yet, efforts are exerted to tailor suitable non-viral
vectors with sufficient gene expression period (Jackson et al., 2006; Yin
et al., 2014). Two main categories of non-viral vectors have been used:
physical and chemical methods.
2. Physical methods of non-viral systems
These methods depend on using physical force in order to destabi-
lize the cellular membrane, therefore facilitating the entry of gene
therapeutic materials into the cells. These methods are simple and
straightforward.
2.1. Electroporation
Electroporation is known as gene electro injection, gene electro
transfer, electrically mediated gene therapy, or electro gene transfer
(Ramamoorth and Narvekar, 2015). It works by applying an electric
field greater than the membrane capacitance into the targeted tissue
cell membrane, resulting in a pore that allows the molecules to pass
through it. As a result, the previously injected DNA can enter into the
cytoplasm and nucleoplasm of the cell (Nayerossadat et al., 2012). This
method is very effective and safe when it applied in vivo in comparison
to other non-viral methods. However, the complexity of surgical pro-
cedures, and high voltage [ > 700 V/cm] applied to the tissues, as well
as the difficulty of reaching some internal tissues makes this method
inappropriate for delivering DNA (Young and Dean, 2015).
2.2. Gene gun
The gene gun (also known as particle bombardment, micro pro-
jectile gene transfer or ballistic DNA) delivers DNA coated heavy metal
particles into the target tissue at a particular speed using high voltage
electronic discharge, spark discharge, or high pressure inert gas, usually
helium (Mali, 2013). The most common metal particles used in this
method are gold, tungsten, and silver, which all typically measure 1 µm
in diameter. Gene transfer is affected by several parameters such as gas
pressure, particle size, and dose frequency. Precise delivery of DNA is
the most important advantage using the gene gun method, and it most
commonly used in gene therapy that targets ovarian cancer cells in vitro
(Ramamoorth and Narvekar, 2015).
2.3. Sonoporation
Sonoporation is a noninvasive technique using ultrasound wave to
permeabilize the cell membrane; thus, allowing the uptake of DNA.
Genetic materials of interest are first administered into the circulation
using microbubbles, followed by the application of the ultrasound
waves. The ultrasound waves cavitate and break up the microbubbles
within the microcirculation of target tissue, leading to the disruption of
the nearby cell membrane that results in targeted transfection of the
therapeutic gene (Omata et al., 2015). The major advantages of sono-
poration include safety, noninvasiveness, and the ability to reach in-
ternal organs without the necessity of surgery; consequently, it is used
in the brain, cornea, kidney, and peritoneal cavity, as well as in muscle
and heart tissues (Ramamoorth and Narvekar, 2015; Ter Haar, 2007).
2.4. Photoporation
This technique works by using a single laser pulse in order to gen-
erate a pore in the cell membrane allowing the DNA to enter into the
cells. The effectiveness of this method depends on the focal point and
pulse frequency of the laser. The major advantage of this approach is its
safety, in which the pore that is formed by the laser can be healed in less
than a second. However, the lack of documented evidence limits the use
of this technique (Li and Huang, 2007).
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International Journal of Pharmaceutics 554 (2019) 224–234
2.5. Magnetofection
The magnetofection technique is based on coupling therapeutic
gene to magnetic nanoparticles, which are then introduced into the cell
culture (Jones et al., 2013). The field gradient is produced by adding
rare, earth electromagnets under the cell culture, which then result in
increasing transfection speed that arises from increasing the complex
sedimentation. The therapeutic gene-magnetic particle complex is ad-
ministered intravenously when it used in vivo. With the help of enzy-
matic cleavage of cross linking molecules, charge interaction, or charge
degradation, the genetic material is released (Plank et al., 2003). This
method is considered to be an alternative for certain primary cells, as
those transfections are difficult when using other techniques.
3. Chemical methods of non-viral systems
Chemical methods of transfection are divided into two categories:
inorganic particles (such as calcium phosphate, silica, and gold parti-
cles); and organic synthetic/natural materials (such as cationic lipid
and cationic polymers). Lipoplexes (cationic lipid and DNA) and poly-
plexes (cationic polymer and DNA) are the most commonly used non-
viral delivery vectors.
4. Gelatin
Gelatin, as a natural polymer, is one of the most effective non-viral
vectors that has been used in the last two decades (Sabet et al., 2017).
Gelatin is extracted from animal collagen either through partial acid
(Type A) or alkaline (Type B) hydrolysis. Cationic gelatin (type A with
an isoelectric point of (IEP) of 7–9), is derived from partial acid hy-
drolysis of pig skin type 1 collagen, and anionic gelatin (type B, IEP
4.8–5) is derived from alkaline bovine collagen (Fig. 1) (Patel et al.,
2008). Alkaline treatment causes respective hydrolysis of asparagine
and glutamine to aspartate and glutamate. Thus, the greater proportion
of carboxylic groups possessed by type B gelatin increases its negative
charge and lowers its IEP (Ninan et al., 2011).
Gelatin is distinguished from other polymers by having amino acid
sequences such as Arg-Gly-Asp (RGD) in its structure. These amino acid
sequences modulate cell adhesion; consequently, they play a significant
role in the final biological performance of gelatin in comparison to
synthetic polymers that lack these cell-recognition sites (Wang et al.,
2012). Gelatin is considered to be GRAS (generally regarded as safe)
according to the United States Food and Drug Administration (FDA)
(Kumar, 2005), and therefore it has been used in various pharmaceu-
tical, cosmetic, and food products for decades (Elzoghby et al., 2012;
Kommareddy et al., 2005a; Lemieux et al., 2000). Gelatin is an am-
phiphilic polymer having both cationic and anionic charges along with
hydrophobic groups present in the approximate ratio of 1:1:1 (Fig. 2).
Gelatin consists of eighteen non-uniformly distributed amino acids with
both positive and negative charges (Samal et al., 2012). This compo-
sition gives rise to the special nature of this polypeptide. Lysine and
arginine represent 13% of gelatin, and both possess a positive charge;
12% of the polymer is comprised of negatively charged glutamic and
aspartic acid groups. The hydrophobic group consists of leucine, iso-
leucine, methionine, and valine, representing 11% of the gelatin
structure. The rest of the chain includes glycine, proline, and hydro-
xyproline. During either acid or alkaline collagen hydrolysis, the posi-
tions where the bonds break determine the molecular weight, the
number of polypeptide chains and the number of each kind of amino
acid residue of the formed gelatin. However, there is no specific bond
proven to be more labile to break during collagen partial hydrolysis.
Multiple bond positions are vulnerable to breakage on a probability
basis, depending on pH and temperature rendering random bond hy-
drolysis which is the main reason of gelatin molecular heterogeneity.
Moreover, the collagen source itself contributes to this variation, since
different animal species, tissue, age, and sex are all factors implicated in
O. Madkhali et al.
Fig. 1. Extraction of gelatin from collagen. The production of Type A (cationic) gelatin is shown on the right of the figure; the production of Type B (anionic) gelatin
is shown on the left of the figure. Reproduced with permission from (Hosseinkhani et al., 2015).
variable collagen amino acid composition. Accordingly, it is a challenge
to control the chemical composition and the overall properties of ge-
latin materials. One of the most significant properties of gelatin is its
versatile structure due to its protein nature that can be modified easily
by modifying its functional groups with different cross-linkers and
targeting-ligands. This property could be very beneficial to improving
and developing potential gene delivery systems, with minimal toxic
effects on host cells (Busch et al., 2003; Wang et al., 2012). Forming
gelatin nanoparticles for the delivery of nucleic acid to cells and the
nucleus is important for several reasons. Firstly, nanoparticles are taken
up more easily and efficiently by cells than large particles (Panyam and
Labhasetwar, 2003). Secondly, nanoparticles have an ability to escape
rapidly from the endosome; consequently, they are protected against
degradation (Labhasetwar, 2005). Additionally, the nano-size range of
these delivery systems allows them to be injected directly into the
systemic circulation without the risk of blocking blood vessels. More-
over, nanoparticles have been proved to improve the transfection
A*
Fig. 2. General chemical structure of gelatin (A), and features of gelatin (B).*Reproduced with permission from (Sahoo et al., 2015).
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International Journal of Pharmaceutics 554 (2019) 224–234
efficiency of plasmid DNA into the nucleus (Prabha et al., 2016). Par-
ticle size of gene therapy vectors is also an important factor that im-
pacts the access and passage of the vector to the targeting site (Jiang
et al., 2007), and it also impacts the stability of colloidal particles in
solution. For efficient endocytosis and gene delivery, the particle size of
the complex should be below 200 nm and compact (Liu et al., 2007).
The particle size depends on many factors, including nucleic acid
concentration, and the order of addition of vector components during
preparation. The particle sizes of polyplexes are closely related to the
overall surface charge of the particles. This review paper will focus on
several modifications that can be applied to gelatin nanoparticulate,
and their effects on gene delivery.
4.1. Cationic gelatin nanoparticles
Gelatin is a polyelectrolyte with a low-charge density that is ap-
preciably changed depending on the solution’s pH. As a result, the
B
Biodegradable
Low cost of production
Physiological tolerance
Easily modified
Biocompatible
Low antigenicity
O. Madkhali et al.
Fig. 3. Chemical structures of molecules used to cationize gelatin.
cationization of gelatin is a significant factor in enhancing the ability of
the polymer to interact with negatively charged cellular membranes or
DNA, thereby obtaining an effective gene delivery vector (Zwiorek
et al., 2004). Cationized gelatin is mainly prepared by introducing
amine residues to the carboxyl groups of gelatin using poly-
ethyleneimine (Mimi et al., 2012), cholamine (Geh et al., 2016;
Zwiorek et al., 2008), ethylenediamine (EDA) (Ishikawa et al., 2012; Xu
et al., 2014; Xu et al., 2008), putrescine, spermidine (Kushibiki et al.,
2006b,c) or spermine (Konat Zorzi et al., 2011) (Fig. 3). Ethylenedia-
mine cationized gelatin can condense plasmid DNA, expressing insulin-
like growth factor (IGF)-1. It has shown a five-fold increase of IGF in
adult articular chondrocytes compared with non-cationized gelatin. In
addition, chondrocytes treated with pIGF using cationized gelatin were
able to maintain stable IGF-1 overexpression when later grown in a
collagen (type II)-glycosaminoglycan (CG) scaffold for up two weeks
and exhibited enhanced biosynthesis (Xu et al., 2008).
Positive charges possessed by gelatin nanoparticles do not only in-
teract electrostatically with the anionic terminal of phospholipid, pro-
teins and glycans on the plasma membrane, but also promote nano-
particle association with the cells inducing cell uptake by cellular
endocytic mechanisms (Bannunah et al., 2014). Among multiple en-
docytosis pathways, clathrin-mediated (CME) (Fröhlich, 2012; Yameen
et al., 2014) and caveolae mediated (Morille et al., 2008) endocytosis
are the major route for cellular uptake for positively charged poly-
plexes. Moreover, recent studies postulate that cellular uptake of posi-
tively charged NPs uptake is related to energy dependent processes like
proteins dynamin and F-actin (Dausend et al., 2008). However, highly
positively-charged NPs could cause perforations in the cellular lipid
bilayer to enter the cells by-passing endocytic pathways (Sadat et al.,
2016). Throughout gelatin cationization, a considerable challenge
aroused to balance between the high positive zeta potential required for
both cell penetration and nanoparticles stability, and the low zeta po-
tential required to avoid opsonization and cytotoxicity where cationic
NPs might interact with proteoglycans on the cell surface, triggering
cellular necrosis and apoptosis (Vega-Villa et al., 2008). For instance,
Chou et al. (Chou et al., 2018) revealed that gelatin nanoparticles with
surface modification of 1.8-kD PEI showed the optimum protein de-
livery to cancer cells. However, 10-kD PEI grafted gelatin was more
cytotoxic while PEI with molecular weight less than 1.8-kD unveiled
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International Journal of Pharmaceutics 554 (2019) 224–234
lower colloidal stability and electric binding efficiency (Chou et al.,
2018; Kuo et al., 2011). PEI is a highly branched polymer with about
25% primary amine groups, 50% secondary amine groups, and 25%
tertiary amine groups. It plays a crucial role in endosomal escape via
‘‘proton-sponge mechanism’’ when grafted on gelatin. The proton-
sponge hypothesis presumes the ability of PEI (due to its secondary
amine groups) to buffer the pH, causing the ATPase enzyme in the
endosomal membrane to influx more protons to reach the desired pH,
resulting in subsequent influx of counter chloride ions which causes
osmotic swelling and rupture of the endosomal membrane (Mimi et al.,
2012; Morille et al., 2008). In an interesting study conducted by Zorzi
et al (Zorzi et al., 2015), cationized gelatin showed lesser cytotoxicity in
comparison with cationized atelocollagen and albumin. Moreover,
among different amines used for cationization, gelatin cationized with
spermine proved to be the optimum nanoparticulate system for pro-
tecting both pDNA and siRNA under the conditions endeavored. Simi-
larly, Kushibiki et al. (2006a) investigate the in vitro transfection effi-
ciency of plasmid DNA for mouse fibroblasts using cationized gelatin
prepared from different types of amine compounds namely; ethylene-
diamine, putrescine, spermidine and spermine. The highest level of
gene expression was observed with spermine grafted gelatin owing to
two main reasons; a) the intrinsic ability of this natural polyamine to
condense and pack DNA into small particles and b) it possesses the
highest buffering effect among all other cationized gelatins used
(Kushibiki et al., 2006c). Hence, it is currently used in commercial
transfection agents like Lipofectamine®, which contains a lipid modified
with spermine (Hawley-Nelson et al., 2008). These results indicate that
cationic gelatin transfection efficiency and cytotoxicity depends on the
type of gelatin modification and the molecular weight of the grafted
molecule.
Various methods have been used to prepare cationic gelatin nano-
particles. Those most commonly used are discussed in the next section.
5. Methods of preparations of cationic gelatin
5.1. Preparation of cationic gelatin using amination method
Cationic gelatin is prepared by introducing a cationic agent directly
to the carboxylic group of gelatin (Fig. 4). Briefly, gelatin Type A (IEP
O. Madkhali et al.
Fig. 4. Schematic diagram of the preparation of cationic gelatin using EDA or spermine and EDC. Adapted with permission from (Samal et al., 2012).
7–9) is completely dissolved in 0.1 M phosphate buffer saline (PBS)
containing potassium dihydrogen phosphate and disodium hydrogen
phosphate. The cationic agent is added, and the pH is subsequently
adjusted to 5. To this solution, 1-ethyl-3- (3-dimethylaminopropyl)
carbodiimide (EDC) is added, and the final volume is made up with
PBS. The solution is then agitated at 37 °C for 18 h and subsequently
dialyzed for two days with 16 changes of water. Following the dialysis
process, the solution is freeze-dried for 4 to 7 days to afford cationized
gelatin (Fukuyama et al., 2006). In this method, no nanoparticles are
formed. There are several methods for preparing cationic gelatin na-
noparticles, which will be described below.
5.2. Preparation of cationic gelatin nanoparticles using two-step desolvation
method
Two-step desolvation approach was adopted to limit the hetero-
geneity of gelatin structure and produce gelatin nanoparticles with a
reduced tendency for aggregation. Gelatin nanoparticles were prepared
using a two-step desolvation method previously described by Coaster
et al. (Coester et al., 2000). In this method, after the first desolvation
step, the low molecular gelatin fractions presented in the supernatant
were removed by decanting, and the high molecular fractions presented
in the sediment were re-desolved (Saxena et al., 2005). In brief, gelatin
type A is dissolved in distilled water under constant heating (45–50 °C).
Desolvating agents such as acetone or ethanol are added as to pre-
cipitate the high molecular weight (HMW) of gelatin. After the super-
natant is discarded, the HMW gelatin is re-dissolved in distilled water
and stir at 600 RPM under a constant heating process. Subsequently, the
pH is adjusted to 2.5 using HCl. Following this, acetone or ethanol is
added drop-wise to form nanoparticles. Glutaraldehyde (GA) 25% (v/v)
is added as a cross-linking agent, which is important to provide
homogenous, stable, and spherical shape nanoparticles, and to enhance
circulation times in vivo (compared to uncrossed-linked nanoparticles
(Elzoghby et al., 2012; Kommareddy et al., 2005a)), and stirred at
600 rpm at room temperature overnight. The following day, the nano-
particles are purified using centrifugation or dialysis membrane prior to
freeze drying. Following the freeze drying process, the gelatin is ca-
tionized by introducing amino residues to the carboxyl group of gelatin
nanoparticles in a manner analogous to the cationization of molecular
gelatin. The gelatin nanoparticles are dispersed in highly purified
water, and then the pH is adjusted between pH 4.5 and pH 5. To this
dispersion 50 mg of amine residue (i.e. spermine, cholamine, etc.) is
added and incubated for five minutes, the same amount of EDC is then
added to the solution and the reaction is left for 1 h in the dark. The
resulting cationized gelatin nanoparticles are purified using cen-
trifugation or dialysis membrane for two days prior to lyophilization.
5.3. Preparation of cationic gelatin using one-step desolvation method
The one-step desolvation method is similar to the two-step method
described above. However, the first step which precipitates the high
molecular weight is omitted (Fig. 5). This method has proved to be
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International Journal of Pharmaceutics 554 (2019) 224–234
robust in the preparation of gelatin nanoparticles (Geh et al., 2016).
5.4. Preparation of cationic gelatin nanoparticles using ionic gelation
technique
Zorzi et al. (Parraga et al., 2009; Zorzi et al., 2011) prepared ca-
tionic gelatin nanoparticles using the ionic gelation technique for an
ocular surface (Fig. 6). In this technique, Zorzi and colleagues used
chondroitin sulfate (CS) and dextran sulfate (DS) to make cationic ge-
latin. Briefly, gelatin was first cationized using spermine hydrochloride
(SPM). The cationized gelatin was then added to triphosphate (TPP)
containing the plasmid and CS or DS. The advantage of this method
includes the fact that no organic solvents such as acetone and methanol
are used, in comparison with one-step or two-step desolvation (Zorzi
et al., 2015). In addition, glutaraldehyde, which is reported to be toxic,
is not added to this method (Gough et al., 2002; Maitra et al., 2006).
Table 1 summarized different methods of preparing cationic gelatin
using different cationic agents and the effect of these methods on the
transfection efficiency and cell viability.
6. PEGylated gelatin nanoparticles
GNPs are mainly engulfed by the cells of the reticuloendothelial
system (RES) upon systemic administration. This leads to weak trans-
fection and gene expression. However, coating the GNPs with poly
(ethylene glycol) (PEG) generates a dense hydrophilic shell of long
chains that conserve the core of GNPs from non-specific hydrophobic
interaction with serum protein. Consequently, this significantly reduces
the effect of RES (Otsuka et al., 2003). Another advantage of PEGyla-
tion is that it may increase the hydrodynamic size of the particles (more
than 30 nm) which leads to a decrease in their clearance from the
kidney, in that renal filtration is dependent on the molecular mass and
volume. These advantages result in an increase in the circulation half-
life of the particles in vivo (Crawford, 2002; Kommareddy et al., 2005b).
PEG capacity for repelling proteins and avoiding macrophages depends
on different considerations such as PEG MW, density, the conformation
and chain flexibility (Vonarbourg et al., 2006). According to multiple
studies, the optimum MW required for decreasing protein adsorption in
vitro is 1.5–3.5 KDa. However, regarding the macrophage uptake, very
long chains of approximately 20 KDa are essential (Mosqueira et al.,
1999). In addition, the existence of PEGylation on the surface of the
GNPs is beneficial in terms of the protection of the particles from di-
gestion by proteinases (Xu et al., 2012).
In vitro studies showed that PEGylated gelatin NPs were internalized
by nonspecific endocytosis pathway where their cargo was delivered to
the peri-nuclear region within 12 h (Kaul and Amiji, 2004). Although
studies claimed that shielding of gelatin cationic groups via PEGylation
decreases both cytotoxicity and efficacy in NPs, yet Hoskins et al.
proved that charge shielding via PEGylation had a small impact on
cellular uptake while greatly decreasing cytotoxicity, whereas obvious
reduction in membrane damage, lipid peroxidation, and oxidative stress
were reported in neuroblastoma cells (Hoskins et al., 2012).
O. Madkhali et al. International Journal of Pharmaceutics 554 (2019) 224–234

Fig. 5. Schematic illustration of the preparation of cationic gelatin nanoparticles using the one-step desolvation method.

Fig. 6. Schematic illustration of the preparation of cationic gelatin nanoparticles using the ionic gelatin technique.

Adding PEGylation to non-condensing type B GNPs has resulted in
an excellent system for effective distribution in solid tumors because of
the presence of hyperpermeable angiogenic blood vessels in such tu-
mors and the enhanced permeability and retention (EPR) effect (Kaul
and Amiji, 2004, 2005). According to Amiji and Kaul (Kaul and Amiji,
2004), PEGylated GNPs favorably targeted tumor mass in Lewis lung
carcinoma (LLC) bearing female mice, and about 4–5% of the injected
dose remained in the tumor for approximately twelve hours after ad-
ministration. Amiji and Kaul also stated that reporter pDNA encoding
for β-galactosidase (pCMV-β) was effectively encapsulated in PEGy-
lated GNPs and showed significant expression in the tumor of LLC with
61% transfection efficiency (Kaul and Amiji, 2005).
Kushibiki et al. (2004) studied the long-circulation property of PE-
Gylated gelatin using 125I-labeled gelatin. They compared unmodified
GNPs and PEGylated 125I-labeled GNPs after I.V. administration
through the tail vein in LLC-bearing mice. They found that PEGylated
GNPs have longer circulating properties in the blood and remained in
the tumor for up to 24 h after administration. In another study thiolated
PEGylated GNPs exhibited prolonged circulation and enhanced tumor
extravasation in vivo in an orthotopic human breast adenocarcinoma
xenograft model (Kommareddy and Amiji, 2007a). In comparison with
the non-PEGylated GNPs, the PEGylated nanoparticles showed longer
circulation with plasma and tumor half-lives of 15.3 and 37.8 h re-
spectively. Generally, the advantages of the existence of PEG in com-
bination with GNPs are summarized as follows: increase in the circu-
lation time in the plasma and tumor mass, stabilization of therapeutic
cargo during transportation, prevention of RES elimination, and the
provision of potential for the conjugation of targeting moieties (Kaul

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O. Madkhali et al. International Journal of Pharmaceutics 554 (2019) 224–234

Table 1
Preparation of cationic gelatin for gene delivery using different cationic agents.
Method Gelatin Type Cationic agent NP* Gene used In vitro/ Cell type Outcomes Ref.
vivo

Two-step Type A Cholamine Yes pCMV-luc In vitro B16F10 Moderate transfection with high cell Zwiorek et al. (2004)
Type A Polyethyleneimine siRNA _ _ viability Zillies and Coester (2005)
pCMV-luc NIH 3 T3 High transfection with high cell Kuo et al. (2011)
In vitro viability
One step Type A and Cholamine Yes _ _ _ _ Geh et al. (2016), Zwiorek
Type B et al. (2008)
Ionic gelation Type A Spermine Yes pMUC5AC In vitro HCE Spermine showed high transfection Konat Zorzi et al. (2011)
Type B Spermine & pDNA & siRNA In vitro IOBA-NHC and gene silencing with adequate cell Zorzi et al. (2015)
Ethylenediamine HCE viability
IOBA-NHC
Aminization Type A Etylenediamine No pCMV-luc In vitro RGM-1 Spermine showed the highest Hosseinkhani et al. (2002a),
Spermine pGL3-Luc In vitro L929 transfection with low cell viability Hosseinkhani et al. (2002b)
Spermidine pGL3-Luc In vivo ddY mice† Spermine showed the highest Kushibiki et al. (2006b, c)
Putrescine transfection with low cell viability Kushibiki and Tabata (2005)

* Nanoparticles formed.
†
6–7 weeks old mice.

and Amiji, 2004, 2005).
6.1. Methods of preparations of PEGylated gelatin
Preparation of PEGylated gelatin is based on using amine reactive
PEG derivatives to form covalent bond with the free amino groups on
gelatin (Fig. 8). Kyung and Youngro were able to synthesize carboxy-
lated derivative of PEG which was further activated using dicyclohex-
ylcarbodiimide (DCC) to couple the PEG to the amino group of gelatin
with a PEGyltion degree (mol/mol%) of 48% (Kim and Byun, 1999).
According to another method described by Kaul and Amiji (Kaul and
Amiji, 2002), PEG-epoxide is used as activated derivative to graft PEG
as illustrated in Fig. 7 where higher PEGyltion degree (mol/mol%) were
attained. Briefly, PEG-epoxide is dissolved in alkaline borate buffer (pH
8.5), gelatin type B is then added, and the reaction is kept under stirring
for 14 h at 40 °C. The product obtained is precipitated with excess
acetone to remove any unreacted PEG-epoxide, dialyzed against dis-
tilled water, and then lyophilized.
An alternative method relies on reacting gelatin with amine reactive
molecules like methoxy PEG-succinimidyl glutarate (Kommareddy and
Amiji, 2007b) and methoxy PEG-succinimidyl succinate (Kushibiki
et al., 2004; Kushibiki and Tabata, 2005) with a degree of PEGylation of
90% and 30–100% respectively. Briefly, gelatin (1.0 µmol) is dissolved
Fig. 7. Schematic illustration of the reaction between PEG-epoxide and gelatin
nanoparticles. Reproduced from (Elzoghby, 2013).
in anhydrous DMSO at room temperature. Methyl ether NHS-PEG with
(4.0 × 10−5 mol) previously dissolved DMSO is slowly added to the
gelatin solution and left stirring at room temperature for 3 h, dialyzed
against deionized water, and then lyophilized.
7. EGFR-targeted gelatin nanoparticles
One of the greatest challenges for gene delivery is the area of tar-
geting. A delivery vector is required to distinguish host cells, evade
nonspecific binding, and resist degradation in the systemic circulation.
After reaching the target cells, the delivery vector should cross the cell
membrane, facilitate the escape of the vector from the endosome, re-
lease nucleic acid from the complex, which can then enter the nucleus
to express the required protein (Xu et al., 2013). Although tumor tar-
geting using PEG surface modified nanoparticles accomplishes some
preferential accumulation in tumor cells and allows for intracellular
delivery, some types of cancer do not have adequate vasculature, or the
nanoparticles may not be able to penetrate deeply into the tumor mass
(Xu et al., 2012). The mutation of the epidermal the growth factor re-
ceptor (EGFR) has been shown to be associated with poor prognosis in
several types of cancers including ovarian cancer (Ciardiello and
Tortora, 2001). Between 33% and 75% of EGFR has reported to be
overexpressed in ovarian cancer, and has been found in both the growth
and the progression of the disease (Sewell et al., 2002).
EGFR is a member of the ErbB/her family of ligand activated re-
ceptor tyrosine kinases (RTKs). It has been recognized as a molecular
target. EGFR consists of an extracellular ligand-binding domain like any
other receptor of tyrosine kinases which are involved in interactions
between receptors within a membrane, and a cytoplasmic domain with
tyrosine kinase activity (Schlessinger, 2002). Accordingly, receptor
mediated endocytosis is the main cellular uptake mechanism for gelatin
NPs decorated with EGFR (Mickler et al., 2012).
To conjugate EGFR targeting peptides to gelatin, either the gelatin
amino groups are converted to thiol groups via reacting with 2-imi-
nothiolane hydrochloride at room temperature for 15 h, (Kommareddy
and Amiji, 2005; Tseng et al., 2008) then left over night to react with
Sulfo-MBS activated EGFR at 4 °C (Tseng et al., 2008) or gelatin is firstly
modified with PEG maleimide which is then conjugated to the peptide
through a spacer containing a sulfhydryl group in its terminal cysteine
residue (Fig. 8) (Magadala and Amiji, 2008).
Consequently, the conjugation of gelatin with targeting EGFR pep-
tide has been shown to improve the transfection efficiency in several
types of cancer cells. EGFR-targeted GNPs carrying plasmid DNA en-
coding for EGFp-N1 obtained the highest transfection efficiency in
Panc-1 pancreatic adenocarcinoma cells in comparison with other

230
O. Madkhali et al.
Fig. 8. Schematic illustration of grafting different ligands to gelatin nanoparticles. Gelatin nanoparticles can be chemically modified by adding -thiol groups, cationic
agents, alendronate, PEG, and conjugating targeting EGFR peptide to PEGylated gelatin nanoparticles.
controls, particularly 48 h after transfection (Magadala and Amiji,
2008). The intravenous injection of EGFR-targeted GNPs to mice
bearing Panc-1 pancreatic adenocarcinoma showed almost a double
efficiency with regard to targeting, in comparison with PEG-GNPs and
unmodified GNPs. Additionally, it accumulated and was sustained for a
longer period in the tumor mass (Xu et al., 2013). Another study by Xu
and Amiji (Xu and Amiji, 2012) used EGFR-targeted thiolated gelatin
nanoparticles to deliver plasmid DNA into Panc-1 pancreatic adeno-
carcinoma cells. The EGFR improved the targeting, and the thiol group
improved the stability of GNPs. The results showed that EGFR-targeted
thiolated GNPs had a small nanoparticle size (150–200 nm) with high
GFP expression, even higher than the positive control lipofectin-com-
plexed DNA, and they obtained high cell viability as well (Xu and Amiji,
2012). Using targeting-ligands with GNPs facilitates the delivery system
‘s recognition of the host cell; as a result, transfection is improved, and
cytotoxicity is reduced, thereby achieving the optimal goal for gene
therapy delivery systems.
8. Thiolated gelatin nanoparticles
The thiol group (-SH) has been considered as a potential addition to
GNPs due to its ability to respond to environmental changes, either
inside or outside the cell. A thiol group is similar to alcohol in its
chemical structure but differs in terms of its chemical properties; thiols
are more nucleophilic, more acidic, and more readily oxidized than
alcohol (Senning, 1997). Adding thiol groups to gelatin leads to the
formation of disulfide bonds (S-S) in an oxidation reaction within the
polymer. This can be beneficial in strengthening the tertiary and
231
International Journal of Pharmaceutics 554 (2019) 224–234
quaternary protein structure of gelatin (Bacalocostantis et al., 2013). In
addition, disulfide bonds can stabilize the nanoparticles during sys-
temic circulation and release the encapsulated payload when they are
broken inside the cell (Kommareddy and Amiji, 2005). Groups of thiols
are easily and rapidly crosslinked; thus, they can be used for the
synthesis of polymeric delivery vectors (Bacalocostantis et al., 2013).
Glutathione (GSH) is a dipeptide, used as an antioxidant to prevent
damage caused by an oxygen species. GSH and peroxide exist in high
concentration inside the cells to a greater extent than they do outside
(100-fold higher), and their concentration is much higher in the cyto-
plasm of tumor cells. As a result, Kommareddy and Amiji (Kommareddy
and Amiji, 2005) introduced a thiol (SH) group into gelatin using a 2-
iminothilane reagent and prepared the nanoparticles by desolvation
using ethanol under adjusted and controlled pH and temperature con-
ditions. The plasmid DNA was then incorporated into the thiolated
gelatin nanoparticles. The thiolated GNP encapsulated DNA showed
high transfection efficiency in NIH-3T3 murine fibroblast cells in con-
trast to unmodified gelatin and lipofectin®-complexed DNA
(Kommareddy and Amiji, 2005). Six hours after transfection, the ex-
pression of the green fluorescent protein was observed. These results
can be interpreted as the disulfide bonds increasing the stability of the
nanoparticles and indicated that thiolated GNPs have the rapid release
of their contents into a highly reducing environment inside the cell
where the high concentration of GSH (Kommareddy and Amiji, 2005,
2007b). Furthermore, the same group evaluated three modifications of
gelatin: PEG-GNPs, thiolated-GNPs, and PEG-modified thiolated GNPs
(Fig. 8) in NIH-3 T3 to deliver plasmid DNA. Of the three formulations
tested, PEG-thiolated GNPs indicated the highest GFP expression to
O. Madkhali et al.
even a greater degree than the positive control lipofectin-complexed
DNA (Kommareddy and Amiji, 2007b). Generally, both PEG-GNPs and
PEG-modified thiolated GNPs demonstrated longer circulation in the
blood and higher accumulation in the tumor cells, in contrast with
unmodified GNPs (Xu et al., 2012).
A new tumor-targeted siRNA delivery system using polymerized
siRNA (poly-siRNA) and thiol-modified gelatin nanoparticles was de-
veloped by Lee et al. (Lee et al., 2013). The poly-siRNA was prepared by
self-polymerization of the thiol group and was encapsulated in the self-
assembled thiolated-GNPs using chemical cross-linking. The results
showed that the siRNA was protected from enzymatic degradation; the
siRNA molecules were released effectively in a reductive condition.
Also, poly-siRNA-thiolated –GNPs demonstrated an excellent accumu-
lation in tumor cells, induced effective target gene-silencing in tumors
after intravenous injection, and demonstrated high cell viability, closer
to 100% compared with lipofectamine and non-thiolated siRNA-GNPs
(Lee et al., 2013). It is apparent that the disulfide bonds formed by the
(thiol) group could play a significant role in the stability of nano-
particles, thereby resulting in effective gene expression and high cell
viability.
9. Alendronate gelatin nanoparticles
Bisphosphonates, like alendronate, are characterized by their high
affinity to hydroxyapatite. Moreover, they maintain their binding cap-
ability even after conjugation to macromolecules (Low and Kopecek,
2012). For targeted gene delivery, Alendronate (ALN) modified gelatin
biopolymers, which could be promising for bone targeting, were syn-
thesized and characterized. An innovative four-component system
composed of alendronate sodium trihydrate (ALN), gelatin, gemini
16–3-16 surfactant and DNA was prepared. For ALN conjugation, ge-
latin is dissolved in a suitable volume of phosphate buffer (pH 4.5 and
heated to 50 °C) and then ALN is added at the required molar ratios.
EDC is added as a last step to the gelatin-ALN solution to limit self-
cross-linking of gelatin molecules (Fig. 8). ALN-gelatin biopolymers
prepared at various alendronate/gelatin ratios were utilized to prepare
nanoparticles and were optimized in combination with DNA and gemini
surfactant for transfecting both HEK-293 and MG-63 cell lines (Mekhail
et al., 2016). The study revealed that not only did the degree of ALN
substitution on gelatin significantly affected the transfection efficiency
of the gelatin based nanocomplexes but also the type of grafted gelatin.
In particular, nanocomplexes formulated using the Type A functiona-
lized gelatin prepared at a 1:4 gelatin: ALN ratio had a higher zeta
potential than those obtained from Type B functionalized gelatin and
thus a higher bone selective transfection efficiency (comparing MG-63
to HEK-293), which was approximately three times that of Lipofecta-
mine 2000® and 1.5 times the transfection efficiency of the gemini/
DNA complexes (Mekhail et al., 2016). The presence of ALN as a tar-
geting moiety facilitated receptor mediated endocytosis for cellular
uptake. Those results revealed that ALN-modified gelatin established a
platform for both drug delivery and a promising vector for the for-
mulation of targeted cationic nanocomplexes for enhanced gene de-
livery to bone tissues.
10. Conclusion
There are numerous advantages of using gelatin as polymer for
fabrication of nanoparticles. Gelatin possesses low cellular toxicity be-
cause it is derived from animal protein and also producing very low-
level allergic reaction. Gelatin can be easily polymerized to form na-
noparticles which are relatively stable and reproducible. Gelatin also
facilitates for future up scaling. Due to low cost of gelatin and its bio-
degradable nature gelatin gain popularity among researcher specially
for commercial scaling. The major issues related with nanomaterials are
about their termination from the body after targeting to the specific
organ, tissue or cell. Another advantage of gelatin is amino acid side-
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International Journal of Pharmaceutics 554 (2019) 224–234
chains of the gelatin matrix molecule which facilitate further mod-
ifications. This may be useful for coupling of ligands for improving the
target diseased tissues and to enhance particular cellular uptake or af-
fect intracellular distribution. Gelatin nanoparticles are promising non-
viral vectors for gene delivery applications. The modifications im-
proved the delivery system and led to more effective transfection effi-
ciency and cell viability. Depending on the final outcome/objective of
the gelatin nanoparticle, various manufacturing methods may be uti-
lized such as cationic, PEGylated, thiolated, EGFR-targeted, and alen-
dronate gelatin nanoparticles. One of the biggest challenges for using
gelatin as a gene delivery system is scaling up, particularly for gelatin
nanoparticles. With scaling up, gelatin loses its properties such as bio-
compatibility and biodegradability and results in more aggregates and
less homogenous and stable nanoparticles, which is more noticeable
with modified gelatin. This problem can be overcome by using cross-
linkers such as glutaraldehyde, which should be removed after pre-
paring nanoparticles due to the risk of toxicity.
Acknowledgements
Funding from the School of Pharmacy, University of Waterloo,
Canada and the Faculty of Pharmacy, Jazan University, Kingdom of
Saudi Arabia is gratefully acknowledged. SDW is funded by the Natural
Sciences and Engineering Research Council of Canada (RGPIN-2016-
04009).
References
Bacalocostantis, I., Mane, V.P., Goodley, A.S., Bentley, W.E., Muro, S., Kofinas, P., 2013.
Investigating polymer thiolation in gene delivery. J. Biomater. Sci. Polym. Ed. 24,
912–926.
Bannunah, A.M., Vllasaliu, D., Lord, J., Stolnik, S., 2014. Mechanisms of nanoparticle
internalization and transport across an intestinal epithelial cell model: effect of size
and surface charge. Mol. Pharm. 11, 4363–4373.
Busch, S., Schwarz, U., Kniep, R., 2003. Chemical and structural investigations of bio-
mimetically grown fluorapatite–gelatin composite aggregates. Adv. Funct. Mater. 13,
189–198.
Cardoso, A.M., Morais, C.M., Silva, S.G., Marques, E.F., de Lima, M.C.P., Jurado, M.A.S.,
2014. Bis-quaternary gemini surfactants as components of nonviral gene delivery
systems: a comprehensive study from physicochemical properties to membrane in-
teractions. Int. J. Pharm. 474, 57–69.
Chou, M.-J., Yu, H.-Y., Hsia, J.-C., Chen, Y.-H., Hung, T.-T., Chao, H.-M., Chern, E.,
Huang, Y.-Y., 2018. Highly efficient intracellular protein delivery by cationic poly-
ethyleneimine-modified gelatin nanoparticles. Materials 11, 301.
Ciardiello, F., Tortora, G., 2001. A novel approach in the treatment of cancer: targeting
the epidermal growth factor receptor. Clin. Cancer Res. 7, 2958–2970.
Coester, C., Langer, K., Von Briesen, H., Kreuter, J., 2000. Gelatin nanoparticles by two
step desolvation a new preparation method, surface modifications and cell uptake. J.
Microencapsul. 17, 187–193.
Crawford, J., 2002. Clinical uses of PEGylated pharmaceuticals in oncology. Cancer Treat.
Rev. 28, 7–11.
Dausend, J., Musyanovych, A., Dass, M., Walther, P., Schrezenmeier, H., Landfester, K.,
Mailänder, V., 2008. Uptake mechanism of oppositely charged fluorescent nano-
particles in HeLa cells. Macromol. Biosci. 8, 1135–1143.
Elzoghby, A.O., 2013. Gelatin-based nanoparticles as drug and gene delivery systems:
reviewing three decades of research. J. Control. Release 172, 1075–1091.
Elzoghby, A.O., Samy, W.M., Elgindy, N.A., 2012. Protein-based nanocarriers as pro-
mising drug and gene delivery systems. J. Controlled Release : Off. J. Controlled
Release Soc. 161, 38–49.
Fröhlich, E., 2012. The role of surface charge in cellular uptake and cytotoxicity of
medical nanoparticles. Int. J. Nanomed. 7, 5577.
Fukuyama, N., Onuma, T., Jujo, S., Tamai, Y., Suzuki, T., Myojin, K., Tabata, Y., Ishihara,
Y., Takano, J., Mori, H., 2006. Efficient preparation of cationized gelatin for gene
transduction. Tokai J. Exp. Clin. Med. 31, 49–52.
Geh, K.J., Hubert, M., Winter, G., 2016. Optimisation of one-step desolvation and scale-up
of gelatine nanoparticle production. J. Microencapsul. 1–10.
Gough, J.E., Scotchford, C.A., Downes, S., 2002. Cytotoxicity of glutaraldehyde cross-
linked collagen/poly (vinyl alcohol) films is by the mechanism of apoptosis. J.
Biomed. Mater. Res. Part A 61, 121–130.
Griffith, F., 1928. The significance of pneumococcal types. Epidemiol. Infect. 27,
113–159.
Hawley‐Nelson, P., Ciccarone, V., Moore, M.L., 2008. Transfection of cultured eukaryotic
cells using cationic lipid reagents. Curr. Protoc. Mol. Biol. 81 9.4. 1-9.4. 17.
Hoskins, C., Wang, L., Cheng, W.P., Cuschieri, A., 2012. Dilemmas in the reliable esti-
mation of the in-vitro cell viability in magnetic nanoparticle engineering: which tests
and what protocols? Nanoscale Res. Lett. 7, 77.
Hosseinkhani, H., Abedini, F., Ou, K.L., Domb, A.J., 2015. Polymers in gene therapy
O. Madkhali et al.
technology. Polym. Adv. Technol. 26, 198–211.
Hosseinkhani, H., Aoyama, T., Ogawa, O., Tabata, Y., 2002a. Ultrasound enhancement of
in vitro transfection of plasmid DNA by a cationized gelatin. J. Drug Target. 10,
193–204.
Hosseinkhani, H., Aoyama, T., Yamamoto, S., Ogawa, O., Tabata, Y., 2002b. In vitro
transfection of plasmid DNA by amine derivatives of gelatin accompanied with ul-
trasound irradiation. Pharm. Res. 19, 1471–1479.
Ishikawa, H., Nakamura, Y., Jo, J.-I., Tabata, Y., 2012. Gelatin nanospheres incorporating
siRNA for controlled intracellular release. Biomaterials 33, 9097–9104.
Jackson, D.A., Juranek, S., Lipps, H.J., 2006. Designing nonviral vectors for efficient gene
transfer and long-term gene expression. Mol. Ther. 14, 613–626.
Jiang, H.-L., Kim, Y.-K., Arote, R., Nah, J.-W., Cho, M.-H., Choi, Y.-J., Akaike, T., Cho, C.-
S., 2007. Chitosan-graft-polyethylenimine as a gene carrier. J. Control. Release 117,
273–280.
Jin, L., Zeng, X., Liu, M., Deng, Y., He, N., 2014. Current progress in gene delivery
technology based on chemical methods and nano-carriers. Theranostics 4, 240.
Jones, C.H., Chen, C.-K., Ravikrishnan, A., Rane, S., Pfeifer, B.A., 2013. Overcoming
nonviral gene delivery barriers: perspective and future. Mol. Pharm. 10, 4082–4098.
Karimi, M., Solati, N., Ghasemi, A., Estiar, M.A., Hashemkhani, M., Kiani, P., Mohamed,
E., Saeidi, A., Taheri, M., Avci, P., 2015. Carbon nanotubes part II: a remarkable
carrier for drug and gene delivery. Exp. Opin. Drug Delivery 12, 1089–1105.
Kaul, G., Amiji, M., 2002. Long-circulating poly (ethylene glycol)-modified gelatin na-
noparticles for intracellular delivery. Pharm. Res. 19, 1061–1067.
Kaul, G., Amiji, M., 2004. Biodistribution and targeting potential of poly(ethylene glycol)-
modified gelatin nanoparticles in subcutaneous murine tumor model. J. Drug Target.
12, 585–591.
Kaul, G., Amiji, M., 2005. Tumor-targeted gene delivery using poly(ethylene glycol)-
modified gelatin nanoparticles: in vitro and in vivo studies. Pharm. Res. 22, 951–961.
Keeler, A.M., ElMallah, M.K., Flotte, T.R., 2017. Gene therapy 2017: progress and future
directions. Clin. Trans. Sci.
Kim, K.J., Byun, Y., 1999. Preparation and characterizations of self-assembled PEGylated
gelatin nanoparticles. Biotechnol. Bioprocess Eng. 4, 210–214.
Kommareddy, S., Amiji, M., 2005. Preparation and evaluation of thiol-modified gelatin
nanoparticles for intracellular DNA delivery in response to glutathione. Bioconjug.
Chem. 16, 1423–1432.
Kommareddy, S., Amiji, M., 2007a. Biodistribution and pharmacokinetic analysis of long-
circulating thiolated gelatin nanoparticles following systemic administration in
breast cancer-bearing mice. J. Pharm. Sci. 96, 397–407.
Kommareddy, S., Amiji, M., 2007b. Poly(ethylene glycol)-modified thiolated gelatin na-
noparticles for glutathione-responsive intracellular DNA delivery. Nanomed.
Nanotechnol. Biol. Med. 3, 32–42.
Kommareddy, S., Shenoy, D.B., Amiji, M.M., 2005a. Gelatin nanoparticles and their
biofunctionalization. Nanotechnol. Life Sci Online.
Kommareddy, S., Tiwari, S.B., Amiji, M.M., 2005b. Long-circulating polymeric nano-
vectors for tumor-selective gene delivery. Technol. Cancer Res. Treat. 4, 615–625.
Konat Zorzi, G., Contreras-Ruiz, L., Parraga, J.E., Lopez-Garcia, A., Romero Bello, R.,
Diebold, Y., Seijo, B., Sanchez, A., 2011. Expression of MUC5AC in ocular surface
epithelial cells using cationized gelatin nanoparticles. Mol. Pharm. 8, 1783–1788.
Kumar, C.S., 2005. Biofunctionalization of nanomaterials. In: Kumar, Challa S.S.R. (Ed.),
Biofunctionalization of Nanomaterials. Wiley-VCH, pp. 377.
Kuo, W.-T., Huang, H.-Y., Chou, M.-J., Wu, M.-C., Huang, Y.-Y., 2011. Surface mod-
ification of gelatin nanoparticles with polyethylenimine as gene vector. J.
Nanomater. 2011, 28.
Kushibiki, T., Matsuoka, H., Tabata, Y., 2004. Synthesis and physical characterization of
poly (ethylene glycol)-gelatin conjugates. Biomacromolecules 5, 202–208.
Kushibiki, T., Nagata-Nakajima, N., Sugai, M., Shimizu, A., Tabata, Y., 2006a. Enhanced
anti-fibrotic activity of plasmid DNA expressing small interference RNA for TGF-β
type II receptor for a mouse model of obstructive nephropathy by cationized gelatin
prepared from different amine compounds. J. Control. Release 110, 610–617.
Kushibiki, T., Tabata, Y., 2005. Preparation of poly(ethylene glycol)-introduced catio-
nized gelatin as a non-viral gene carrier. J. Biomater. Sci. Polym. Ed. 16, 1447–1461.
Kushibiki, T., Tomoshige, R., Iwanaga, K., Kakemi, M., Tabata, Y., 2006b. Controlled
release of plasmid DNA from hydrogels prepared from gelatin cationized by different
amine compounds. J. Controlled Release: Off. J. Controlled Release Soc. 112,
249–256.
Kushibiki, T., Tomoshige, R., Iwanaga, K., Kakemi, M., Tabata, Y., 2006c. In vitro
transfection of plasmid DNA by cationized gelatin prepared from different amine
compounds. J. Biomater. Sci. Polym. Ed. 17, 645–658.
Labhasetwar, V., 2005. Nanotechnology for drug and gene therapy: the importance of
understanding molecular mechanisms of delivery. Curr. Opin. Biotechnol. 16,
674–680.
Lacerda, L., Russier, J., Pastorin, G., Herrero, M.A., Venturelli, E., Dumortier, H., Al-
Jamal, K.T., Prato, M., Kostarelos, K., Bianco, A., 2012. Translocation mechanisms of
chemically functionalised carbon nanotubes across plasma membranes. Biomaterials
33, 3334–3343.
Lee, S.J., Yhee, J.Y., Kim, S.H., Kwon, I.C., Kim, K., 2013. Biocompatible gelatin nano-
particles for tumor-targeted delivery of polymerized siRNA in tumor-bearing mice. J.
Control. Release 172, 358–366.
Lemieux, P., Vinogradov, S.V., Gebhart, C.L., Guerin, N., Paradis, G., Nguyen, H.K.,
Ochietti, B., Suzdaltseva, Y.G., Bartakova, E.V., Bronich, T.K., St-Pierre, Y., Alakhov,
V.Y., Kabanov, A.V., 2000. Block and graft copolymers and NanoGel copolymer
networks for DNA delivery into cell. J. Drug Target. 8, 91–105.
Li, S.-D., Huang, L., 2007. Non-viral is superior to viral gene delivery. J. Controlled
Release: Official J. Controlled Release Soc. 123, 181.
Liu, C., Gong, C., Pan, Y., Zhang, Y., Wang, J., Huang, M., Wang, Y., Wang, K., Gou, M.,
Tu, M., 2007. Synthesis and characterization of a thermosensitive hydrogel based on
233
International Journal of Pharmaceutics 554 (2019) 224–234
biodegradable amphiphilic PCL-Pluronic (L35)-PCL block copolymers. Colloids Surf.,
A 302, 430–438.
Low, S.A., Kopecek, J., 2012. Targeting polymer therapeutics to bone. Adv. Drug Deliv.
Rev. 64, 1189–1204.
Lundstrom, K., Boulikas, T., 2003. Viral and non-viral vectors in gene therapy: technology
development and clinical trials. Technol. Cancer Res. Treat. 2, 471–485.
Magadala, P., Amiji, M., 2008. Epidermal growth factor receptor-targeted gelatin-based
engineered nanocarriers for DNA delivery and transfection in human pancreatic
cancer cells. Am. Assoc. Pharm. Sci. J. 10, 565–576.
Maitra, A., De, T.K., Mitra, S., 2006. Hydrogel nanoparticles: applications in drug de-
livery. second ed. pp. 2821–2837.
Mali, S., 2013. Delivery systems for gene therapy. Indian J. Human Genet. 19, 3.
Mekhail, G.M., Kamel, A.O., Awad, G.A., Mortada, N.D., Rodrigo, R.L., Spagnuolo, P.A.,
Wettig, S.D., 2016. Synthesis and evaluation of alendronate-modified gelatin biopo-
lymer as a novel osteotropic nanocarrier for gene therapy. Nanomed. Nanotechnol.
Biol. Med. 11, 2251–2273.
Mickler, F.M., Möckl, L., Ruthardt, N., Ogris, M., Wagner, E., Bräuchle, C., 2012. Tuning
nanoparticle uptake: live-cell imaging reveals two distinct endocytosis mechanisms
mediated by natural and artificial EGFR targeting ligand. Nano Lett. 12, 3417–3423.
Mimi, H., Ho, K.M., Siu, Y.S., Wu, A., Li, P., 2012. Polyethyleneimine-based core-shell
nanogels: a promising siRNA carrier for argininosuccinate synthetase mRNA knock-
down in HeLa cells. J. Control. Release 158, 123–130.
Morille, M., Passirani, C., Vonarbourg, A., Clavreul, A., Benoit, J.-P., 2008. Progress in
developing cationic vectors for non-viral systemic gene therapy against cancer.
Biomaterials 29, 3477–3496.
Mosqueira, V.C.F., Legrand, P., Gref, R., Heurtault, B., Appel, M., Barratt, G., 1999.
Interactions between a macrophage cell line (J774A1) and surface-modified poly (D,
L-lactide) nanocapsules bearing poly (ethylene glycol). J. Drug Target. 7, 65–78.
Nayerossadat, N., Maedeh, T., Ali, P.A., 2012. Viral and nonviral delivery systems for
gene delivery. Adv. Biomed. Res. 1.
Ninan, G., Jose, J., Abubacker, Z., 2011. Preparation and characterization of gelatin ex-
tracted from the skins of rohu (Labeo rohita) and common carp (Cyprinus carpio). J.
Food Process. Preserv. 35, 143–162.
Omata, D., Negishi, Y., Suzuki, R., Oda, Y., Endo-Takahashi, Y., Maruyama, K., 2015.
Nonviral Gene Delivery Systems by the Combination of Bubble Liposomes and
Ultrasound. Adv. Genet. 89, 25–48.
Otsuka, H., Nagasaki, Y., Kataoka, K., 2003. PEGylated nanoparticles for biological and
pharmaceutical applications. Adv. Drug Deliv. Rev. 55, 403–419.
Panyam, J., Labhasetwar, V., 2003. Biodegradable nanoparticles for drug and gene de-
livery to cells and tissue. Adv. Drug Deliv. Rev. 55, 329–347.
Parraga, J.E., Zorzi, G.K., Sanchez, A., Seijo, B., 2009. Gelatin nanoparticles for ocular
gene delivery, Human gene therapy. Mary Ann Liebert Inc 140 Huguenot Street, 3rd
FL, New Rochelle, NY 10801 USA. pp. 1545–1545.
Patel, Z.S., Yamamoto, M., Ueda, H., Tabata, Y., Mikos, A.G., 2008. Biodegradable gelatin
microparticles as delivery systems for the controlled release of bone morphogenetic
protein-2. Acta Biomater. 4, 1126–1138.
Plank, C., Schillinger, U., Scherer, F., Bergemann, C., Rémy, J.-S., Krötz, F., Anton, M.,
Lausier, J., Rosenecker, J., 2003. The magnetofection method: using magnetic force
to enhance gene delivery. Biol. Chem. 384, 737–747.
Prabha, S., Arya, G., Chandra, R., Ahmed, B., Nimesh, S., 2016. Effect of size on biological
properties of nanoparticles employed in gene delivery. Artif. Cells Nanomed.
Biotechnol. 44, 83–91.
Ramamoorth, M., Narvekar, A., 2015. Non viral vectors in gene therapy-an overview. J.
Clin. Diagn. Res.: JCDR 9, GE01.
Sabet, S., George, M.A., El-Shorbagy, H.M., Bassiony, H., Farroh, K.Y., Youssef, T.,
Salaheldin, T.A., 2017. Gelatin nanoparticles enhance delivery of hepatitis C virus
recombinant NS2 gene. PLoS One 12, e0181723.
Sadat, S.M., Jahan, S.T., Haddadi, A., 2016. Effects of size and surface charge of poly-
meric nanoparticles on in vitro and in vivo applications. J. Biomater.
Nanobiotechnol. 7, 91.
Sahoo, N., Sahoo, R.K., Biswas, N., Guha, A., Kuotsu, K., 2015. Recent advancement of
gelatin nanoparticles in drug and vaccine delivery. Int. J. Biol. Macromol. 81,
317–331.
Samal, S.K., Dash, M., Van Vlierberghe, S., Kaplan, D.L., Chiellini, E., Van Blitterswijk, C.,
Moroni, L., Dubruel, P., 2012. Cationic polymers and their therapeutic potential.
Chem. Soc. Rev. 41, 7147–7194.
Saxena, A., Sachin, K., Bohidar, H., Verma, A.K., 2005. Effect of molecular weight het-
erogeneity on drug encapsulation efficiency of gelatin nano-particles. Colloids Surf.,
B 45, 42–48.
Schlessinger, J., 2002. Ligand-induced, receptor-mediated dimerization and activation of
EGF receptor. Cell 110, 669–672.
Senning, A., 1997. A review of:“An Introduction to Organosulfur Chemistry.
Sewell, J., Macleod, K., Ritchie, A., Smyth, J., Langdon, S., 2002. Targeting the EGF re-
ceptor in ovarian cancer with the tyrosine kinase inhibitor ZD 1839 (‘Iressa’). Br. J.
Cancer 86, 456–462.
Stone, D., 2010. Novel viral vector systems for gene therapy. Mol. Divers. Preserv. Int.
1002–1007.
Ter Haar, G., 2007. Therapeutic applications of ultrasound. Prog. Biophys. Mol. Biol. 93,
111–129.
Trabulo, S., Cardoso, A.L., Mano, M., De Lima, M.C.P., 2010. Cell-penetrating pepti-
des—mechanisms of cellular uptake and generation of delivery systems.
Pharmaceuticals 3, 961–993.
Tseng, C.-L., Wu, S.Y.-H., Wang, W.-H., Peng, C.-L., Lin, F.-H., Lin, C.-C., Young, T.-H.,
Shieh, M.-J., 2008. Targeting efficiency and biodistribution of biotinylated-EGF-
conjugated gelatin nanoparticles administered via aerosol delivery in nude mice with
lung cancer. Biomaterials 29, 3014–3022.
O. Madkhali et al.
Vega-Villa, K.R., Takemoto, J.K., Yáñez, J.A., Remsberg, C.M., Forrest, M.L., Davies, N.M.,
2008. Clinical toxicities of nanocarrier systems. Adv. Drug Deliv. Rev. 60, 929–938.
Vonarbourg, A., Passirani, C., Saulnier, P., Benoit, J.-P., 2006. Parameters influencing the
stealthiness of colloidal drug delivery systems. Biomaterials 27, 4356–4373.
Wang, H., Boerman, O.C., Sariibrahimoglu, K., Li, Y., Jansen, J.A., Leeuwenburgh, S.C.,
2012. Comparison of micro-vs. nanostructured colloidal gelatin gels for sustained
delivery of osteogenic proteins: bone morphogenetic protein-2 and alkaline phos-
phatase. Biomaterials 33, 8695–8703.
Wirth, T., Parker, N., Ylä-Herttuala, S., 2013. History of gene therapy. Gene 525,
162–169.
Xu, J., Amiji, M., 2012. Therapeutic gene delivery and transfection in human pancreatic
cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles. J.
Visualized Exp: JoVE, e3612.
Xu, J., Ganesh, S., Amiji, M., 2012. Non-condensing polymeric nanoparticles for targeted
gene and siRNA delivery. Int. J. Pharm. 427, 21–34.
Xu, J., Gattacceca, F., Amiji, M., 2013. Biodistribution and pharmacokinetics of EGFR-
targeted thiolated gelatin nanoparticles following systemic administration in pan-
creatic tumor-bearing mice. Mol. Pharm. 10, 2031–2044.
Xu, J., Singh, A., Amiji, M.M., 2014. Redox-responsive targeted gelatin nanoparticles for
delivery of combination wt-p53 expressing plasmid DNA and gemcitabine in the
treatment of pancreatic cancer. BMC Cancer 14, 75.
Xu, X., Capito, R.M., Spector, M., 2008. Delivery of plasmid IGF-1 to chondrocytes via
cationized gelatin nanoparticles. J. Biomed. Mater. Res. Part A 84, 73–83.
234
International Journal of Pharmaceutics 554 (2019) 224–234
Yameen, B., Choi, W.I., Vilos, C., Swami, A., Shi, J., Farokhzad, O.C., 2014. Insight into
nanoparticle cellular uptake and intracellular targeting. J. Control. Release 190,
485–499.
Yin, H., Kanasty, R.L., Eltoukhy, A.A., Vegas, A.J., Dorkin, J.R., Anderson, D.G., 2014.
Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541.
Young, J.L., Dean, D.A., 2015. Chapter three-electroporation-mediated gene delivery.
Adv. Genet. 89, 49–88.
Zillies, J., Coester, C., 2005. Evaluating gelatin based nanoparticles as a carrier system for
double stranded oligonucleotides. J. Pharm. Pharm. Sci.: Publ. Can. Soc. Pharm. Sci.
Societe canadienne des Sci. Pharm. 7, 17–21.
Zorzi, G.K., Parraga, J.E., Seijo, B., Sanchez, A., 2015. Comparison of different cationized
proteins as biomaterials for nanoparticle-based ocular gene delivery. Colloids Surf. B
Biointerfaces 135, 533–541.
Zorzi, G.K., Párraga, J.E., Seijo, B., Sánchez, A., 2011. Hybrid nanoparticle design based
on cationized gelatin and the polyanions dextran sulfate and chondroitin sulfate for
ocular gene therapy. Macromol. Biosci. 11, 905–913.
Zwiorek, K., Bourquin, C., Battiany, J., Winter, G., Endres, S., Hartmann, G., Coester, C.,
2008. Delivery by cationic gelatin nanoparticles strongly increases the im-
munostimulatory effects of CpG oligonucleotides. Pharm. Res. 25, 551–562.
Zwiorek, K., Kloeckner, J., Wagner, E., Coester, C., 2004. Gelatin nanoparticles as a new
and simple gene delivery system. J. Pharm. Pharm. Sci.:Publ. Can. Soc. Pharm. Sci.
Societe canadienne des Sciences Pharm. 7, 22–28.