Synthesis and characterization of pectin-chitosan conjugate for biomedical application

Journal Pre-proof Synthesis and characterization of pectin-chitosan conjugate for biomedical application

Lijun Tian, Anudwipa Singh, Akhilesh Vikram Singh PII:

S0141-8130(20)30550-X

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.313

Reference:

BIOMAC 14930

To appear in:

International Journal of Biological Macromolecules

Received date:

17 January 2020

Revised date:

24 February 2020

Accepted date:

26 February 2020

Please cite this article as: L. Tian, A. Singh and A.V. Singh, Synthesis and characterization of pectin-chitosan conjugate for biomedical application, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2020.02.313

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© 2018 Published by Elsevier.

Journal Pre-proof

Synthesis and Characterization of Pectin-chitosan Conjugate for biomedical application Lijun Tian1, Anudwipa Singh2, Akhilesh Vikram Singh3

Shanxi Provincial People's Hospital, Taiyuan, China

2

Bharat Institute of Technology, Hyderabad, Telangana, India

3

Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam, India

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Corresponding address:

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Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam, India Email: [email protected] Contact Number: +91-9100940734

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Journal Pre-proof ABSTRACT

Background: Polysaccharides are extensively used in drug delivery systems due to their ability to undergo a broad range of chemical and enzymatic reactions, forming new molecules. Among these, chitosan (CS) and pectin (PEC) are widely used for designing new conjugates and biopolymers. Methods: In this study, we synthesized pectin-chitosan conjugate (PEC-CS) by using

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carbodiimide crosslinking chemistry. Pectin-N-hydroxysuccinimide ester, formed in the

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presence of dicyclohexylcarbodiimide under anhydrous conditions, was conjugated with chitosan by linking the free carboxyl group of PEC with the primary amino group of chitosan.

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Results: Fourier-transform infrared spectroscopy confirmed the formation of the PEC-CS

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conjugate. X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis,

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and scanning electron microscopy analyses showed that the PEC-CS conjugate is amorphous in nature, has high thermostability than those of native polymers, and has no cytotoxicity.

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Conclusion: Our results indicated that PEC-CS conjugate can be further developed for use in

Keywords:

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drug delivery systems.

Pectin,

chitosan,

bioconjugate,

carbodiimide

induced

cross-linking,

pectin-chitosan conjugate

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Journal Pre-proof 1. Introduction

Natural polymers such as polysaccharides, proteins, and lipoproteins have gained prominence due to the development of novel drug delivery systems [1]. Natural polymers are renewable resources obtained from plants and animals that show relatively lesser toxicity and immunogenicity than those of synthetic polymers. Their physicochemical properties such as swelling, dissolution or viscoelasticity depend on pH, enzymes, temperature, ion

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concentration, and can be exploited in novel drug delivery systems [2]. These naturally

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occurring polymers can also be conjugated to one another to design novel materials with specific properties required for the targeted drug delivery system. Many such bioconjugate

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show increased solubility and stability, biocompatibility, biodegradability, mechanical

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strength, in vivo targeting, and stimulated release [3]. Bioconjugates are applied in diverse

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areas such as biosensors, nanoelectronics, conducting polymers, and photonics[4].

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Polysaccharides are extensively used in drug delivery systems due to their ability to undergo a wide range of chemical and enzymatic reactions, forming new molecules. Among these,

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chitosan (CS) and pectin (PEC) are widely used for designing new conjugates and biopolymers [5,6].CS is a cationic polymer obtained from the shells of marine animals. It consists of repeating units of N-glucosamine and N-acetyl-glucosamine. It is a linear polysaccharide containing reactive hydroxyl and amino groups and retains a positive charge in the acidic environment due to the protonation of RNH3+ groups. The primary aliphatic amino group renders it reactive to typical amino reactions such as N-acylation and Schiff reaction [7]. PEC is obtained mainly from the skin of citrus fruits and consists of repeating units of D-galacturonic acid monomers that aid in conjugation reactions[8].It is a lyophilic colloid with a negative charge and is stabilized by hydration[9].One of the main characteristics of PEC is its gelling tendency, which is dependent on its degree of 3

Journal Pre-proof esterification [7].

Depending on their charge distribution, CS and PEC have been crosslinked into polyelectrolyte film or networks, [9-11] to form inserts, [12] microgels [13,14], nanocarriers [15], buccal patches [16], and tablet coatings for colonic drug delivery [14,17]. However, pectin-chitosan (PEC-CS) bioconjugates have not yet been studied for their suitability in drug delivery systems. Polymers are generally bioconjugated by polymerization, coupling,

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ligation, and solid-phase synthesis, or copolymerization reactions. Apart from the standard

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coupling reactions, bioconjugates are also synthesized using peptide reaction, ‘click’ reactions and biotin-avidin coupling. In this study, we attempt to synthesize and characterize

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a PEC-CS bioconjugate using the carbodiimide crosslinking method by coupling PEC with

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CS.

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2.1 Materials

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2. Materials and Methods

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Chitosan (medium viscosity, deacetylation degree >85%), N-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) were purchased from Sigma Aldrich (St. Louis, MO, USA). All other chemicals/reagents used were of analytical grade. PEC was purchased from Yarrow Chem (Mumbai, India). Dimethyl sulfoxide (DMSO) was procured from Thermo Fisher Scientific (Waltham, MA, USA).

2.2 Methods

2.2.1 Synthesis of the pectin-chitosan conjugate (PEC-CS)

The N-hydroxysuccinimide ester of pectin (NHS-PEC) was prepared following the procedure 4

Journal Pre-proof described in the reaction scheme (Fig.1). In brief, PEC (1.0 g) was dissolved in a mixture of anhydrous dimethyl sulfoxide (DMSO, 40 ml) and triethylamine (TEA, 0.5 ml), with continuous

stirring

in

the

dark

overnight

under

anhydrous

condition.

Dicyclohexylcarbodiimide (DCC, 0.5 g) and N-hydroxysuccinimide (NHS, 0.5 g), was added to the PEC solution and stirred in the dark for 24 h to activate the carboxyl groups of PEC. The precipitated byproduct dicyclohexylurea (DCU) was removed by filtration. The filtered product (NHS-PEC) was then washed three times with Millipore water and finally

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lyophilized.

Chitosan was purified as described by Zu et al. [18]. Lyophilized NHS-PEC (1.50 g) was

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dissolved in DMSO (150 mL) using ultrasonicator (Soniclean, Australia). Afterward, CS

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(1.00 g) was added into the NHS-PEC solution and stirred for 4 h at 60°C. The mixture was

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precipitated by centrifugation (12,000 rpm, 5 min) at room temperature, and the pellet NHSPEC was washed with DMSO. Finally, the pellet was washed with deionized water to remove

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residual DMSO and PEC-CS was obtained by freeze-drying.

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2.2.2 Fourier-transform infrared spectroscopy (FTIR)

The change in the functional groups after conjugation was confirmed using Fourier transform infrared spectroscopy (FT-IR). The FT-IR spectra were recorded on Thermo-Nicolet 6700FTIR/Raman spectrophotometer using the KBr pellet method. Measurements were carried out in a 4,000–400 cm-1 spectrum range for all the samples.

2.2.3 X-ray diffraction spectra (XRD) study

Physical characterization of PEC, CS, and PEC-CS bioconjugate was carried out using XPERTPro (PANalytical, Philips, The Netherlands) X-ray diffractometer (XRD). XRD 5

Journal Pre-proof spectra of all the samples were recorded at 27℃ using monochromatic Cu-Kα radiation (k =1.54056 nm).

2.2.4 Thermogravimetric study The STA 409 (NETZSCH, Germany) equipped with PROTEUS® software was used for the thermogravimetric analysis. The measurements were carried out in the temperature range of

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30–800℃ at a heating rate of 10℃/min in an argon environment. About 2.5 mg of each

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2.2.5 Differential scanning calorimetry (DSC) study

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sample was used in the study.

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Glass transition temperature (Tg) and melting temperature (Tm) of native PEC, CS and the

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synthesized bioconjugate, PEC-CS was determined by differential scanning calorimetry with METTLER DSC822e thermal analyzer and its STARe software. Samples (2.5 mg each) were

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placed on aluminum pans under nitrogen and heated to 200°C, at a rate of 10°C/min.

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2.2.6 Morphological characterization

Surface topography analysis was carried out using scanning electron microscopy (SEM) Quanta-200 (FEI®, USA). The samples were prepared on a silica plate and coated with sputtered gold and studied at 500X resolution at 12.5 kV.

2.2.7 Biocompatibility assay

The biocompatibility of PEC, CS and PEC-CS conjugate was determined using MTT assay on the HeLa cell line. The cells were seeded at a density of 2000 cells/well and cultured for 12 h in 96 well plates in DMEM supplemented with 10% fetal calf serum. The cells were 6

Journal Pre-proof then treated with CS, PEC, and PEC-CS conjugates at different concentrations. After 24 h post-treatment, 20 μL of MTT dye (5 mg/mL) was added to each well and incubated for 4 h at 37°C. About 50 μL of DMSO was added to each well and the absorbance of treated and untreated samples was read at 570 nm. Statistical analysis (ANOVA) used to calculate the p values.

3. Results and Discussion

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3.1 Synthesis of bioconjugate

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The bioconjugate of PEC and CS was synthesized by the reaction between activated carboxyl groups of PEC and amino groups of CS. The carboxyl groups of pectin were activated by

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adding a mixture of NHS/DCC underanhydrous condition. The bioconjugate was formed

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when CS added to PEC with activated carboxyl groups (NHS-PEC) replaced NHS to form

but soluble in DMSO.

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amide bonds. The synthesized conjugated material was white in color and insoluble in water

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3.2 Changes in FT-IR spectra of the conjugate

The formation of PEC-CS bioconjugate was confirmed by comparing the FT-IR spectra of the native and conjugated polymer. Characteristic infrared peaks due to the stretching vibrations

of NH, C=O (amide) and C-N of chitosan were

at

3421 cm-1,

1657 cm-1, 1600 cm-1 (Fig. 2) [19,20]. FT-IR spectra of PEC showed characteristic absorption peaks at 1745 cm-1, and 1641 cm-1 corresponding to carboxyl ester and carboxylate group respectively (Fig.2). It also exhibited characteristic bands in the fingerprint region [21]. The main changes observed in the conjugate (Fig.2) were in the region of 1750–1500 cm-1. Several additional peaks were observed at 1709 cm-1, 1641 cm-1, 1554 cm-1, 1509 cm-1,1371 7

Journal Pre-proof cm-1, 1232 cm-1, 1158 cm-1in the conjugate. The amine band at 3400 cm-1 seen in chitosan was no longer sharp, thus signifying a change in the environment of the amine nitrogen. The peaks in the region 1709 cm-1 and 1641 cm-1correspondedto weak carbonyl stretch due to Hbonding and the amide-I band of the formed conjugate. Also seen in the FT-IR spectra was a peak

belonging

to

an

amide-II

band

of

secondary

amides

at

1554 cm-1 and C~C ring stretch at 1509 cm-1. The pyranose ring structural fingerprint at

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3.3 X-ray diffraction spectra (XRD) of the conjugate

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915–630 cm-1wasobserved in all the spectra [16,21].

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XRD analysis was carried out to investigate the polymorphism of PEC and CS after

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conjugation. XRD profiles of the PEC, CS and PEC-CS conjugate are shown in Fig. 3. The characteristic sharp peak of CS was observed at 2θ of 20°. The diffraction pattern of PEC

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showed characteristics peaks at 12.6°, 14.4°, 16.9°, 18.3°, 19.6°, 22.8° and 28.13° confirming

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its crystalline structure. However, PEC-CS showed no prominent diffraction peak in the XRD analysis except for the one at 20.64°. Thus, the novel PEC-CS conjugate exhibits amorphous

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characteristics, differing from both its parent polymers.

3.4 Thermogravimetric analysis(TGA)

Thermal stability analysis of CS, PEC and its conjugate system (PEC-CS) showed the conjugate to have better thermal stability than its parent polymers. The TGA curve for CS shows three events of weight loss with an increase in temperature (Fig. 4).The first loss occurred at 50℃and continued up to 275℃ with a 3 % weight reduction due to loss of moisture. The second event of weight loss occurred between 300℃and 400℃ with 15.5 % weight loss that corresponds to the decomposition of CS. The TGA curve for PEC shows two 8

Journal Pre-proof significant weight loss events; the first one occurs at 0–350℃ with a weight loss of up to 41% and the second weight loss (13.45%) was at 350–400℃. The conjugate, however, shows significantly low weight loss till 300℃ (23.6 %) as compared to PEC. Second and third weight loss events were noted at 300–700℃ (43.09%) and at 700–800℃ (9.8 %) respectively. The above results thus show that PEC-CS conjugate has higher thermal stability

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3.5 Differential scanning calorimetric (DSC) analysis

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as compared to its native precursors.

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The comparative thermogram data of PEC, CS and PEC-CS conjugate (Fig. 5) shows changes in the endothermic peak. The thermogram shows an endothermic peak at 86℃ for

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CS indicating moisture loss; while at 76.8℃ for PEC indicating melting of the polymer and a

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mild peak at 74.2℃ for PEC-CS indicated endothermic transition due to the loss of moisture

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from the conjugate system. It is to be noted is the decrease in the intensity of the endothermic peak of PEC in the PEC-CS conjugate system, which confirms that there is a reduction in the

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rigid crystalline structure. The glass transition temperature (Tg) of PEC was 69.55℃, while that of PEC-CS was 55.13℃ (Table 1). As changes in Tg is considered to be an indicator of change in molecular structure, we can conclude that the conjugate is more amorphous. This result also confirms that the movement of molecules in PEC-CS is unhindered than in PEC.

3.6 Scanning electron microscopy (SEM) study

Scanning electro-micrographs of the synthesized PEC-CS conjugate (Fig.6) showed no evidence of the known crystalline structure of PEC, but a more amorphous structure after conjugation. This amorphous structure of PEC-CS bioconjugate observed under SEM lends support to our conclusions from the results obtained in X-ray diffraction analysis. 9

Journal Pre-proof 3.7 Biocompatibility analysis

Chitosan is approved by the USFDA for application only in wound dressings and not for use in a drug delivery system [22]. In order to ascertain the biocompatibility of the conjugate, we did MTT assay on Hela cell line treated with PEC, CS, and PEC-CS at concentrations ranging from 10-7 to 10-4g/mL. Our results showed no significant difference (p<0.05) in the cytotoxicity between the conjugate and the parent polymer (Fig. 7). Similarly, another study

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has shown that chitosan exhibits low toxicity on BHK-21 cells, if used below 0.1 % w/v [23].

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Our findings are supported by another study that showed that chitosan is relatively non-toxic at concentrations of up to 1 mg/mL when tested on L929 fibroblast cell line [24]. Also, an in

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vivo study on mice showed that 10% of dietary chitosan is non-toxic [25]. Hence, the novel

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PEC-CS conjugate material synthesized is biocompatible at the concentrations used in our

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4. Conclusion

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study.

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In conclusion, we report the successful synthesis of a PEC-CS conjugate using carbodiimide crosslinking chemistry. We activated the carboxyl groups of PEC by adding a mixture of NHS/DCC, underanhydrous conditions. FT-IR and XRD confirmed the formation of a conjugated product of PEC and CS. The DSC and TGA analyses established this conjugate as a thermostable material as compared to its native precursors. XRD analysis further showed that compared to PEC which has a crystalline structure, its conjugate with chitosan is more amorphous having a semi-crystalline structure which was further confirmed with SEM. In addition, the conjugate showed no difference in its cytotoxic property when compared with its precursors. The PEC-CS thus has the potential to be used as carriers in drug delivery system. However, further studies to ascertain its half-life, stability, mechanical strength, 10

Journal Pre-proof renewability, biodegradability, targeting ability has to be undertaken to establish it as a drug delivery system.

Disclosure statement The authors confirm that there is no conflict of interest. Funding source

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This research received no specific grant from any funding agency in the public, commercial,

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or not-for-profit sectors.

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Author contributions

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Akhilesh Singh, Anudwipa Singh, and LijunTian conceptualized the study design, performed

manuscript.

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Acknowledgement

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data curation, and analysis. Akhilesh Singh and LijunTian wrote and reviewed the

The authors are thankful to ClinBiotic Solutions for providing language editing support.

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pectin interacted with chitosan for targeted and sustained drug delivery. Colloid Surface

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Journal Pre-proof system.Chromatographica 57 (2004) 779–782. 22. T. Kean, M.Thanou. Biodegradation, biodistribution and toxicity of chitosan.Adv. Drug Del. Rev. 62 (2010) 3–11. 23. Ariani MD, Yuliati A, Adiarto T. Toxicity testing of chitosan from tiger prawn shell waste on cell culture. Dental journal (Maj.Ked.Gigi), 2009, 42(1), 15–20. 24. S. Mao, X.Shua, F. Unger, M. Simon, D. Bi, T.Kissel. The depolymerization of chitosan: effects on physicochemical and biological properties. Int. J. Pharm. 281 (2004)

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(1968) 89–94.

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Journal Pre-proof Table1: Thermal transition observed during DSC study for the pectin-chitosan conjugate

Tg (°C)

ΔCp (J/gK)

J/g

Chitosan

53.3

0.427

−64.17

Pectin

64.1

8.08

−164.96

Pectin–chitosan conjugate

54.9

0.231

−174.53

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Sample

Tian:

Methodology,

Software

and

Writing-Reviewing.

Anudwipa Singh:

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Lijun

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Author Statement

Conceptualization, Methodology, Data curation, Writing- Original draft preparation.

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Akhilesh Vikram Singh: conceptualization, Methodology, Visualization, Investigation, Data

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curation, Formal analysis, Validation, Writing- Reviewing and Editing

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7