Modified gelatin nanoparticles for gene delivery

Accepted Manuscript Review Modified Gelatin Nanoparticles for Gene Delivery Osama Madkhali, George Mekhail, Shawn D. Wettig PII: DOI: Reference:

S0378-5173(18)30819-6 https://doi.org/10.1016/j.ijpharm.2018.11.001 IJP 17893

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

30 July 2018 31 October 2018 1 November 2018

Please cite this article as: O. Madkhali, G. Mekhail, S.D. Wettig, Modified Gelatin Nanoparticles for Gene Delivery, International Journal of Pharmaceutics (2018), doi: https://doi.org/10.1016/j.ijpharm.2018.11.001

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Review Paper Modified Gelatin Nanoparticles for Gene Delivery Osama Madkhali1,2 George Mekhail1,3 Shawn D. Wettig1,4* 1

School of Pharmacy, University of Waterloo, Waterloo ON, N2L 3G1, Canada 2 Faculty of Pharmacy, Jazan University, Jizan, Kingdom of Saudi Arabia 3 Department of Pharmaceutics and Industrial Pharmacy, School of Pharmacy, Ain Shams University, Cairo, Egypt 4 Waterloo Institute of Nanotechnology, University of Waterloo, Waterloo ON, N2L 3G1, Canada

*Corresponding Author: Shawn Wettig Email: [email protected]

Address: School of Pharmacy, University of Waterloo, 200 University Ave. W., Waterloo, ON N2L 3G1 CANADA

All authors contributed equally to this work.

1

Abstract: Gelatin nanoparticles (GNPs) are one of the most extensively used natural polymers for gene therapy. With advantages of being biodegradable, biocompatible, low cost and easily modified, gelatin holds great promise as a non-viral system for gene delivery. This review examines various methods of preparation of modified gelatin nanoparticles and considers how these modifications apply to gene delivery. The article discusses cationic gelatin, 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. Keywords: gelatin, gene delivery, modification, method

2

1.1 Introduction

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 (Xlinked SCID), emphysema, retinitis pigmentosa, sickle cell anemia, hemophilia, Duchenne Muscular Dystrophy (DMD), some autosomal dominant disorders, vascular disease, neurodegenerative disorders, polygenic disorders, 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 substance 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 functional 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 significant 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)) require a delivery vehicle in order to efficiently travel through extra- 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 3

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-associated 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 longterm expression through chromosomal integration (Lundstrom and Boulikas, 2003; Nayerossadat et al., 2012). Yet viral vectors have several concerns regarding 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-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. 1.2 Physical Methods of Non-viral Systems These methods depend on using physical force in order to destabilize the cellular membrane, therefore facilitating the entry of gene therapeutic materials into the cells. These methods are simple and straightforward. 1.2.1 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

4

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 procedures, and high voltage [>700V/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). 1.2.2 Gene gun The gene gun (also known as particle bombardment, micro projectile 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). 1.2.3 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 5

advantages of sonoporation include safety, noninvasiveness, and the ability to reach internal 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). 1.2.4 Photoporation This technique works by using a single laser pulse in order to generate 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). 1.2.5 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 administered intravenously when it used in vivo. With the help of enzymatic 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. 1.3 Chemical Methods of Non-viral Systems Chemical methods of transfection are divided into two categories: inorganic particles (such as calcium phosphate, silica, and gold particles); and organic synthetic/natural materials 6

(such as cationic lipid and cationic polymers). Lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA) are the most commonly used non-viral delivery vectors. 1.4 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 hydrolysis 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 pharmaceutical, cosmetic, and food products for decades (Elzoghby et al., 2012; Kommareddy et al., 2005a; Lemieux et al., 2000). Gelatin is an amphiphilic polymer having both cationic and anionic charges along with hydrophobic groups present in the approximate 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 7 left of the figure. Reproduced with permission from (Hosseinkhani et al., 2015)

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 composition 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, isoleucine, methionine, and valine, representing 11% of the gelatin structure. The rest of the chain includes glycine, proline, and hydroxyproline. During either acid or alkaline collagen hydrolysis, the positions 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 hydrolysis 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 variable collagen amino acid composition. Accordingly, it is a challenge to control the chemical composition and the overall properties of gelatin 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

8

allows them to be injected directly into the systemic circulation without the risk of blocking blood vessels. Moreover, nanoparticles have been proved to improve the transfection efficiency of plasmid DNA into the nucleus (Prabha et al., 2016). Particle size of gene therapy vectors is also an important factor that impacts 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.

Fig. 2. General chemical structure of gelatin (A), and features of gelatin (B). *Reproduced with permission from (Sahoo et al., 2015) 1.4.1 Cationic gelatin nanoparticles Gelatin is a polyelectrolyte with a low-charge density that is appreciably changed depending on the solution’s pH. As a result, the 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 polyethyleneimine (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). Ethylenediamine 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 9

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 interact electrostatically with the anionic terminal of phospholipid, proteins and glycans on the plasma membrane, but also promote nanoparticle association with the cells inducing cell uptake by cellular endocytic mechanisms (Bannunah et al., 2014). Among multiple endocytosis 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 polyplexes. Moreover, recent studies postulate that cellular uptake of positively charged NPs uptake is related to energy dependent processes like proteins dynamin and Factin (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 potential 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 delivery to cancer cells. However, 10-kD PEI grafted gelatin was more cytotoxic while PEI with molecular weight less than 1.8-kD unveiled 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

10

‘‘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 protecting both pDNA and siRNA under the conditions endeavored. Similarly, Kushibiki et al., (Kushibiki et al., 2006a) investigate the in vitro transfection efficiency of plasmid DNA for mouse fibroblasts using cationized gelatin prepared from different types of amine compounds namely; ethylenediamine, 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 nanoparticles. Those most commonly used are discussed in the next section.

Fig. 3. Chemical structures of molecules used to cationize gelatin. 11

12

1.5 Methods of preparations of cationic gelatin 1.5.1 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 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 ºC for 18 hours 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 nanoparticles, which will be described below.

Fig. 4. Schematic diagram of the preparation of cationic gelatin using EDA or spermine and EDC. Adapted with permission from (Samal et al., 2012)

1.5.2 Preparation of cationic gelatin nanoparticles using two-step desolvation method Two-step desolvation approach was adopted to limit the heterogeneity 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

13

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 ºC). Desolvating agents such as acetone or ethanol are added as to precipitate the high molecular weight (HMW) of gelatin. After the supernatant is discarded, the HMW gelatin is redissolved 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 crosslinking 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 nanoparticles are purified using centrifugation or dialysis membrane prior to freeze drying. Following the freeze drying process, the gelatin is cationized 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 hour in the dark. The resulting cationized gelatin nanoparticles are purified using centrifugation or dialysis membrane for two days prior to lyophilization. 1.5.3 Preparation of cationic gelatin using one-step desolvation method

14

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 robust in the preparation of gelatin nanoparticles (Geh et al., 2016).

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

1.5.4 Preparation of cationic gelatin nanoparticles using ionic gelation technique Zorzi et al., (Parraga et al., 2009; Zorzi et al., 2011) prepared cationic 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 gelatin. 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.

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

15

Table 1 Preparation of cationic gelatin for gene delivery using different cationic agents Method

Gelatin Type

Cationic agent

Type A

Cholamine

Two-step

One step

Ionic gelation

Aminization

Type A

Polyethyleneimine

Type A and Type B

Cholamine

Type A

Spermine

Type B

Spermine & Ethylenediamine

Type A

Etylenediamine Spermine Spermidine Putrescine

*Nanoparticles formed.

†

NP*

Gene used

In vitro / vivo

Cell type

Outcomes

Ref.

Yes

pCMV-luc siRNA

In vitro _

B16F10 _

Moderate transfection with high cell viability

(Zwiorek et al., 2004) (Zillies and Coester, 2005)

pCMV-luc

In vitro

NIH 3T3

High transfection with high cell viability

(Kuo et al., 2011)

_

_

_

_

(Geh et al., 2016; Zwiorek et al., 2008)

pMUC5AC

In vitro

HCE IOBANHC

pDNA & siRNA

In vitro

HCE IOBANHC

pCMV-luc

In vitro

RGM-1

pGL3-Luc pGL3-Luc

In vitro In vivo

L929 ddY mice†

Yes

Yes

No

Spermine showed high transfection and gene silencing with adequate cell viability

(Konat Zorzi et al., 2011)

(Zorzi et al., 2015)

Spermine showed the highest transfection with low cell viability Spermine showed the highest transfection with low cell viability

(Hosseinkhani et al., 2002a; Hosseinkhani et al., 2002b) (Kushibiki et al., 2006b, c) (Kushibiki and Tabata, 2005)

6 – 7 weeks old mice.

1.6 PEGylated gelatin nanoparticles GNPs are mainly engulfed by the cells of the reticuloendothelial system (RES) upon systemic administration. This leads to weak transfection 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 PEGylation 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;

16

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 digestion 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 and cytotoxicity, whereas obvious reduction in membrane damage, lipid peroxidation, and oxidative stress were reported in neuroblastoma cells (Hoskins et al., 2012). 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 tumors 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 administration. Amiji and Kaul also stated that reporter pDNA encoding for β-galactosidase (pCMV-β) was effectively encapsulated in PEGylated GNPs and showed significant expression in the tumor of LLC with 61% transfection efficiency (Kaul and Amiji, 2005).

17

Kushibiki and colleagues (Kushibiki et al., 2004) studied the long-circulation property of PEGylated gelatin using PEGylated

125

125

I-labeled gelatin. They compared unmodified GNPs and

I-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 hours 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 hours respectively. Generally, the advantages of the existence of PEG in combination with GNPs are summarized as follows: increase in the circulation 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 and Amiji, 2004, 2005) . 1.6.1 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 carboxylated derivative of PEG which was further activated using dicyclohexylcarbodiimide (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 14h at 40 oC. The product

18

obtained is precipitated with excess acetone to remove any unreacted PEG-epoxide, dialyzed against distilled 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 PEGsuccinimidyl 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 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 3h, dialyzed against deionized water, and then lyophilized.

Fig.7. Schematic illustration of the reaction between PEG-epoxide and gelatin nanoparticles. Reproduced from (Elzoghby, 2013)

1.7 EGFR-targeted gelatin nanoparticles One of the greatest challenges for gene delivery is the area of targeting. 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, release nucleic acid from the complex, which can then enter the nucleus to express the required protein (Xu et al., 2013). Although tumor targeting 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 receptor (EGFR) has been shown to be associated with poor prognosis in several types of cancers including ovarian cancer (Ciardiello and Tortora, 2001). Between 19

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 receptor 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-iminothiolane hydrochloride at room temperature for 15 hours, (Kommareddy and Amiji, 2005; Tseng et al., 2008) then left over night to reacted with Sulfo-MBS activated EGFR at 4 oC (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, 2008b). Consequently, the conjugation of gelatin with targeting EGFR peptide has been shown to improve the transfection efficiency in several types of cancer cells. EGFR-targeted GNPs carrying plasmid DNA encoding for EGFp-N1 obtained the highest transfection efficiency in Panc-1 pancreatic adenocarcinoma cells in comparison with other controls, particularly 48 h after transfection (Magadala and Amiji, 2008a). 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

20

thiolated gelatin nanoparticles to deliver plasmid DNA into

Panc-1 pancreatic

adenocarcinoma 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-complexed 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. 1.8 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 quaternary protein structure of gelatin (Bacalocostantis et al., 2013). In addition, disulfide bonds can stabilize the nanoparticles during systemic 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 cytoplasm 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 21

adjusted and controlled pH and temperature conditions. 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 contrast to unmodified gelatin and lipofectin®-complexed DNA (Kommareddy and Amiji, 2005). Six hours after transfection, the expression 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-3T3 to deliver plasmid DNA. Of the three formulations tested, PEG-thiolated GNPs indicated the highest GFP expression to even a greater degree than the positive control lipofectincomplexed DNA (Kommareddy and Amiji, 2007b). Generally, both PEG-GNPs and PEGmodified 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 developed 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 accumulation 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

22

disulfide bonds formed by the (thiol) group could play a significant role in the stability of nanoparticles, thereby resulting in effective gene expression and high cell viability. 1.9 Alendronate gelatin nanoparticles Bisphosphonates, like alendronate, are characterized by their high affinity to hydroxyapatite. Moreover, they maintain their binding capability 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 synthesized 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, gelatin 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). ALNgelatin 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 functionalized 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 Lipofectamine 2000® and 1.5 times the transfection efficiency of the gemini/DNA complexes (Mekhail et al., 2016). The presence of ALN as a targeting moiety facilitated receptor mediated endocytosis for cellular uptake. Those results revealed that ALN-modified

23

gelatin established a platform for both drug delivery and a promising vector for the formulation of targeted cationic nanocomplexes for enhanced gene delivery to bone tissues.

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.

1.10 Conclusion There are numerous advantages of using gelatin as polymer for fabrication of nanoparticles. Gelatin possesses low cellular toxicity because it is derived from animal protein and also producing very low-level allergic reaction. Gelatin can be easily polymerized to form nanoparticles which are relatively stable and reproducible. Gelatin also facilitates for future up scaling. Due to low cost of gelatin and its biodegradable 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-chains of the gelatin matrix molecule which facilitate further modifications. This may be useful for coupling of ligands for improving the target diseased tissues and to enhance particular cellular uptake or affect intracellular distribution. Gelatin nanoparticles are promising non-viral vectors for gene delivery applications. The modifications improved the delivery system and led to more effective transfection efficiency and cell viability. Depending on the final outcome/objective of the gelatin nanoparticle, various manufacturing methods may be utilized such as cationic, PEGylated, thiolated, EGFR-targeted, and alendronate 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 biocompatibility 24

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 crosslinkers such as glutaraldehyde, which should be removed after preparing 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 (RGPIN2016-04009).

25

References Bacalocostantis, I., Mane, V.P., Goodley, A.S., Bentley, W.E., Muro, S., Kofinas, P., 2013. Investigating polymer thiolation in gene delivery. Journal of Biomaterials Science, Polymer Edition 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. Molecular pharmaceutics 11, 4363-4373. Busch, S., Schwarz, U., Kniep, R., 2003. Chemical and structural investigations of biomimetically grown fluorapatite–gelatin composite aggregates. Advanced Functional Materials 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 interactions. International journal of pharmaceutics 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 Polyethyleneimine-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. Clinical Cancer Research 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. Journal of microencapsulation 17, 187-193. Crawford, J., 2002. Clinical uses of pegylated pharmaceuticals in oncology. Cancer Treatment Reviews 28, 7-11. Dausend, J., Musyanovych, A., Dass, M., Walther, P., Schrezenmeier, H., Landfester, K., Mailänder, V., 2008. Uptake mechanism of oppositely charged fluorescent nanoparticles in HeLa cells. Macromolecular bioscience 8, 1135-1143. Elzoghby, A.O., 2013. Gelatin-based nanoparticles as drug and gene delivery systems: reviewing three decades of research. Journal of Controlled Release 172, 1075-1091. Elzoghby, A.O., Samy, W.M., Elgindy, N.A., 2012. Protein-based nanocarriers as promising drug and gene delivery systems. Journal of controlled release : official journal of the Controlled Release Society 161, 38-49. Fröhlich, E., 2012. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. International journal of nanomedicine 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. Journal of Microencapsulation, 1-10. Gough, J.E., Scotchford, C.A., Downes, S., 2002. Cytotoxicity of glutaraldehyde crosslinked collagen/poly (vinyl alcohol) films is by the mechanism of apoptosis. Journal of Biomedical Materials Research Part A 61, 121-130. Griffith, F., 1928. The significance of pneumococcal types. Epidemiology & Infection 27, 113-159. Hawley‐ Nelson, P., Ciccarone, V., Moore, M.L., 2008. Transfection of cultured eukaryotic cells using cationic lipid reagents. Current protocols in molecular biology 81, 9.4. 1-9.4. 17. Hoskins, C., Wang, L., Cheng, W.P., Cuschieri, A., 2012. Dilemmas in the reliable estimation of the in-vitro cell viability in magnetic nanoparticle engineering: which tests and what protocols? Nanoscale research letters 7, 77. 26

Hosseinkhani, H., Abedini, F., Ou, K.L., Domb, A.J., 2015. Polymers in gene therapy technology. Polymers for Advanced Technologies 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. Journal of drug targeting 10, 193204. Hosseinkhani, H., Aoyama, T., Yamamoto, S., Ogawa, O., Tabata, Y., 2002b. In vitro transfection of plasmid DNA by amine derivatives of gelatin accompanied with ultrasound irradiation. Pharmaceutical research 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. Molecular therapy 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. Journal of Controlled 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. Molecular pharmaceutics 10, 40824098. 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. Expert opinion on drug delivery 12, 1089-1105. Kaul, G., Amiji, M., 2002. Long-circulating poly (ethylene glycol)-modified gelatin nanoparticles for intracellular delivery. Pharmaceutical research 19, 1061-1067. Kaul, G., Amiji, M., 2004. Biodistribution and targeting potential of poly(ethylene glycol)modified gelatin nanoparticles in subcutaneous murine tumor model. Journal of drug targeting 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. Pharmaceutical research 22, 951961. Keeler, A.M., ElMallah, M.K., Flotte, T.R., 2017. Gene therapy 2017: Progress and future directions. Clinical and Translational Science. Kim, K.J., Byun, Y., 1999. Preparation and characterizations of self-assembled PEGylated gelatin nanoparticles. Biotechnology and Bioprocess Engineering 4, 210-214. Kommareddy, S., Amiji, M., 2005. Preparation and evaluation of thiol-modified gelatin nanoparticles for intracellular DNA delivery in response to glutathione. Bioconjugate chemistry 16, 1423-1432. Kommareddy, S., Amiji, M., 2007a. Biodistribution and pharmacokinetic analysis of longcirculating thiolated gelatin nanoparticles following systemic administration in breast cancerbearing mice. Journal of pharmaceutical sciences 96, 397-407. Kommareddy, S., Amiji, M., 2007b. Poly(ethylene glycol)-modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery. Nanomedicine : nanotechnology, biology, and medicine 3, 32-42. Kommareddy, S., Shenoy, D.B., Amiji, M.M., 2005a. Gelatin nanoparticles and their biofunctionalization. Nanotechnologies for the Life Sciences:Online. Kommareddy, S., Tiwari, S.B., Amiji, M.M., 2005b. Long-circulating polymeric nanovectors for tumor-selective gene delivery. Technology in cancer research & treatment 4, 615-625.

27

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. Molecular pharmaceutics 8, 1783-1788. Kumar, C.S., 2005. Biofunctionalization of nanomaterials. Biofunctionalization of Nanomaterials, by Challa SSR Kumar (Editor), pp. 377. ISBN 3-527-31381-8. Wiley-VCH, November 2005., 377. Kuo, W.-T., Huang, H.-Y., Chou, M.-J., Wu, M.-C., Huang, Y.-Y., 2011. Surface modification of gelatin nanoparticles with polyethylenimine as gene vector. Journal of Nanomaterials 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. Journal of controlled release 110, 610-617. Kushibiki, T., Tabata, Y., 2005. Preparation of poly(ethylene glycol)-introduced cationized 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. Journal of controlled release : official journal of the Controlled Release Society 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. Current opinion in biotechnology 16, 674680. 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 nanoparticles for tumor-targeted delivery of polymerized siRNA in tumor-bearing mice. Journal of Controlled 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. Journal of drug targeting 8, 91-105. Li, S.-D., Huang, L., 2007. Non-viral is superior to viral gene delivery. Journal of controlled release: official journal of the Controlled Release Society 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 biodegradable amphiphilic PCL-Pluronic (L35)-PCL block copolymers. Colloids and Surfaces A: Physicochemical and Engineering Aspects 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. Technology in cancer research & treatment 2, 471-485. Magadala, P., Amiji, M., 2008a. Epidermal growth factor receptor-targeted gelatin-based engineered nanocarriers for DNA delivery and transfection in human pancreatic cancer cells. American Association of Pharmaceutical Scientists Journal 10, 565-576. 28

Magadala, P., Amiji, M., 2008b. Epidermal growth factor receptor-targeted gelatin-based engineered nanocarriers for DNA delivery and transfection in human pancreatic cancer cells. The AAPS journal 10, 565. Maitra, A., De, T.K., Mitra, S., 2006. Hydrogel nanoparticles: applications in drug delivery. Second Edition (pp. 2821-2837). SomasundaramP and HubbardA, Eds. Encyclopedia of Surface and Colloid Science 2. Mali, S., 2013. Delivery systems for gene therapy. Indian journal of human genetics 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 biopolymer as a novel osteotropic nanocarrier for gene therapy. Nanomedicine : nanotechnology, biology, and medicine 11, 2251-2273. ic ler . . c l, L., Ruthardt, N., Ogris, M., agner . r chle, C., 2012. Tuning nanoparticle uptake: live-cell imaging reveals two distinct endocytosis mechanisms mediated by natural and artificial EGFR targeting ligand. Nano letters 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 knockdown in HeLa cells. Journal of Controlled 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, Llactide) nanocapsules bearing poly (ethylene glycol). Journal of drug targeting 7, 65-78. Nayerossadat, N., Maedeh, T., Ali, P.A., 2012. Viral and nonviral delivery systems for gene delivery. Advanced biomedical research 1. Ninan, G., Jose, J., Abubacker, Z., 2011. Preparation and characterization of gelatin extracted from the skins of rohu (Labeo rohita) and common carp (Cyprinus carpio). Journal of Food Processing and Preservation 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. Advances in Genetics 89, 25-48. Otsuka, H., Nagasaki, Y., Kataoka, K., 2003. PEGylated nanoparticles for biological and pharmaceutical applications. Advanced drug delivery reviews 55, 403-419. Panyam, J., Labhasetwar, V., 2003. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Advanced drug delivery reviews 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 protein2. Acta biomaterialia 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. Biological chemistry 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. Artificial cells, nanomedicine, and biotechnology 44, 83-91. Ramamoorth, M., Narvekar, A., 2015. Non viral vectors in gene therapy-an overview. Journal of clinical and diagnostic research: JCDR 9, GE01.

29

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 polymeric nanoparticles on in vitro and in vivo applications. Journal of Biomaterials and Nanobiotechnology 7, 91. Sahoo, N., Sahoo, R.K., Biswas, N., Guha, A., Kuotsu, K., 2015. Recent advancement of gelatin nanoparticles in drug and vaccine delivery. International journal of biological macromolecules 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. Chemical Society Reviews 41, 7147-7194. Saxena, A., Sachin, K., Bohidar, H., Verma, A.K., 2005. Effect of molecular weight heterogeneity on drug encapsulation efficiency of gelatin nano-particles. Colloids and Surfaces B: Biointerfaces 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 Introd ction to Organos lf r Chemistry”. Sewell, J., Macleod, K., Ritchie, A., Smyth, J., Langdon, S., 2002. Targeting the EGF receptor in ovarian cancer with the tyrosine inase inhibitor ZD 1839 (‘Iressa’). ritish journal of cancer 86, 456-462. Stone, D., 2010. Novel viral vector systems for gene therapy. Molecular Diversity Preservation International. 1002-1007. Ter Haar, G., 2007. Therapeutic applications of ultrasound. Progress in biophysics and molecular biology 93, 111-129. Trabulo, S., Cardoso, A.L., Mano, M., De Lima, M.C.P., 2010. Cell-penetrating peptides— mechanisms of cellular uptake and generation of delivery systems. Pharmaceuticals 3, 961993. 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. 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. Advanced drug delivery reviews 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 phosphatase. 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. Journal of visualized experiments : JoVE, e3612. Xu, J., Ganesh, S., Amiji, M., 2012. Non-condensing polymeric nanoparticles for targeted gene and siRNA delivery. International journal of pharmaceutics 427, 21-34. Xu, J., Gattacceca, F., Amiji, M., 2013. Biodistribution and pharmacokinetics of EGFRtargeted thiolated gelatin nanoparticles following systemic administration in pancreatic tumor-bearing mice. Molecular pharmaceutics 10, 2031-2044. 30

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. Journal of Biomedical Materials Research Part A 84, 73-83. Yameen, B., Choi, W.I., Vilos, C., Swami, A., Shi, J., Farokhzad, O.C., 2014. Insight into nanoparticle cellular uptake and intracellular targeting. Journal of Controlled 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. Nature Reviews Genetics 15, 541. Young, J.L., Dean, D.A., 2015. Chapter Three-Electroporation-Mediated Gene Delivery. Advances in genetics 89, 49-88. Zillies, J., Coester, C., 2005. Evaluating gelatin based nanoparticles as a carrier system for double stranded oligonucleotides. Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques 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 and surfaces. 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. Macromolecular bioscience 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 immunostimulatory effects of CpG oligonucleotides. Pharmaceutical research 25, 551-562. Zwiorek, K., Kloeckner, J., Wagner, E., Coester, C., 2004. Gelatin nanoparticles as a new and simple gene delivery system. Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques 7, 22-28.

31

32

A*

B Biodegradable Low cost of production Physiological tolerance Easily modified Biocompatible Low antigenicity

33

34

35

36

37

38