Bifunctional Polysulfone-Chitosan Composite Hollow Fiber

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Bifunctional polysulfone-chitosan composite hollow fiber membrane for bioartificial liver Rohit S Teotia, Dhrubajyoti Kalita, Atul K Singh, Surendra K Verma, Sachin S Kadam, and Jayesh Bellare ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ab500061j • Publication Date (Web): 20 Apr 2015 Downloaded from http://pubs.acs.org on April 25, 2015

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ACS Biomaterials Science & Engineering

Bifunctional polysulfone-chitosan composite hollow fiber membrane for bioartificial liver

Rohit S. Teotia1, Dhrubajyoti Kalita2, Atul K. Singh3, Surendra K. Verma2, Sachin S. Kadam4, Jayesh R. Bellare2*

1

Department of Biosciences and Bioengineering, Institute of Technology-Bombay, Mumbai-

400076, India. 2Department of Chemical Engineering, Indian Institute of Technology-Bombay, Mumbai-400076, India. 3Centre for Research in Nanotechnology & Science, Indian Institute of Technology-Bombay, Mumbai-400076, India. 4Defence Institute of Advanced Technology, (Deemed University) , Girinagar, Pune 411025, India.

*Correspondence Author: Jayesh R. Bellare Department of Chemical Engineering Indian Institute of Technology-Bombay Mumbai-400076, India E-mail: [email protected] Phone: +91 22 2576 7207 Fax: +91 22 2572 6895 or +91 22 2572 3480

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ACS Paragon Plus Environment


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Abstract Hollow fiber membranes are widely used as assist devices for bioartificial liver application. Asymmetric porous polysulfone and polysulfone-Tocopheryl polyethylene glycol succinate (PsfTPGS) composite hollow fiber and flat membranes were prepared by phase inversion procedure and subsequently surface modified with chitosan using sulfonation with concentrated sulphuric acid. Sulfonation induces negative charge on the prepared membrane surface and facilitates the attachment of chitosan amine groups by electrostatic interaction. The surface modification of membrane is stable at room temperature as dictated by presence of nitrogen in XPS analysis and amide linkages in FT-IR spectra. Further, biological studies of the membranes were performed using HepG2 cell line. Chitosan is biocompatible and shows structural similarity to glycosaminoglycans, a native liver ECM component. The chitosan modified composite Psf and Psf-TPGS membranes have shown enhanced attachment and proliferation of HepG2 cells on outer surface as confirmed by the cell counting, DNA content, confocal microscopy and SEM micrographs. The cells form a 3D multicellular spheroid structure on the chitosan modified membranes in significantly larger number as seen in the SEM micrographs. Also, the hemocompatibility of the modified composite membranes were comparable to the unmodified membranes. Thus, the chitosan modified composite membranes we have developed are bifunctional and have potential to be used in bioartifical liver application.

Keywords: hollow fiber, bioartificial liver, chitosan, TPGS, HepG2, spheroid.

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1. INTRODUCTION Liver is the largest internal multifunctional organ of body. According to World Health Organisation (WHO) 10% of the world’s population is suffering from chronic liver disease1. The ultimate treatment for all chronic liver diseases is liver transplantation from heart beating cadaver donor. However, inadequate supply of donors is a major limitation for the extension of this therapy. With the large number of patients awaiting liver transplant, there is a quest for temporary liver support system that could be used until the liver for transplantation is available or patients own liver regains its function, as liver is regenerative even after 70% damage 2. The temporary liver support can be artificial or bioartificial. The artificial support devices aim for the detoxification purpose only, whereas the bioartificial liver (BAL) support devices are more promising with a biomaterial and cellular components for both detoxification and synthetic function. Though effective, these BAL devices still needs further improvements as seen in the outcome of clinical trials3. Many of these devices in the clinical trials are hollow fiber membrane (HFM) based due to their simplicity to separate cells from blood and high surface area provided by HFM. Polysulfone (Psf), polyethersulfone, cellulose acetate, polyacrylonitrile and polypropylene are most commonly used material for HFM preparation. Polysulfone has used for the BAL application in clinical trials in some of the BAL devices like HepatAssist and AMC-BAL1. Psf occupies a large market of the HFM due to its desirable membrane forming ability, mechanical strength, chemical inertness, mechanical and thermal properties, but the concern is hydrophobicity of Psf HFMs. Most of the HFM devices for BAL are resultant of devices for blood purification or hemodialysis, wherein the major concern is about the hemocompatibility of lumen side for the flow of plasma or blood. Modification of Psf HFM by additives like PEG,

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PVP and MPC are done in order to make it hydrophilic and enhance biocompatibility4. Although, hemocompatibility is a major factor for BAL devices as the source of nutrients for the cells comes from blood or plasma, but there is a need for a consideration of favourable environment for cellular attachment of cells on outer side of BAL devices. Therefore, a bifunctional hollow fiber that is hemocompatible on the lumen side and cytocompatible outer surface would be an ideal choice for the development of devices for BAL. Hepatic cell attachment on double porosity Psf membranes have been studied without any material modification to Psf5. In our previous work, we have reported an increase in cell adhesion and proliferation of NIH3T3 cells along with the improvement in hemocompatibility of Psf when making composite HFM of Psf-TPGS6. Many synthetic and natural polymers have been tried to enhance the functionality of the liver cells7. Chitosan is a naturally derived polymer from the partial N-deacetylation of chitin. It is biodegradable, biocompatible and shows structural similarity to glycosaminoglycans, which is a native liver ECM component8. Increase in viability and function of hepatocyte due to its architecture and extensive cell–cell communication using chitosan/gelatin microcarriers were reported9. HL-7702 cells formed spheroids on TiO2/chitosan composite scaffolds with prolonged viability and improved liver-specific functions10. Even with improvements in functions well documented, there are no reports on chitosan coated HFMs support device for bioartificial liver application. There are not many reports on the use of the Psf/chitosan HFMs for the use in bioartificial liver. The aim of this work was to develop the hollow fiber based extracorporeal BAL with enhanced cellular and hemocompatible material properties. The bifunctional device was prepared by facile method with sulfonation of the preformed HFM to allow for the attachment of the chitosan on outer surface. The composite HFM has a hemocompatible surface on the inner side in which the

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blood will flow, and the outer surface has a natural polymer coating forenhanced attachment and functionality of HepG2 cells.Experimental Procedures Materials Polysulfone (UDEL P-3500 LCD MB7-BULK) was procured from M/s. Solvay Advanced Polymers, USA. Vitamin E TPGS (NF grade) was generously gifted by M/s. Isochem SA, France and used without any further purification. Chitosan from sigma aldrich was used as coating material. N-methyl-2-pyrrolidone (NMP), sulfuric acid, acetic acid, glutraldehyde were obtained from Merck. Dextran for solute rejection and standard curve procured from M/s. Pharmacosmos, Denmark. BD Vacutainer® Plus plastic plasma tubes containing 150 USP units sodium heparin (spray-coated) anticoagulant was used for blood collection. For cell culture Dulbecco’s modified eagle’s medium (DMEM) (Gibco, Invitrogen, USA) containing 2 mM L-glutamine (SigmaAldrich, USA), supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml) (SigmaAldrich, USA) and 10% fetal calf serum (Invitrogen, USA) was used. The plastic ware for cell culture was procured from BD. Urea and albumin assay kit for biochemical quantification was purchased from R.K Diagnostics (India). PicoGreen DNA Quant-iT™ PicoGreen® dsDNA reagent kit (Invitrogen) was used for DNA quantification. Experimental Design Preparation of Psf and Psf-TPGS hollow fiber membranes Polysulfone was dried in vacuum oven for 24 h at 90˚C for the removal of absorbed moisture. NMP was used as solvent for dope preparation. The dope compositions used for HFMs preparation were 20/80 (Psf/NMP) designated as P and 20/10/70 (Psf/TPGS/NMP) designated as PT in the manuscript. Dope solution was degassed prior to the start of spinning in order to remove dissolved gas in the solution. Indigenously developed HFM spinning pilot plant was used

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for making fibers11. The process parameters used for HFM spinning are listed in Table 1. The prepared fibers were kept in water for a day to remove the residual solvent and used for further studies. The flat membranes (FM) were also prepared of the same dope solution by casting the flat film over a grease free clean glass plate and dipping in the water for coagulation. Flat membrane was used for the confocal studies and contact angle, while HFMs were used for all other experiments. Table 1: Process parameters used for hollow fiber membrane preparation. Ambient Temperature (ºC)

25

Relative humidity (%)

50-60

Dope solution composition

P: Psf 20/NMP 80 PT: Psf 20/TPGS 10/NMP 70

Bore solution composition

Deionized water

Dope solution temperature (ºC)

25

Bore solution temperature (ºC)

25

Dope flow rate (ml/min)

1

Bore flow rate (ml/min)

5

Spinneret ID/OD (mm)

0.8/1.4

Air gap (cm)

45

Coagulation bath composition

RO Water

Rinse bath composition

RO Water

Coagulation bath temperature (ºC)

25

Rinse bath temperature (ºC)

35

Take-up drum velocity (m/min)

9 6


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Hollow fiber morphology by SEM Morphology studies of HFMs were carried out using scanning electron microscope (SEM). Hollow fibers were freeze fractured in liquid nitrogen for preventing structure damage and dried. HFMs were coated with platinum by sputter coating using Auto fine coater JFC-1600 (JEOL, Japan). Samples were observed under scanning electron microscope at 5-10 kV (JSM-7600F, Jeol). Solute Rejection Cross -flow filtration system setup and methodology explained in our previous study was used for the collection of samples for solute rejection11. Briefly, hollow fiber modules consisting of 10 HFMs of 25 cm length are potted with araldite resin. A feed solution of mixed dextran fractions in deionized (DI) water was pumped in the lumen of HFM module. The retentate and permeate were recycled through module the for 30 min and then sampled12. These samples were analyzed using gel permeation chromatography (GPC). The calibration was done using standard dextran fractions concentrations (Table 2) ranging from 1-500 kDa. GPC chromatograph of dextrans in retentate and permeate is obtained in terms of RI (refractive index) signal verses retention time (min). The rejection curve is obtained using procedure described in articles13. The rejection was calculated from the following equation,

 SA p ( MWi )   R( MWi ) = 1 − SAr ( MWi )  

(1)

Where SAp(MWi) and SAr(MWi) are the slice area of permeate and retentate respectively at molecular weight MWi.

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Table 2: Feed composition of dextran fractions used for solute rejection Dextran

MW

Suppliers

Concentration

Fraction

(kDa)

T1

1

Pharmacosmos(Holbaek,Denmark) 0.74

T4

4

Serva(Frankfurt,Germany)

T10

10


T40

40


T70

70


T500

500


(gm/lit)

1.22

Modification of Prepared Psf and Psf-TPGS composite membrane The sulfonation of the prepared Psf and Psf-TPGS hollow fiber membrane (HFM) and flat membrane of same dope composition was done using the conditions previously reported by Smithar et. al14 with some modification. The reaction procedure is schematically shown in Figure 1(A). The ends of the prepared P and PT HFMs were heat sealed. The sealed membranes were then soaked in 60 wt% sulfuric acid and rotated using Rolymax (Tarson, India) for a period of 4h. Then module was prepared by potting one end only of the bundle of HFMs. Picture of one end open module is shown in Figure 1 (B).. The lumen of the module was rinsed by connecting the module in the solute rejection setup described earlier and pumping water at a flow rate of 100ml/min for a time interval of 3-5 minutes. Chitosan 1% solution used for outer coating was prepared in distilled water with overnight stirring using 0.1% glacial acetic acid. The membranes were dipped in the chitosan solution and put for rotation in Rolymax for 3 h. The membranes were rinsed on inner side as described earlier with one end potted module and hang dried at 45˚C 8


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overnight. There is possibility of sulfuric acid leading to sulfonation in the lumen. We have done the immediate rinsing of lumen after sulfuric acid treatment to remove acid. Although, there may be possibility of sulfonation at inner lumen, but the sulfonation does not induce hemolysis15. The chitosan will not be entering into the lumen, due to the viscosity of the chitosan solution. The coated membranes were labeled as PC for polysulfone/chitosan composite membrane and PTC for polysulfone/TPGS/chitosan membrane. The abbreviations used in further manuscript for different membrane nomenclature are listed in Table 3.

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Figure 1: Schematic of the reaction adduct between polysulfone and chitosan for surface modification of HFM (A) and picture of module potted at one end for free flow of water through lumen (B)

Table 3: Abbreviations used for different membrane nomenclature. Abbreviation

Membrane Type

P

Polysulfone

PT

Polysulfone/TPGS

PC

Polysulfone- Chitosan

PTC

Polysulfone/TPGS- Chitosan

Surface composition by ATR-FTIR The ATR (MTR) spectra of the membranes were measured using an FTIR system 2000 (Perkin Elmer Corp., USA); 256 scans were signal averaged at data resolution of 4cm-1. The attenuated total reflection (ATR) spectra were obtained using a KRS-5 prism with an incident angle of 45. X-ray photoelectron spectroscopy (XPS) The surface chemistry of modified HFM was analyzed by X-ray photoelectron spectroscopy (XPS) using an X-ray photoelectron spectroscope ESCA LAB 220I spectrometer (Thermo VG Scientific, Beverly, MA) with an monochromatic Al Kα (1486.6 eV) X-ray source operated at 100 W. Wide scan survey spectra were obtained using a binding energy (BE) at step size of 0.5 eV. Contact angle Water contact angle of all the membranes was measured to study the effect of surface coating on wettability of the membranes. Samples were kept on a sample stage and 20 µl of distilled water 10


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was dropped at three different sites with different time intervals and the contact angle was measured using Digidrop GBX (GBX Instruments, Romans, France).

Hemocompatibility Test The procedure for hemolysis as described in our previous work6 was followed in accordance with extant institutional guidelines. Briefly, the packed erythrocytes were washed three times with normal saline solution (NSS) (0.9% w/v NaCl). 50 % hematocrit was prepared by adding NSS to the erythrocytes. The HFMs were rinsed thoroughly with NSS before being transferred into 24 well plates. For outer side hemolysis, 5 pieces of the test membranes of 5cm were soaked in diluted blood solution. For hemolysis of inner side, 35µl of the blood was put into the lumen of the membrane and ends were heat sealed. Both the inner and outer hemolysis samples were kept at 37°C for 60 min. The blood in inner lumen side was collected and diluted appropriately. Distilled water and normal saline were used as positive and negative control respectively. The blood solutions along with controls were aspirated and centrifuged at 1000×g for 5 min. The absorbance of the supernatant was measured at 542 nm. The hemolysis ratio (HR) was obtained by the equation  AS − AN  HR =   × 100  AP − AN 

(2)

Where, AS is the absorbance of sample supernatant, AP and AN are absorbance of the positive controls and the negative control respectively16. Biocompatibility Evaluation of Hollow Fiber Membrane Cell Attachment HepG2 (human hepatocellular carcinoma) cells were procured from National Centre for Cell Science, Pune, India. HepG2 cells were cultured in complete DMEM media at 37°C in a 11



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humidified atmosphere of 5% CO2. Hollow fibers samples for cell culture were prepared by cutting two cm length capillary and then sterilized by dipping in 70% ethanol followed by exposing to UV-light for 20 min17. Samples were washed with sterile phosphate buffer saline (PBS) to remove excess of ethanol. These sterile samples were placed into 2ml tubes with 1×105 cells/ml. Samples were stirred in the shaker inside the CO2 incubator (Thermoscientific) for 30 min and then the fibers were taken out and placed in 6-well tissue culture plate containing cell culture medium. After allowing for the attachment for 24 h, the HFMs were transferred to a new 24 well plate and incubated for a period of 2 and 5 days. Scanning Electron Microscopy (SEM) Study At day 2 and 5, growth medium from the 24 well was discarded and fibers containing cells were washed with PBS. Cells were fixed in 2.5% glutaraldehyde for 2 h at room temperature, there after washed with PBS and dehydrated with graded ethanol for 10 min each. Samples were dried overnight and sputter coated with gold (Quorum Technologies Ltd, UK) and observed under SEM. Confocal Microscopy HepG2 cells were seeded over flat membranes as described above. At day 3 media was discarded and materials containing cells were washed with PBS. Fixation of cells was done with 4% paraformaldehyde for 5 min at 4°C followed by permeabilization using 0.1% triton-x solution. To visualize the attached cells, nucleus were stained with DAPI (4’,6-diamidoino-2phenylindole) (Sigma-Aldrich) and mounted over glass-slide using Vectashield mounting medium (Vector laboratories, Burlingame, CA). Images were captured using a confocal laser scanning microscope (CLSM) (Olympus Fluoview, FV500, Tokyo, Japan).

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DNA Estimation Picogreen assay was done to study the cell proliferation on HFMs as per the protocol described previously18. Samples were harvested at indicated time points, and the DNA contents were measured by using PicoGreen reagent kit. The DNA was quantified in relation to a standard curve with HepG2. Determination of Urea and Albumin Samples of the culture medium from the HepG2 cells, grown on the different membrane material were collected and stored at -20˚ C until needed. Quantification of urea synthesis and albumin secretion kits was done as per the manufactures protocol. The data were normalized with the cell number. Statistics Data are expressed as mean ± SEM unless otherwise indicated. The data obtained in experiments were subjected to one-way ANOVA followed by Tukey’s multiple comparison test of significance using GraphPad

Prism

3.02 software. Qualitative observations have been

represented following assessments made by three individuals blinded to the experiments.

3. RESULTS AND DISCUSSION Asymmetric concentric hollow fiber membranes were prepared by using the dry wet phase inversion process. The figure 2 shows overview of cross section, side view of cross section and inner skin layer of the native polysulfone (Psf) and the composite Psf/TPGS HFMs. Both the hollow fiber membranes are uniform in structure as seen in figure 2a and 2d. The hollow fiber has an inner dense skin with nanopores in both the HMFs as seen in the Figure 2c and 2f. The inner skin layer determines the separation performance followed by the porous support layer.

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The Psf/TPGS HFM is more porous in structure (Figure 2e) compared to the Psf HFM (Figure 2b) as seen by the presence of continuous macrovoids in the micro structure. This is due to the leaching of the PEG moiety of the TPGS in the Psf/TPGS membrane, thereby enhancing the porosity. The increase in the porosity by leaching of PEG additive is reported19. The asymmetric structure is ideal for the dual functionality membranes for BAL application. The inner skin should act as barrier transport with small pore sizes, whereas the outer membrane should be highly porous so as to have a very low or no mass transfer limitation on to the outer surface20.

Figure 2: The SEM micrograph of the prepared Psf HFM. a and d shows the overview of cross section; b and e shows the side view of the cross section; c and f show the inner pore structure of Psf and Psf/TPGS membranes. Psf/TPGS membrane appears to be more porous compared to naive Psf. Molecular weight cut off Nominal molecular weight cut off (NMWCO) is the molecular weight of solute corresponding to 90% rejection. The MWCO is a very important parameter for the BAL functioning. The 14


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membrane should be semi-permeable allowing for the passage of nutrients and growth factors and albumin (70k Da) but at the same time should act as a barrier for the transport for the immunoglobulin IgG (150k Da) across the membrane. The MWCO profile of membranes prepared is shown in Figure 3. The MWCO of 125-135kDa for both P and PT membranes are comparable and well suited for HFM in BAL application.

Figure 3: Solute rejection plot of prepared P and PT HFMs measured by gel permeation chromatography. Both membranes have similar MWCO profiles suitable for BAL application. FTIR: The presence of TPGS and chitosan at outer surface of HFM was confirmed by ATR-FTIR spectroscopy. ATR-FTIR spectra of different membranes and pure compounds are shown in Figure 4. Polysulfone membranes have shown bands in the finger-print region of spectrum below 1700 cm-1. The Psf consists of a backbone made up of diaryl sulfone (Ar-SO2-Ar) and diaryl ether (Ar-O-Ar) groups showing strong absorption peaks at 1151 and 1242 cm–1 respectively. The band at 1488, and 1586 cm–1 belongs to CH3-C-CH3 stretching and stretching vibration of 15



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the aromatic C=C in Psf molecule respectively. PT membranes introduced new peaks at 2873, 1745 and 948 cm-1, which were ascribed to the CH2 symmetric stretch, C=O stretch (ester group in TPGS) and C-C stretch of TPGS respectively6. The absorption band at 3400–3650 cm-1 is attributed to the terminal hydroxyl group in pure TPGS21. The pure chitosan (C) has strong absorption peaks at 1,658 and 1,322 cm-1, which are the characteristic of chitosan and have been reported as amide I and III peaks, respectively. Another broad peak at 3,447 cm-1 is caused by amine N–H vibration of chitosan

22

. In the chitosan modified membranes of the PC and PTC,

broad peak at 3,447 cm-1 signifies the presence of chitosan on modified membranes.

Figure 4: ATR FTIR spectra of different HFM of P, PT, PC, PTC and pure compounds of TPGS and chitosan (C), spectra show the integration of compound in the modified membranes. 16


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XPS: XPS is a powerful technique for characterizing the chemical composition of the top few atomic layers at the surface of the membrane. XPS analysis was performed to confirm the chemical composition at the outer surface of the P, PT and chitosan modified HFMs. Figure 5 shows individual elemental characteristic binding energy peaks of P, PT, PC and PTC HFMs. The individual scan of the nitrogen in Figure 5d shows the presence of nitrogen only in the chitosan modified membranes thereby confirming the coating of chitosan on the membrane surface.

Figure 5: The XPS scan of individual elements is shown in the figure a) represents the carbon (C1s), b) oxygen (O1s), c) sulfur (S2p) and d) shows the individual nitrogen (N1s) scan of the different membranes. Spectra show the characteristic peaks of individual elements with nitrogen peaks appearing only in chitosan modified composite PC and PTC membranes

All the membranes show the presence of carbon, sulfur and oxygen in all the membranes in the different ratios as shown in the Table 4. Area ratio of the element peaks was calculated using the origin software. The carbon content in the chitosan modified membranes PC and PTC decreased due to masking of polysulfone carbon backbone by comparatively bigger polysaccharide units, which is detectable to fewer depths by XPS. The increase in the sulfur and oxygen in the 17



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chitosan modified membranes can be attributed to the linking sulphonate moiety in the sulfonated adduct of the membrane during the modification. An increase in the oxygen content in the PTC than PC dictates the proper coating of TPGS in the earlier one. Polysulfone membranes are more favorable for chitosan coating as seen by the higher area percentage of nitrogen in the P membranes compared with the PT membranes. Table 4: The percentage of elemental composition of the different HFMs. Membrane Type

Carbon (%)

Oxygen (%)

Sulfur (%)

Nitrogen (%)

1

P

54.15

40.05

5.8

0

2

PT

51.16

44.38

4.45

0

3

PC

33.72

49.01

13.02

4.25

4

PTC

34.54

50.76

11.66

3.03

Contact Angle: The hydrophilicity of membranes was examined by measuring their water contact angle. The images of the contact angle of different membranes is shown in Figure 6 and the same has been tabulated in Table 5. The contact angle of the Psf membrane was 74 and is reduced to 70˚ with the addition of the TPGS to the membrane. The contact angle was further reduced by the addition of the chitosan to the membranes. Chitosan is hydrophilic in nature and provides the surface hydrophilicity to the membrane, thereby reducing the contact angle. The TPGS also has the hydrophilic PEG moiety which also helps in making the membrane hydrophilic, thereby reducing the contact angle of P from 74 to 70˚ in the PT membranes. The contact angle was lowest in the case of the PTC membrane due to the combined effect of the TPGS and chitosan. A similar decrease in the contact angle of the Psf was also seen when the chitosan was cross-linked as a thin layer over the Psf membrane23. 18


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Table 5: The contact angle of different HFMs. S.No.

Sample

Contact Angle (˚)

1

P

74.4 + 3.5

2

PT

69.75 + 3

3

PC

65.5 + 4

4

PTC

51.95 + 3.5

Figure 6: The contact angle of the different HFMs with the drop and nozzle. Native P is hydrophobic but tends to shift towards hydrophilicity upon modification.

Hemocompatibility: For bioartificial liver assist devices, the blood will flow through the lumen of the HFM. Blood normally consists of 48% erythrocyte volume fraction. Interaction of erythrocytes with hollow fiber is essential to study the release of hemoglobin (called hemolysis). In vitro hemolysis test was performed on P, PT, PC, PTC HFMs at both inner and outer side, results are shown in Figure 7. The hemolysis ratio of the inner side is lower in all the membranes and lowest in the PT membranes. The PT membranes have lower hemolysis ration than the P membrane due to the presence of TPGS. Hemolysis ratio on outer side of PT membrane was lower with respect to P membrane. The hemolysis ratio of chitosan modified PC and PTC composite membranes was significantly higher in comparison to native P membranes. This is due to the presence of chitosan on the outer side of the membrane. Previous studies indicates that chitosan does not promote surface induced hemolysis24. Chitosan only induce the adhesion of 19



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erythrocytes but not seriously damage the cell membrane. The acetic acid used to dissolve chitosan can be a factor in the induction of the hemolysis25. However, the hemolysis ratio was less than 5% in all the different HFMs in both outer and inner side. According to ASTM F-75608 standard 5% is regarded as non-toxic and well within the permissible limit, hence proving the hemocompatibility of both native and modified composite membranes.

Figure 7: Hemolysis ratio of the inner side is shown in (A), and (B) shows the outer surface hemolysis for all the different membranes. All the membranes are within permissible limits. Values are expressed as mean ± SEM; **p < 0.01vs. P as determined by one way ANOVA followed by Tukey’s multiple comparison test. Cell Attachment: The HepG2 cell line from hepatocellular carcinoma performs many metabolic and synthetic functions of liver. The cell count was done to determine the number of cells attaching on the fiber surface at the end of day 2 and day 5(Figure 8A). There was an increase in cell population at the day 2 and 5. The cell count was modest but significantly higher in chitosan modified PC and PTC HFMs over the native P HFMs at day 2. But at day 5, cell count 20


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significantly increased in the case of PTC and PC membranes in comparison to P membranes. The cell count was comparable between P and PT membranes at both days with marginally higher in PT membranes. This is due to presence of TPGS, which is a synthetic amphiphile that undergoes enzymatic cleavage to deliver the lipophilic antioxidant, α-tocopherol (vitamin E) to cell membranes26. The attachment was higher in the case of the chitosan modified membrane due to the roughness provided by the attachment of the chitosan particles on the membrane surface, thereby enhancing the cellular attachment of the HepG2 cells. Increase in the roughness of the surface even in nanometer scale enhances attachment of cells27. The cell number at day 5 in PC is more than PTC as area occupied by the chitosan on PC membrane surface is more compared to the PTC membranes.

Figure 8: (A) Cell count and (B) DNA content of cells attached on the outer surface of different HFMs at day 2 and 5. Both cell count and DNA significantly increases in composite membranes. Values are expressed as mean ± SEM; *p < 0.05 and **p < 0.01 vs. P, $p < 0.05 vs. PT as determined by one way ANOVA followed by Tukey’s multiple comparison test. The DNA content was higher in the day 5 compared to day 2 in cells on all the different HFMs as seen in Figure 8(B). The amount of the DNA content is proportional to the cellular growth. 21



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The cellular proliferation was higher in the chitosan modified membranes as compared to the native membranes. The DNA content was significantly higher in chitosan modified PC and PTC HFMs over the native P HFMs on both days. SEM Study: HepG2 cells were seeded on HFM as described above. At day 2 and 5, the SEM micrographs of the grown cells were taken and are shown in Figure 9. The spheroid formation and cell attachment on opaque HFM surface can be effectively studied using SEM due to large depth of field, resolution and view area28. The coating of the chitosan on the modified membranes on HFM at Day 0 was evidently seen by SEM micrographs in PC and PTC membranes. Chitosan particles provide roughness on membrane surface which aid in the attachment of the cells on the membrane surface as was observed in cell count of chitosan modified membranes. In human liver, hepatocytes are organized in a three-dimensional lobular structure. Monolayer 2D culture system fails to provide the microenvironment and does not truly represent the organization of cells29. The hollow fiber membranes provide a 3-D environment for the growth of the cells and have been used for the expansion and differentiation of embryonic liver cells into hepatocytes30. HepG2 cells on 3-D matrices aggregate to form multicellular structures called spheroids31. As can be seen in the SEM images of day 5, there is aggregation of cells to form multicellular spheroids on all the different membranes with shapes being either oval spheroid, polyhedron or tightly packed. When comparing the different membrane types, the highest tightly packed spheroid aggregation can be seen in the case of the PTC followed by PC HFMs. This is due to presence of chitosan in the composite membranes. Organization of HepG2 cells into multicellular spheroids on chitosan films was also shown by Verma et al32. Similarly many researchers have also reported the stem cells spheroid formation on chitosan surfaces33.

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Figure 9: SEM micrographs of HFM overview: HFM at day 0 and HFM seeded with HepG2 cells over a period of 2 and 5 days. Coating of chitosan on fiber surface is observed in PC and PTC membranes, providing surface roughness. All HFM supported the cellular attachment of HepG2 cells. Spheroid formation was seen in all different membranes type at day 5.

Confocal Study: The confocal images of the different flat membranes are shown in Figure 10. The cell nucleus is stained by DAPI dye. The cellular attachment was seen on all the different HFMs. The cellular 23



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attachment was increased in both chitosan modified PC and PTC membranes. The nucleus is more in number in the case of the chitosan coated membranes due to increase number of multi cellular spheroid formation. As the spheroids are multicellular and 3 D in nature, the nucleus distribution is not planar on the membrane surface.

Figure 10: Confocal microscopy images of cell nucleus stained with DAPI on the different flat membranes at day 3. Cell attachment is enhanced on chitosan modified PC and PTC membranes. Urea synthesis and albumin secretion: Urea synthesis and albumin secretion are key markers for functionality testing of the devices used for BAL applications. The urea synthesis and albumin concentration increased from day 2 24


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to day 5 in all the different membranes type as seen in Figure 11. Urea synthesis and albumin secretion were significantly higher in case of chitosan modified membranes on day 5 with respect to native P membranes. This may be due to enhancement of HepG2 cells organization into multicellular spheroids on chitosan surface, resulting in more differentiated and functionally active state of the cells comparable to the native membranes. Similar consistent increase of liver specific function in terms of albumin secretion and urea synthesis by HepG2 cells when cultured on chitosan microfibers and film due to spheroid formation is reported32, 34. The increase in the values of both urea and albumin at day 14 was marginal with respect to day 5. This might be due to formation of large spheroids of cells at day 14. Also, it has been reported that the HepG2 cells do not proliferate once it has gained the spheroid morphology. The cells continue to increase size of spheroids and there is decrease in the functionality of cells due to transport limitation at centre core region leading to necrosis35.

Figure 11: Biochemical activity of cell attached on HFMs (A) Urea synthesis and (B) Albumin secretion of cells attached on outer surface of different HFMs at day 2, 5 and 14. Both biochemical activities are enhanced at day 5 with very marginal increase at day 14.Values are expressed as mean ± SEM (n =3); *p < 0.05 value was found out by comparing chitosan coated

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(PC and PTC) membranes vs. the native polysulfone (P) membrane. One way ANOVA followed by Tukey’s multiple comparison test was used.

Conclusion In modern technological era, there is always a need to fine-tune the existing technology as per the requirement. We have developed bifunctional Psf-chitosan composite hollow fiber with inner surface that has good hemocompatibility. The rough outer surface due to chitosan coating further supports cell growth, proliferation and the expression of the liver-specific function. These results demonstrate that the coating of preformed hemocompatible HFMs is a facile method for making the HFMs bifunctional. HepG2 cells cultured on chitosan modified HFMs, formed multicellular spheroids more efficiently than unmodified membranes. Further study is needed to construct a composite hollow fiber bioreactor and to evaluate its mass transfer and immunobarrier properties. Although this work is reported for the BAL application here, but this will also have an equal impact on other application like stem cells expansion, cell line testing and biochemical and biomaterial production.

Acknowledgements: The authors are thankful to Centre for Research in Nanotechnology and Science (CRNTS) and IRCC for characterization facility, Department of Chemical Engineering for Contact angle and GPC facility and Department of Physics for XPS facility of Indian Institute of Technology Bombay. Funds: We thank Department of Biotechnology (DBT) for supporting grant. Authors acknowledge research fellowship grants from the MHRD (RST), DST-INSPIRE (AKS) and CSIR (SKV).

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Graphic for manuscript 188x115mm (150 x 150 DPI)


Bifunctional Polysulfone-Chitosan Composite Hollow Fiber

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