Castor oil-derived monomer ricinoleic acid based biodegradable unsaturated polyesters

Journal Pre-proof Castor oil-derived monomer ricinoleic acid based biodegradable unsaturated polyesters P. Rajalakshmi, J. Margaret Marie, A. John Maria Xavier PII:

S0141-3910(19)30344-1

DOI:

https://doi.org/10.1016/j.polymdegradstab.2019.109016

Reference:

PDST 109016

To appear in:

Polymer Degradation and Stability

Received Date: 27 August 2019 Revised Date:

7 October 2019

Accepted Date: 22 October 2019

Please cite this article as: Rajalakshmi P, Marie JM, Maria Xavier AJ, Castor oil-derived monomer ricinoleic acid based biodegradable unsaturated polyesters, Polymer Degradation and Stability (2019), doi: https://doi.org/10.1016/j.polymdegradstab.2019.109016. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

Castor oil-derived monomer ricinoleic acid based biodegradable unsaturated polyesters P. RAJALAKSHMIa, J. MARGARET MARIEb, A. JOHN MARIA XAVIERa* a

PG& RESEARCH DEPARTMENT OF CHEMISTRY, LOYOLA COLLEGE, UNIVERSITY

OF MADRAS, CHENNAI, INDIA b

DEPARTMENT OF CHEMISTRY, WOMEN’S CHRISTIAN COLLEGE, UNIVERSITY OF

MADRAS, CHENNAI, INDIA *[email protected] KEYWORDS Castor oil, ricinoleic acid, biodegradable polymer, unsaturated polyester, drug delivery

Abstract Castor oil-based monomers and derivatives are favorable starting materials for the synthesis of polymers for biomedical applications. A series of polymers containing ricinoleic acid (extracted from cold-pressed castor oil) as a prime monomer along with glutaric acid and various linear aliphatic α, ω-diols were synthesized through self-catalyzed melt polycondensation method. The unsaturated prepolymers formed were cured under various temperature conditions to obtain cross-linked polymers. The polyesters were characterized by FTIR, 1HNMR, and

13

C NMR

spectral techniques. Their molecular weights were determined using GPC and the thermal

1

properties were studied using TGA and DSC techniques. These polyesters possess wide range of elastic modulus from 0.24 Mpa to 5.15 Mpa. The biodegradability, in vitro hydrolytic degradation and cell viability in HepG2 human liver cells display promising properties as tissue scaffolds or drug delivery vehicles.

Graphical Abstract

1

INTRODUCTION Monomers derived from biomass feedstock are always of paramount interest for polymer

researchers. Vegetable oils are the most remarkable candidate as renewable feedstock for biopolymer productions. Among the various vegetable oils, castor oil (obtained from the seeds of the plant Ricinus communis) and its derivatives are versatile starting material for the synthesis of renewable monomers and polymers. Biodegradable, environmental friendly and widely abundant nature of castor oil makes it a potent candidate for a wide array of applications in the biomedical field, as well in the preparation of elastomers and packaging materials [1, 2]. Castor oil has been

2

known for its medicinal value since ancient days. India is the leading producer of castor oil and it covers 90 % of the total production followed by China and Brazil [35]. Castor oil is distinctive among other vegetable oils as it is the main commercial source for trifunctional, unsaturated fatty acid monomer called ricinoleic acid [4]. Castor oil contains approximately 85-90 w/w of triglycerides of ricinoleic acid [5]. Ricinoleic acid could provide additional natural chemical functionality for modifications, cross-linking or polymerization [6]. The hydrophobic nature of ricinoleic acid could enhance the drug encapsulating capacity of the polymer for longer period and will act as controlled drug carrier [7]. The presence of single cis double bond can act as a potential site for further modifications such as cross-linking and grafting [8] Castor oil and ricinoleic acid has been proven to enhance the transdermal penetration of other chemicals hence correct utilization of this renewable feedstock require appropriate choice of comonomers and synthetic strategy [ 9,10] A vast array of copolymers is viable when castor oil (or ricinoleic acid) is combined with other monomers. Materials with varied properties could be obtained by tweaking the chemistry of these copolymers. Therefore, a wide range of biomaterials, from solid implants to in situ injectable hydrophobic gels are made from castor oil/ ricinoleic acid polyesters. Altering of comonomer compositions leads to polyesters with controlled mechanical, thermal, viscoelastic, and degradation profiles. Biodegradable polyesters and polyanhydrides have been modified to be more hydrophobic, cross-linked, pliable or pasty by simply copolymerization with small amounts of castor oil or/and RA or its lactone [11]. In this paper, ricinoleic acid extracted from highly pure cold-pressed castor seed oil by saponification was used for polyester synthesis using simple catalyst and solvent free melt polycondensation method. The chosen comonomers (glutaric acid and linear aliphatic α, ω-diols)

3

are safe for use in biomaterial applications and are used in the synthesis of ricinoleic acid polymers for the first time [12]. The unsaturated polyesters with varying mechanical and degradation profiles have been synthesized. These polymers have tremendous scope for further functionalization and fabrications.

2

EXPERIMENTAL SECTION

2.1.

Materials High purity glutaric acid, 1,2-ethanediol, 1,3-propanediol, 1,4-butanediol, 1,5-

pentanediol, 1,6- hexanediol, 1,8-occtanediol, 1,10-decanediol, 1,12-dodecanediol were all purchased from Sigma Aldrich and Spectrochem Chemicals. High purity castor oil was obtained from traditional cold pressed (chekku) oil extraction store.

2.2.

Monomer ricinoleic acid extraction from Castor oil

Ricinoleic acid was extracted from chekku castor oil by saponification reaction followed by acidification reaction [13]. Potassium hydroxide pellets (24 g) were accurately weighed and transferred in to a 500 mL round bottomed flask. An amount of 150 mL of rectified sprit was added followed by the addition of 200 mL of castor oil. Then the round bottomed flask was set up to the Liebig condenser and was kept on magnetic stirrer coupled with heating. The temperature was checked regularly so that it does not exceed 100 °C. The whole set up was run for almost 10 hours with periodical checking.

On completion of the reaction, the entire mixture was transferred into a beaker containing ice cubes. Acidification was done by adding 30 mL of 2M HCl and was kept for 30 min under

4

mechanical stirring. The fatty acid was extracted from the acidified solution using diethyl ether as solvent. The organic phase was washed three times with hot distilled water in order to remove the inorganic impurities and glycerol. The organic phase was collected and on evaporation of solvent a dense light yellowish liquid of ricinoleic acid obtained whose structure was confirmed by IR [14] and NMR [15] spectroscopy.

2.3.

Representative synthesis of Poly (diol-co-ricinoleate-co-glutarate) prepolymer and cured polyesters Ricinoleic acid (R) and glutaric acid (G) were reacted with various aliphatic diols of

different chain length. In the first step, a calculated quantity of glutaric acid was taken in a three necked round bottom flask and heated to 120 °C. The molar stoichiometric ratios of monomers taken for all polymers are presented in Table 1. The reactivity ratio R (OH/COOH) was maintained at closer to one. The reaction was carried out at 150 °C in a thermostatic oil bath for 12-16 hours under inert atmosphere. On the completion of the reaction time, the highly viscous prepolymer was dissolved in 1, 4-dioxane, the viscous polymer was transferred into a Teflon petri dish, without further purification, for stepwise curing to complete the cross-linking process. The mild curing conditions were maintained at 90 °C for 24 h, and further at 110 °C for 24 h. The tougher curing was done by gradually increasing the temperature from 110 °C to 130 °C for 48 h and finally at 150 °C for 24 hours [16].

Table 1 Monomer ratios and R values for different ricinoleic acid based polyesters containing glutaric acid as another common monomer and varying diols Polymer

Monomer moles

Concentration

in Total moles of functional group

R ratio = OH /

5

COOH

Diol

Ricinoleic acid (R)

Glutaric acid (G)

Diols

OH

COOH

RGE

Ethanediol

0.05

0.05

0.05

0.15

0.15

1

RGP

Propanediol

0.05

0.05

0.05

0.15

0.15

1

RGB

Butanediol

0.05

0.05

0.05

0.15

0.15

1

RGPe

Pentanediol

0.05

0.05

0.05

0.15

0.15

1

RGH

Hexanediol

0.05

0.05

0.05

0.15

0.15

1

RGO

Octanediol

0.035

0.035

0.035

0.105

0.105

1

RGD

Decanediol

0.035

0.035

0.035

0.105

0.105

1

RGDd

Dodecanediol

0.035

0.035

0.035

0.105

0.105

1

2.3.

FTIR spectroscopy Infrared spectra of ricinoleic acid monomer and various synthesized prepolymers were

recorded on a Bruker-Tensor II FT-IR spectrophotometer. The spectrum of cured polymers was recorded using Attenuated total reflectance mode (ATR-IR) in a spectrophotometer. The structures of the polymers were confirmed by scanning in the range of 4000-400 cm-1. 2.4.

NMR spectroscopy The 1H and 13C NMR for the synthesized monomer Ricinoleic acid and prepolymers were

recorded at room temperature in a Bruker Ascend 400 MHz NMR spectrometer. The samples were all dissolved in deuterated chloroform and the chemical shifts in parts per million (ppm) for the 1H NMR were taken using Tetramethylsilane as an internal reference. 2.5.

Gel permeation chromatography (GPC) The weight average and number average molecular weight of the synthesized

prepolymers were determined using Shimadzu’s prominence gel permeation chromatography system equipped with a LC -20 AD pump and RID-10A refractive index detector. A CTO-20A

6

column oven was maintained to 40 °C. A PLgel 5 µm mixed-D column from polymer labs in tetrahydrofuran at a flow rate of 1 mL/min was used. Linear poly (styrene) standards with a narrow polydispersity index were used for calibration. (Agilent technologies, United States), Shimadzu 2.6.

LC

Solution

software

was

used

to

collect

and

analyze

the

data.

Physical characteristics Prepolymers were tested for their solubility in various solvents. In a 25 mL vial, 0.5 g of

prepolymer and 5 mL of each solvent were taken and stirred well at room temperature. The inherent viscosities of prepolymers were determined in 1, 4-dioxane solvent at concentration of 0.5g/dL at 30 °C using Ubbelohde glass capillary viscometer. The measurements were repeated for n=3 times and reproducible results were obtained. 2.7.

Thermal studies The thermal properties of the cured polyesters were determined using DSC and TGA

techniques under nitrogen atmosphere (from 30 °C to 800 °C for TGA and -50 °C to 250 °C for DSC) with a temperature gradient of 10 °C min-1 and scans were recorded using a TGA Q 50 (V20.6 Build 31). 2.8.

Sol content and degree of swelling measurements Percentage of swelling was calculated by placing a known dimension (discs of

dimensions 10 mm × 10 mm) and weight (W1) of cured RG series of samples (n=3 per macromer in solvent) in 20 mL of hydrophilic solvent DMF. This solvent was chosen because of its ability to swell the polymeric membranes and not to dissolve the polymers at room temperature. The samples were allowed to swell for 24 hours at room temperature. After 24 hours, samples were taken out, wiped using filter papers and weighed (W2). The samples were dried again for 24

7

hours to constant weight and their weights were noted (W3). The percentage of swelling and sol content were calculated using the following formulas. Percentage of swelling =

× 100

Sol content in percentage = 2.9.

× 100

Mechanical Properties Tensile mechanical testing was conducted using Hounsfield model H50KS universal

testing machine (UTM) with QMAT software. The tensile tester was equipped with 10 KN load cell. Dog-bone shaped polymer films (n=3 per macromer) were cut from the cross-linked membranes. The samples were pulled at a speed of 5mm/min and elongated to failure at room temperature. From the slope of stress-strain curve, Young’s modulus E was calculated. Crosslinking density was calculated using the equation

= E 3RT R represents the universal gas

constant (8.314 J mol-1 K-1) and T denotes the absolute temperature in Kelvin (300 K). 2.10.

Contact angle measurements The contact angles of Ricinoleic acid based polymeric membranes were measured in a

goniometer via the standard sessile drop technique with the measurement range of 0-180 °C. The measurements were done using the contact angle instrument model HO-IAD-CAM-01. The deionized water droplets were carefully placed on the surface of membrane using a tiny needle syringe at five different locations. (n=5 times per macromer) and observed through a microscope. The contact angles of the polyesters were determined by measuring the angle made with tangent using a calibrated angular scale. The average and standard deviation was calculated and tabulated for each macromer in RG series. 2.11.

Hydrolytic Degradation of polymers

8

The cross-linked polymeric membranes were cut into small pieces of 1.5 cm × 1.5 cm area and their initial weights were noted as W1. These cut pieces of polymeric membranes were transferred into four different 100 mL beakers containing 30 mL of distilled water, 30mL of 2% NaOH, 5% NaOH and 7% NaOH solutions respectively. At denoted time points, the samples were taken out, wiped with filter paper and moved to hot air oven at 50 °C for drying until constant weight was obtained. The weight was noted as W2. The percentage of weight loss was calculated using the formula Percentage of weight loss = 2.12.



× 100

In Vitro Degradation Studies The cured polymer membranes were cut in to pieces of 15 mm length, 10 mm in width

and 1mm of thickness. These samples were weighed (W1) and immersed in 40 mL of 0.1 M phosphate buffered saline solution adjusted to pH of 7.4. The PBS solution was periodically changed with fresh buffer solution. This was done to avoid the saturation of PBS solution. At each evaluation point, the samples were taken out, rinsed in double distilled water and dried at 50 °C until a constant weight was obtained.(W2).The hydrolysis of polymers were measured by weight loss and percentage of degradation was calculated using the formula Percentage of degradation =

× 100

Where W1 is the weight of polymer before degradation and W2 is the weight of polymer after degradation at specified time t. 2.13.

In Vitro Biocompatibility studies The effect of synthesized prepolymers on the viability of Human HepG2 cells was

analyzed by MTT assay.

9

Cell culture: HepG2 cells derived from human liver (NCCS Pune, India) were maintained in DMEM containing 10% heat inactivated fetal bovine serum albumin (FBS), 100 IU/mL penicillin, 0.1 µg/mL streptomycin, and non-essential amino acid in humidified atmosphere of 5% CO2 at 37 °C. The cells were passaged by trypsinization (0.25% (w/v) trypsin in 0.53m M EDTA) and they were used for the analysis. All the analysis was done in a clean atmosphere. MTT assay: The cell cytotoxicity was determined by MTT assay (Borenfreund et al., 1988). Cells were harvested at the exponential growth phase, seeded into 96-well plates at 3 × 104 cells/well in 100 µl, and incubated for 24 h. Test cells were with synthesized polymer compounds at selected concentrations (n=3 per dilution). These solutions were prepared with serum-free DMEM containing 0.1% DMSO (v/v). After 48 h, 50 µl of MTT (5 mg/mL) was added to the medium in each well, and the plates were incubated at 37 °C for 3 h. The formazan product was removed and added with 100 µl/well of DMSO. The MTT formazan absorbance in each well was measured at 492 nm using a 96-well plate reader. The percentage viability of synthesized prepolymers of RG series was calculated using the following formula: Cell viability in percentage =

3.

(

) – (

( )

!)

× 100

RESULTS AND DISCUSSIONS The synthesis of cross-linked unsaturated polyesters was accomplished by solvent and

catalyst- free melt condensation technique. This synthetic route was adopted to evade the toxicity produced by catalysts and solvents so as to enhance their application in biomedical field. The reaction temperature was maintained at 150 °C for 16-24 hours. The presence of secondary hydroxyl group in ricinoleic acid and notable steric hindrance due to lateral aliphatic chain of ricinoleic contributed to the prolonged time requirement for the synthesis. A schematic

10

representation of polymer synthesis and plausible structures of all polyesters are given in scheme 1. Scheme 1. Schematic reaction for polymer synthesis O

O OH +

HO

HO

R

OH

linear aliphatic α,ω diols

Glutaric Acid

O R

+ HO

O

O

O O

R

O

O R

Synthesised Polyester OH Ricinoleic Acid

O

R = (C2,C3,C4,C5,C6,C8,C10 & C12)

All the synthesized prepolymers were highly viscous, transparent, and faint yellow to dark brown in color. The prepolymers were tested for their miscibility in wide range of organic solvents at room temperature. Their solubility parameter was observed and results were tabulated in Table 2. All prepolymers show good solubility in various solvents such as toluene, dichloromethane, chloroform, tetrahydrofuran, 1, 4-dioxane, etc. The solubility of prepolymers increases from non-polar to mid polar solvents. The polymers were partially soluble in methanol and toluene. They were all found to be insoluble in polar solvent such as water. The polymeric films were obtained by solubilizing the prepolymers in a known quantity of 1,4-dioxane and left in vacuum oven on Teflon petri dish for step wise curing at 5 mm Hg vacuum for prescribed time. The cured polymeric films were insoluble in all set of solvents denoting that these polymers were all cross-linked [17]. Homopolymerisation of ricinoleic acid resulted in highly viscous yellow liquid which when subjected to curing conditions remained as a

11

sticky, light brown liquid and hence was not studied further. The inclusion of glutaric acid and diols resulted in completely cured, non-sticky stable polymeric membranes without the use of any plasticizers or additives.

Table 2. Miscibility test of synthesized prepolymers Polymers Tested Solvent Used RGE RGP RGB RGPe RGH RGO

RGD

RGDd

n-hexane

+

+

+

+

+

+

+

+

Toluene

+++

+++

+++

+++

+++

+++

+++

+++

Diethyl-ether

+++

+++

+++

+++

+++

+++

+++

+++

Dichloro methane

+++

+++

+++

+++

+++

+++

+++

+++

THF

+++

+++

+++

+++

+++

+++

+++

+++

Chloroform

+++

+++

+++

+++

+++

+++

+++

+++

Ethyl acetate

+++

+++

+++

+++

+++

+++

+++

+++

1,4-Dioxane

+++

+++

+++

+++

+++

+++

+++

+++

Methanol

++

++

++

+

++

++

++

++

Acetone

+++

+++

+++

+++

+++

+++

+++

+++

Acetonitrile

+++

+++

+++

++

++

++

++

++

Acetic acid

+++

+++

+++

+++

+++

+++

+++

+++

DMF

+++

+++

+++

+++

+++

+++

+++

+++

DMSO

+++

+++

+++

++

+++

++

+++

+++

Distilled water

---

---

---

---

---

---

---

---

12

+++ denotes highly soluble; ++ denotes partially soluble; + denotes least soluble; --- denotes insoluble.

The inherent viscosities of the prepolymers were determined in 1, 4- dioxane solvent using Ubbelohde glass capillary viscometer in a constant temperature bath (Table 3). All the polyesters gave satisfactory inherent viscosity ranging from 0.02 to 0.22 dL/g. The value increased with increase in molecular weight of polymers for short chain diols i.e., from1, 2-ethanediol to 1,6hexanediol (n = 2, 3, 4, 5, 6) whereas with higher members of diols such as 1,8-octanediol, 1,10decanediol and 1,12-dodecanediol the values were much lower. This tendency upturned due to steric hindrance raised by an increase in length of aliphatic diols.

Table 3. Inherent viscosity measurement of synthesized polymers:

3.1.

Polymer code

Inherent viscosity in dL/g

RGE

0.0235

RGP

0.1310

RGB

0.1287

RGPe

0.1419

RGH

0.2271

RGO

0.0804

RGD

0.0775

RGDd

0.0977

Fourier Transform-Infrared Spectroscopy (FT-IR)

13

For all the synthesized monomer, prepolymers and cured polymers, FT-IR and ATR-IR spectra were recorded. The synthesis of ricinoleic acid from cold-pressed castor oil was confirmed by the shift of vibrational frequency of ester carbonyl group from 1744 cm-1 to acid carbonyl group at 1711cm-1 and by disappearance of C-O stretching at 1166 cm-1 (Figure 1a and 1b). All the prepolymers and cured polymers with different chain lengths possess almost same set of characteristic peaks and had given positive characteristic peaks for the formed product. It has been shown in Figure 1c and 1d. For the prepolymer, broad band at 3460-3440 cm-1 appeared due to hydroxyl stretching of free OH group. A set of bands at around 2930 cm-1 and 2855 cm-1 were obtained because of methylene group C-H stretching vibrations. The absorption band at 1730-1725 cm-1 confirmed the carbonyl group of the formed polyester. The peaks at 1180-1172 cm-1 and peaks at 1082-1022 cm-1 denotes the C-O stretching vibrations of ester group with primary and secondary alcoholic OH respectively. In case of cured polyesters, the broad band at 3460-3440 cm-1 due to unreacted -OH was completely absent. It confirms the completion of curing reaction. All other characteristic peaks continued to be present in the cured polyester [18].

1a

1b

14

1c

1d

Figure 1. a) FTIR of castor oil b) FTIR of ricinoleic acid; c) FTIR of ricinoleic acid based prepolymers; d) ATR-IR of cured polymers. 3.2.

Nuclear magnetic resonance spectra The figure 2a and 2b confirms the synthesis of ricinoleic acid from castor oil. In the 1H

NMR spectra of synthesized ricinoleic acid, the peak at 0.87 ppm shows the presence of terminal

15

methyl protons. Methylene protons in the center part of ricinoleic acid appear as a high intense peak at 1.3 ppm. The methylene proton attached to free OH group appears at 3.6 ppm. The pre terminal methylene protons attached to functional groups shows peak at 1.6 ppm. The terminal protons bonded with carbonyl group appear at 2.26 ppm. In

13

C NMR spectra the solvent peak

appears at 77 ppm. The peak at 14.09 ppm corresponds to terminal methyl carbon. The methylene carbon attached to free OH had shown peak at 71 ppm. The acid carbonyl group was confirmed at 179 ppm. The double bonded carbon atoms had given peak at 125 and 133 ppm respectively. The methylene carbon next to functional group attached carbon appears in the range of 31-36 ppm. The methylene carbons in center of molecule could be seen in the range of 25-30 ppm. All the above mentioned peaks confirmed the structure of ricinoleic acid. The Figure 2c and 2d represents the NMR spectra of ricinoleic acid based prepolymer. The incorporation of all monomers in product has been confirmed from their characteristic peaks. Some peaks were overlapped, some peaks were common to a particular functional group in different components and some had shown slightly different chemical shift. As a consequence, monomer ratio was also not obtainable quantitatively [19, 20]. The solvent peak appears at 7.3 ppm for CDCl3. The terminal methyl protons of ricinoleic acid appear at 0.86 ppm. The unsaturated alkenyl protons in ricinoleic acid appeared in polymer at 5.35 ppm and 5.5 ppm respectively. The central methylene group of ricinoleic acid backbone with 16 protons appears at 1.26 ppm [21]. Owing to the poor reactivity nature of secondary OH group of ricinoleic acid, in some prepolymers, partially unreacted OH peak appears at 1.44 ppm. Correspondingly the methylene group attached to free OH appears at 3.6 ppm. If the secondary OH is involved in polyester formation, no free OH peak appears and methylene proton attached to esterified OH appears at 4.8 ppm [22]. The most deprotected methylene group of α, ω-diols due to their

16

location next to oxygen atom of ester bond appears at 4.06 ppm. No characteristic primary OH peak of free α, ω-diol appears at 2.6 ppm. The pre terminal methylene group of diol and glutaric acid appears at 1.66 ppm and 1.9 ppm respectively. The terminal methylene groups attached to carbonyl group of ester molecules appear at 2.4 ppm. The figure 2e denotes the

13

C NMR spectra of synthesized prepolymers. It further

verifies the structure of synthesized polyesters. The carbonyl carbon of polyester molecule had given peak at 173 ppm. The peaks at 133 and 124 ppm were attributed due to alkenyl carbon. The peak at 64 ppm confirmed the ester linkage in molecules. It was given by the carbon attached to oxygen atom in( -C-O-C-).The peaks raised at 71 ppm and 74 ppm attributed to the structure of carbon attached to free OH and OR respectively. This confirms the incompletion of reaction in prepolymers. Solvent peak was shown at 77 ppm. All the spectra matched with the proposed chemical structure of monomer and polymers. The individual proton and carbon peaks were labeled in each spectrum. The spectral studies confirmed the success of synthesis of monomers and prepolymers. Owing to less reactive nature of secondary alcoholic group in ricinoleic acid, some polymers had given peak attributed to OH. This is also in agreement with FT-IR spectra. On curing the free OH group was involved in cross-linking and no characteristic OH peak was obtained in ATR-IR. Both 1H and

13

C spectra confirmed the presence of double bond in prepolymers. The

presence of free OH group and double bond in synthesized polyesters could lead to further functionalization of these functional groups.

17

2a

2b

18

2c

2d

19

2e

Figure. 2a) 1H NMR of Ricinoleic acid. 2b) 13C NMR of Ricinoleic acid. 2c & d) 1H NMR of Ricinoleic acid based prepolymers. 2e) 13C NMR of Ricinoleic acid based prepolymers. 3.3.

GPC analysis The molecular weight of the prepolymers was determined using GPC (Table 4). In case

of prepolymers with short chain diols from RGE to RGH, the molecular weight increases with increase in number of methylene group in diol unit. In case of the number average molecular weight and weight average molecular weights range in 4543-2446 g/mol and 16525-3789 g/mol respectively. The molar mass dispersity index ranges in 3.63-1.55. From this wider range of dispersity index it could be concluded that the polymers formed in random manner and could show their influence on many properties of polymeric materials [23]. As the cured polymers

20

were found to be insoluble in various solvents, the molecular weight of cured polymeric films was not determined.

Table 4 GPC Results of Ricinoleic acid based prepolymers Prepolymer

Number average Weight average Molar mass Degree of molecular weight molecular weight dispersity index polymerization (Mn) (g/mol) (Mw) (g/mol) (Mw / Mn ) for prepolymer

RGE

3042

6111

2.01

6.6

RGP

3402

7350

2.16

7.2

RGB

3978

9080

2.28

8.2

RGPe

4301

14851

3.45

8.6

RGH

4543

16525

3.63

8.9

RGO

3172

5173

1.63

5.8

RGD

2582

4533

1.76

4.5

RGDd

2446

3789

1.55

4.1

3.4.

Mechanical properties Tensile strength, young’s modulus and cross-link density results of all the cured

polymeric membranes (n=3 per sample) were calculated and tabulated in Table 5. The influence of chain length of various aliphatic diols on RG series of polyesters was evaluated.

Table 5 Mechanical properties of ricinoleic acid based cured polymers Polymer Tensile Strength

Strain to failure in %

Young’s Modulus E

σ in Mpa in Mpa

Cross-link Density ρᵡ = E / 3RT ( moles m-3 )

21

RGE

0.058 ± 0.02

12.60 ± 0.52

0.576 ± 0.114

77 ± 15.16

RGP

0.067 ± 0.01

26.2 ± 0.43

0.244 ± 0.063

33 ± 8.44

RGB

0.060 ± 0.01

2.77 ± 0.24

2.288 ± 0.253

306 ± 33.75

RGPe

0.058 ± 0.009

4.25 ± 0.31

1.573 ± 0.122

210 ± 16.28

RGH

0.063 ± 0.003

1.22 ± 0.08

5.151 ± 0.246

688 ± 32.89

RGO

0.071 ± 0.01

5.72 ± 0.24

1.201 ± 0.058

160 ± 7.68

RGD

0.063 ± 0.006

2.24 ± 0.11

2.842 ± 0.167

377 ± 22.36

RGDd

0.180 ± 0.020

24.09 ± 4.26

2.075 ± 0.315

277 ± 42.13

In general there is a direct relationship between molecular weight of the diol and mechanical strength of the polymer.

As the chain length and molecular weight of 1, 12-

dodecanediol is highest in case of polymer RGDd, it has maximum tensile strength of 0.18 Mpa. Among the series, RGH has maximum young’s modulus with high cross-link density. As a consequence of very high cross-link density, strain at break is very low showing its brittle nature. Polyester with 1, 3-propanediol is found to be softest material with low modulus and cross-link density. Elongation increases with decrease in cross-link density. In case of above set of polymers there is no much difference in the mechanical strength from RGE to RGD. The values are in the range of 0.058- 0.071 Mpa. These polyesters possess wide range of elastic modulus from 0.24 Mpa to 5.15 Mpa. In addition to molecular weight, large numbers of structural and external factors also control the mechanical property of the polymers [24]. Various studies on mechanical properties of polyesters had concluded that mechanical behavior of a polymer depends heavily on reaction condition, reaction time, temperature, curing time, curing condition, chain orientation, and monomer ratio. By tuning these parameters, the RG series of polyester with particular cross-link density and elastic modulus will surely find application in tissue

22

engineering scaffolds for different body parts which differ over a varied range of elastic modulus [25]. The stress vs strain curve of RG series of cured polyesters are presented in Figure 3.

RGP

0.05 0 0

5

10

15

Stress in Mpa

Stress in Mpa

RGE 0.07 0.05 0.03 0

Strain in %

0 1

2

3

Strain in %

Stress in Mpa

Stress in Mpa

0.05

0.1 0.05 0 0

4

6

RGO

0.1 0.05 0 1

Strain in %

1.5

Stress in Mpa

Stress in Mpa

2 Strain in %

RGH

0.5

30

RGPe

0.1

0

20

Strain in %

RGB

0

10

0.1 0.05 0 0

2

4

6

Strain in %

23

RGDd

0.08

Stress in Mpa

Stress in Mpa

RGD 0.06 0.04 0.02 0 0

1

2

Strain in %

3

0.3 0.2 0.1 0 0

10

20

30

40

Strain in %

Fig. 3. Represents stress - strain curve of cured ricinoleic acid based polymers 3.5.

Contact angle measurements The contact angle of polymeric membranes (n=5 replicates per sample) of known

dimension was measured using sessile method. The average and standard deviation of measured values are tabulated in Table 6. Study on surface property of cured polymer is essential to find their ability of interaction with the biological molecules. The wettability behaviour of cured polyesters was determined using contact angle measurements. In general except 1, 2-ethanediol, all other α, ω-diols are considered as hydrophobic structure maker molecules [26]. In addition to that the hydrophobic nature of vegetable oil based monomer ricinoleic acid also reduces the hydrophilic character of polyesters and made them to possess mid-values required for their cell adhesion and drug carrying activity. Among the synthesized ricinoleic acid based polyesters, except RGE, in all other polymers the contact angle increases with increase in diol length. Their contact angles were shown in the range of 66° to 82°. In general the contact angle less than 90° means wetting of surface area is favorable [27]. As the number of methylene groups in the monomeric diol units increases, their hydrophilic nature decreases. The hydrophobic nature of RGE is shown by its contact angle measurement as 98°. Owing to hydrophilic nature of 1, 2ethanediol it was expected that, with 1, 2-ethanediol content the polyester would offer more

24

hydrophilic OH group to the polymeric surface and thereby decrease the contact angle. Remarkably the polyester surface showed highest contact angle. This suggests that in RGE owing to small length of ethane diol, almost all the free primary hydroxyl groups might completely involve in polyester formation. Cross-linking also plays an important role in determining hydrophobicity. Compare to its next member RGP, it has high cross-link density. These combined factors lead to rise in contact angle. On the whole, these polymers are sufficiently hydrophilic in nature. This moderate hydrophilic aspect of membranes will surely favors the attachment, growth and spread of cells in tissue engineering scaffolds. Table 6 Contact angle of RG series cured polymers

3.6.

Polymer

Contact angle ( ° )

RGE

98.1 ± 0.61

RGP

66.84 ± 0.66

RGB

67.77 ± 1.10

RGPe

68.65 ± 0.66

RGH

75.28 ± 1.14

RGO

75.91 ± 0.96

RGD

82.02 ± 0.63

RGDd

82.97 ± 0.63

Thermal Studies The thermal stability of cured RG series of polyesters was studied by TGA and DSC. The

Td and Tg data of different polymers are listed in Table 7. All the cured polyesters have a good thermal stability under 300 °C. They revealed two decomposition stages that lead to the complete degradation beyond 480 °C.

25

Table 7 Thermal Analysis of Ricinoleic acid based cured polymers Polymer

Td5 °C

Td30 °C

Ts °C

Tg °C

RGE

279.22

374.99

164.98

116

RGP

330.95

387.28

178.73

72

RGB

292.80

365.8

164.93

102

RGPe

275.7

364.06

161.07

91

RGH

306.84

371.31

169.30

125

RGO

313.26

372.86

171.02

5.6

RGD

229.02

395.63

161.20

106

RGDd

316.26

382.56

174.46

16

The Td5% degradation percentage ranges between 229 °C to 330 °C. To further specify the thermal stability of polymeric membranes, the statistical heat resistance index Ts was calculated using Td5% (5% decomposition temperature) and Td30% (30 % decomposition temperature) values obtained from TGA thermogram [28,29]. Ts = 0.49 [Td5% + 0.6 (Td30% − Td5%)]. The statistical heat resistance values ranges between 161-178 °C. The char temperature of thermosetting Ricinoleic acid based polyesters starts at 450 °C. The maximum weight loss of 9598 % occurs at this temperature. Overall thermal stability of polyesters is not much affected by diol length. The thermograms of the polyester are presented in Figure 4.

26

Fig. 4. TGA -Thermogram of Ricinoleic acid based cured polymers In DSC thermogram (figure in supporting document) at -50 °C to 250 °C of cured polyesters crystalline temperature and melting point were not found. It clearly indicates that polymers were cross-linked as the linear polymer would show melting transition. On curing, the network of uncross-linked loose chains disappeared. Cross-linking prevented the chain rearrangement and resulted in the formation of amorphous polyesters. The glass transition temperature (Tg) of polymers was taken from the midpoint of transition. Tg value of RG series spread over a range of 125 °C and 5 °C. In general for cross-linked polymers, the Tg depends on its average composition along with its chain length, molecular weight and degree of cross-linking [30]. Cross-linking restrict the mobility of the molecules by that means they increase the Tg. For the polyesters with short chain diols, (n=2, 3, 4, 5, 6), Tg increased with increase in cross-link density. In case of polyesters with long chain diols(n=8,10,12), with increase in number of carbon atom in diol unit, the chain flexibility increased and correspondingly Tg value decreased

27

[31]. The polymers RGO and RGDd possessed Tg less than physiological temperature thus making them suitable for biological application. 3.7.

Swelling Studies Swelling percentage of polymer network in DMF (n = 3) are tabulated in Table 8.

Percentage of swelling symbolizes the change of dimension in polyester and represents the affinity and exchange of enthalpy between two phases. The swelling behaviour of polymer depends on hydrophilic/hydrophobic nature of the polymer, solvent and cross-link density.

A

highly cross-linked polymer delivers less degree of swelling [32]. Among the synthesised set of polyesters, RGH had shown very less percentage of swelling 33.75 ± 6.51 with highest cross-link density value. As mentioned earlier in contact angle measurements, except RGE, the hydrophilic nature of polymer decreases with increase in diol length. As a consequence of this, swelling percentage was shown to be less than 70% in case of RGD and RGDd. The swelling percentage of rest of the molecules also varies with respect to their hydrophilic nature and cross-link density. Table 8. Swelling percentage of RG Series cured polymers Polymer

Swelling % in DMF

RGE

94.56 ± 7.42

RGP

83.34 ± 1.66

RGB

86.00 ± 3.30

RGPe

83.78 ± 3.30

RGH

33.75 ± 6.51

RGO

94.06 ± 3.79

RGD

67.60 ± 0.58

28

RGDd

3.8.

62.16 ± 1.66

Sol Content In general any set of cross-linked polymers may possess some fraction of free polymer

that may not attach to the network (Table 9). During swelling this sol fraction of macromeres diffuse out of the membrane to the solvent bath. In order to find application of polymers in biomedical field, the study of diffusion of loose macromolecules in swollen state plays an important role. Table 9 Sol content percentage of RG Series cured polymers

3.9.

Polymer

Sol content in percentage

RGE

65.40 ± 2.77

RGP

28.00 ± 0.30

RGB

31.92 ± 6.78

RGPe

29.60 ± 0.93

RGH

62.97 ± 10.41

RGO

80.90 ± 0.32

RGD

24.86 ± 1.83

RGDd

26.33 ± 3.33

In Vitro degradation Studies in Phosphate Buffer Solution: In vitro degradation of ricinoleic acid based

polyesters in phosphate buffer solution was studied in a simulated physiological condition for 175 days. The percentage weight loss of the polymeric membranes was calculated in regular

29

interval of time (Figure 5). In an aqueous medium the polymers starts its degradation through hydrolysis reaction. Initially the water molecules penetrate inside the matrix and carryout hydrolytic cleavage of ester bonds. The initial degradation products enhance the rate of further degradation by auto catalytic reaction. Polymers with long chain diols were found to be more stable than polymer with short chain diols. Cross-linked polymers formed from short chain α, ω diols (n=2, 3, 4, 5, 6) were all completely degraded in a day of time. The polymers from long chain diols (n=8, 10, 12) were found to be stable and undergone degradation in a slow rate. The hydrophobic nature and steric effect caused by voluminous alkyl group of polymeric membranes with long chain linear aliphatic α, ω diols (n=8, 10, 12) hinders the entry of water molecules inside the matrixes and by this way decreases the rate of hydrolysis reaction. Among the three long chain diol polymers, RGO with lowest cross-link density showed the highest weight loss percentage of 86% and on the other hand RGD with highest cross-link density had shown lowest weight loss of 51%. The increase in cross-link density increases the hydrophobic nature of polymeric membrane. This hydrophobic behavior decreases the degradation rate of polymer in hydrophilic medium. The weight losses of macromers are inversely proportional to their cross-

Weight Loss in %

link density.

100 90 80 70 60 50 40 30 20 10 0

86 76 51 175th Day

RGO

RGD

RGDd

30

Fig. 5. in vitro degradation study on Ricinoleic acid based cured polymers 3.10.

Hydrolytic Degradation Studies Among the various degradation studies, hydrolytic degradation study of polymeric

membranes is significant while considering their application in tissue engineering and drug delivery systems. The study was done in distilled water over a period of 160 days and in alkaline solution (2% NaOH, 5% NaOH, and 7% NaOH solutions) of different concentration at room temperature. Figure 6 represents the change of weight of polymeric membranes in distilled water. It represents the overall weight percentage change of 2% to 16% for the cured polymeric membranes. The less change in weight percentage indicates that penetration of pure distilled water inside the membranes is difficult and slow. In case of phosphate buffer, the presence of salt act as driving force for their penetration through polymeric matrixes. 120 160th Day

Change in Weight %

100 80 60 40 20 0 RGE RGP RGB RGPe RGH RGO RGD RGDd RGE Series

Fig. 6. Hydrolytic degradation study of Ricinoleic acid based polymer in distilled water

31

Figure 7 denotes the degradation study of ricinoleic acid based polymeric membranes in alkaline solution. In NaOH solution, the stable carboxylate anion formed acts as a driving force and boost the degradation rate. By increasing the strength of NaOH, pH value increases and degradation occurs in a rapid manner. The polymeric membranes formed from short chain linear aliphatic α, ω-diols (n=2, 3, 4, 5, 6) at all concentrations of NaOH (2%, 5% & 7%) undergone complete degradation in 24 h. The polymers with long chain diols took 4 days of time for their complete degradation. The degradation follows two step mechanisms. In the primary step, the uptake of water by polymeric membranes occurs and in the next step the cleavage of ester linkage followed by diffusion of low molecular weight products out of the membrane takes place. . 100 90

Weight Loss in %

80 70 60 50 RGO

40

RGD

30

RGDd

20 10

2% NaOH

5% NaOH

72Hours

48Hours

24Hours

72Hours

48Hours

24Hours

72Hours

48Hours

24Hours

0

7% NaOH

Fig. 7. Alkaline degradation study on Ricinoleic acid based cured polymers with long chain α,ω diols ( n = 8,10,12 )

32

3.11.

Cell viability assay Cell viability of ricinoleic acid based prepolymers was quantitatively determined by

colorimetric MTT assay. It is a rapid and primary method used to estimate the cytotoxic effect of polymers. The water soluble MTT reduced to blue formazan dye due to oxidoreductase of the viable cells was measured. The ricinoleic acid based polyesters had no effect on the viability of Human HepG2 cells even at the concentration maximum. (n=5 different concentration). The MTT assays were done at triplicate measurements. All ricinoleic acid based polyesters were observed to be more compatible with cells. The cells were healthy and maintained their viability at various increasing concentration. Cisplatin was fixed as positive control. The nonlinearity of toxicity-dose curve response in Figure 8 necessitates selection of mid-point cytotoxicity concentration IC50 [33]. The non-toxic effects of synthesised polyesters are shown by their IC50 values in Table 10. Table 10. Cell viability assay of Ricinoleic acid based polymers Test items

R2

IC50 (µg/mL)

RGE

0.5588

169.9114

RGP

0.7042

157.2936

RGB

0.4024

144.3886

RGPe

0.7485

121.16583

RGH

0.2994

225.2903

RGO

0.5940

169.86658

RGD

0.9897

155.56584

RGDd

0.6856

170.08829

33

Fig. 8. Cell viability graph of Ricinoleic acid based polymers

4.

CONCLUSIONS Biodegradable polyesters from plant oil-based starting material via simple synthetic

strategy were synthesized. The present work includes the design, synthesis, physico-chemical and in vitro biological characterization of castor oil-based polyesters. The monomer, ricinoleic acid was derived from the low cost and readily available raw material cold pressed castor oil which is a renewable resource. Glutaric acid and α, ω-diols as other comonomers were used in the synthesis of prepolymers which were fabricated into thin films. The synthesised polyesters showed wide range of tuneable mechanical properties. It has been reported earlier that ricinoleic acid could enhance the transdermal penetration of other chemicals and so the elastomeric films could be suitable materials as wound dressing materials, drug delivery vehicles or tissue engineering scaffolds. The presence of unsaturation and free functional groups in the cured polymers make them viable materials for further functionalization and modifications. Further,

34

the medicinal property of ricinoleic acid could supplement the application of this material as tissue scaffolds or drug delivery vehicles. Acknowledgements This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: