Physical Properties and Stability of Plasmid DNA-Loaded Chitosan-TPP Nanoparticle

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 479-484

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Physical Properties and Stability of Plasmid

DNA-Loaded Chitosan-TPP Nanoparticle

S. R. Naeimi Torshizi ¹^,², H. Ofoghi *, A. Jangjou , S. Taghizadeh , M. Kianirad

¹Department of cellular and molecular, Nour Danesh Institute of Higher Education, Mymeh, Isfahan

Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran

Department of Emergency Medicine, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran:

Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical

Sciences, Shiraz, Iran:

Received: 12/05/2019

Accepted: 22/08/2019

Published: 24/08/2019

Abstract

Chitosan (CS) is a biodegradable natural polymer that has shown potential for gene delivery. Although a number

of in vitro studies showed that chitosan and its derivatives have emerged as promising vehicles for efficient non-viral

gene and plasmid DNA (pDNA) vaccine delivery, the stability of chitosan/plasmid nanoparticle remain insufficient.

In the present study, ionically crosslinked chitosan nanoparticles were formulated with plasmid DNA using the ionic

gelation technique with sodium tripolyphospate (TPP) as a crosslinking agent. We investigate the stability of

chitosan/pDNA nanoparticles which was synthesized by this method. Optimization study showed that chitosan to TPP

ratios of 1:0.4(w/w) results in the reproducible formation of nanoparticles with good production yields. SEM and DLS

analyses revealed a circular shape of the CS/TPP nanoparticles with an average size diameter of 173 nm. The zeta

potential of the nanoparticles was + 10.8 mv. In vitro study of pDNA release from CS/TPP nanoparticles revealed no

DNA release following incubation of chitosan/pDNA nanoparticles for up to 1 month, in mediums of PBS and acetic

acid at pH 4 and pH 7.4. According to the results, ionically crosslinked CS/TPP nanoparticles have the potential to be

used as a biocompatible non-viral gene delivery system with strong stability.

Keywords: Chitosan, Gene delivery, Nanoparticles, Ionic gelation

Introduction

features such as non-toxicity, biodegradation, and

biocompatibility as well as have a highly chemically

reactive structure render it highly useful for the

pharmaceutical application. A number of therapeutic

effects of CS have been reported, including wound

healing (17), anticancer (18), promotion of hemostasis

and epidermal cell growth (19). These properties have

attracted interest in the application of this substance in

biomedical various fields, such as drug delivery and

targeting (20), wound dressing (21), and tissue

engineering (22).

A number of in vitro studies showed that chitosan

and its derivatives have emerged as promising vehicles

for efficient non-viral gene and plasmid DNA (pDNA)

vaccine delivery (23, 24). In addition to the fact that

CS has a high positive charge and low toxicity, it also

has a high mucus binding affinity, which makes it able

Although macrobiomolecules have great potential

as therapeutic agents, but the potential has yet to be

completely exploited and have been addressed through

the development of proper nanocarriers. As surface

functionalization of nanoparticle are involved in

medical applications, structural materials, catalysts, as

well as in cleaning and purification systems. In this

context, nanoparticles have emerged as one of the

most exciting tools, due to the increased surface-to-

volume ratio, which enable the encapsulated

molecules to retain their biological activity, from the

production steps to the final release.(1-16) Chitosan

(

CS) is an interesting natural linear chain poly-amino

saccharide composed of D-glucosamine residues

linked by β (1→4) glycosidic bonds. Its desirable

Corresponding author: H. Ofoghi, Department of Biotechnology, Iranian Research Organization for Science and Technology,

Tehran, Iran. E-mail: e-mail: ofoghi@irost.ir.

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 479-484

to increase the penetration of large molecules across a

mucosal surface. Today, it has been proven that

chitosan-based delivery systems can be useful for

nasal delivery of siRNA and gene interference in the

lung mucosa (25). As a non-viral vector for gene

delivery, chitosan nanoparticles (CNPs) have many

advantages because viral systems may make it

possible to make recombinant recombination and

oncogenic effects and immunological reactions

leading to potentially serious complications.

CNPs are usually made up by ionic gelation

method, which describes the crosslinking reaction of

CS with sodium tripolyphosphate (TPP). This

technique involves the addition of a crosslinking

agent, i.e. TPP, into the aqueous phase containing

chitosan, thus leading to the formation of chitosan

nanogels (26, 27). This technique has been previously

adapted for the encapsulation of peptides and proteins

2.2 physicochemical

characterization of CS/TPP nanoparticles

After optimization, ionically crosslinked

Preparation

and

nanoparticles based on medium molecular weight CS

were formulated with plasmid DNA (pDNA). For this

formulation, the ionic gelation technique was used.

Chitosan solution is prepared at the concentration of

0.05% (W/V) in acetate buffer solution (pH 4.5). The

sample was stirred overnight and then filtered. The

TPP is dissolved in double-distilled water at a

concentration of 0.20 mg/mL. For formation of

chitosan-TPP nanoparticles containing plasmid in

their matrix, following procedure was performed: I)

the solution of plasmid is added to the previously

prepared solution TPP (0.20 mg/mL), II) nanoparticles

were formed instantaneously upon the dropwise

addition of a volume of plasmid/TPP solution to a

volume of chitosan solution (1:0.4) under magnetic

stirring at room temperature. Chitosan nanoparticles

were recovered by ultracentrifugation (Hettich®,

200R, Tuttlingen, Germany) 12,000 rpm, at 10 ºC for

20 min. All these steps are shown in schematic Figure

1. The particle size distribution and zeta potential were

obtained by the DLS technique, using a Zetasizer

Nano ZS 3600(Malvern Instruments). The

morphology of the nanoparticles was examined via

scanning electron microscope (KYKYEM3200) at an

operating voltage of 25 kV.

(

28). As compared to the other methods used for DNA

association, the nanoparticle formation is not only

determined by the electrostatic interactions between

chitosan and DNA but simultaneously also by physical

entrapment upon the ionic crosslinking induced by

TPP. This process results in the controlled gelation of

chitosan in the form of spherical, homogeneous and

compact nanoparticles, characteristics that are

expected to benefit the performance of the system both

in vitro and in vivo (29-33). It has advantages of not

necessitating sonication and organic solvents for its

preparation, therefore minimizing possible damage to

DNA

during

complexation.

Although

the

1.1 Loading capacity of CS/TPP nanoparticles

Encapsulation efficiencies of pDNA were

calculated from the amount of non-encapsulated

material recovered in the supernatant samples

collected upon centrifugation of the nanoparticles

(10000 RPM, 100C, 20 min). The amount of recovered

DNA was determined by spectrophotometer

(Biochrom®, U.K). Additionally, the association of

DNA to the nanoparticles was also determined by gel

electrophoresis assays (1% agarose containing safe

nucleic acid stain, Safe-green®, 80 V, 40 min).

experimental evidence suggests that in this strategy

chitosan can easily bind or encapsulate DNA and

protect it eﬀ ectively from DNases, but the stability of

chitosan/plasmid nanoparticle still needs to be

elucidated. The aim of this study was to investigate the

stability of chitosan/pDNA nanoparticles which was

synthesized by the method of ionic gelation. The

physical–chemical

properties

chitosan

nanoparticles were optimized by studying the

influence of several key parameters including CS

concentration, CS molecular weight (i.e. 125 kDa vs.

00 kDa), CS/TPP polymer ratio, and DNA loading.

1.1 In vitro release of DNA

In vitro release of pDNA was determined by

incubating the nanoparticles in acetate buffer and

phosphate buffered saline (PBS) (pH 4 and 7.4), (37

ºC, horizontal shaking). At time intervals of 1 day and

Materials and methods

.1 Materials

Chitosan with 75-85% deacetylation degree and

, 2, 3, 4 weeks, individual samples were isolated by

medium molecular weight was purchased from Sigma-

Alderich. Plasmid DNA (pDNA) encoding green

fluorescent protein (PKScGFP). tripolyphosphate

centrifugation (10,000 RPM, 20 min). The supernatant

of samples was analyzed by agarose gel

electrophoresis using un-encapsulated PKScGFP

plasmid as the positive control sample.

(TPP) was obtained from Sigma–Aldrich (Madrid,

Spain). One kBp DNA ladder was obtained from

Merck Millipore Bioscience. All other solvents and

chemicals were of the highest grade commercially

available.

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 479-484

Figure 1: Schematic figure of chitosan/pDNA nanoparticle synthesis by ionic gelation method

Results and discussion

.1 Formation and physicochemical characterization

of CS/pDNA nanoparticles

Initial studies aiming at the optimization of the

nanoparticle formation indicated that chitosan to TPP

ratios of 1:0.4(w/w) results in the reproducible

formation of nanoparticles with good production

yields. The average size of the nanoparticles prepared

of medium molecular weight chitosan (MMW

CS/TPP) was 173 nm. Polydispersity Index (P.I.) of

nanoparticles was 0/292 (Table1). Also, Zeta potential

of chitosan/pDNA was +10.8 mV (Figure 2). The

morphology and structure of CNPs are demonstrated

by SEM. The results showed that the majority of CNPs

were circular in shape with an only limited degree of

aggregation (Figure 3).

Table1: Physical–chemical characteristics of CS/TPP

nanoparticles loaded with pDNA (i.e. pKScGFP).

Figure 3: SEM micrograph of synthesized chitosan/pDNA

nanoparticles

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 479-484

buffer pH 4 and pH 7.4 and phosphate buffered saline

(PBS) pH 4 and 7.4 ), and assayed in agarose gel

retardation assays. The results showed no DNA

release following incubation of chitosan/pDNA

nanoparticles for up to 1 month, in all of types

incubation medium (Fig. 4a and b). These results

indicate that pDNA is very firmly associated with

CS/TPP nanoparticles. However, plasmid DNA could

be released from the nanoparticles following

incubation in lysozyme solution (Fig 5).

Figure 2: Particle size distribution of the CS/pDNA nanoparticles

.2 Entrapment of plasmid DNA

According to research conducted by Carrillo et al

(

34), for loading process, 14µg/ml of plasmid DNA

was included in TPP solution prior to nanoparticle

formation. In this study, chitosan showed high

efficiency for the encapsulation of pDNA, reaching

almost 100% (Table 1). Previous studies have shown

that the entrapment of the pDNA within the CS/TPP

nanoparticles has several advantages over absorbance

of pDNA onto preformed CNPs, such as the more

effective protection of pDNA from decomposition

when administered In vivo, the easier surface

modification of nanoparticles to improve their

interaction with biological surfaces, and more

controllable of the pDNA release process.

.3 In vitro release of pDNA from CS/TPP

nanoparticles

In order to study the stability of chitosan/pDNA

Figure 5: Agarose gel assays following incubation of CS/pDNA

nanoparticles with lysozyme

nanoparticle and the pDNA release properties of

CS/TPP nanoparticles, they were incubated in 2 type

of release media with 2 different pH values (acetate

Figure 4: In vitro pDNA release studies from CS/pDNA nanoparticles incubated for (a) 1 day and (b) 1month. The studies were performed at pH 4

and 7.4 acetate buffer and PBS at 37 ◦C.( Line 1: naked pDNA, Line 2: CNP treated with PBS pH 7.4, Line 2: CNP treated with acetate buffer pH

, Line 4: CNP treated with acetate buffer pH 7.4, Line 5: CNP treated with distilled water)

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 3, Pages: 479-484

montmorillonite K10 as catalyst. Monatshefte für Chemie-

Chemical Monthly. 2009;140(5):547-52.

1. Rostamizadeh S, Amani AM, Mahdavinia GH, Shadjou N.

Conclusion

In this investigation, we have successfully

synthesized

ionically

crosslinked

CS/TPP

Silica

supported ammonium dihydrogen phosphate

nanoparticles as an interesting delivery system for

nucleic acids such as plasmid DNA. Nanoparticles are

spherical in shape with a mean particle size of 173 nm.

Due to strong electrostatic interaction, the

chitosan/pDNA nanoparticles are highly stable and no

pDNA was released after one month in nanoparticles

treated with a solution of PBS and acetate buffer in pH

(NH4H2PO4/SiO2): A mild, reusable and highly efficient

heterogeneous catalyst for the synthesis of 14-aryl-14-H-

dibenzo [a, j] xanthenes. Chinese Chemical Letters.

009;20(7):779-83.

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