. Aug 13;16(8):e. doi: 10./journal.pone.

Poly(amino acid) based fibrous membranes with tuneable in vivo biodegradation

Kristof Molnar

Kristof Molnar

1Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary 2Department of Food, Agricultural and Biological Engineering, College of Food, Agricultural, and Environmental Sciences, The Ohio State University, Wooster, OH, United States of America Conceptualization, Formal analysis, Visualization, Writing &#; original draft Find articles by Kristof Molnar 1,2, Constantinos Voniatis

Constantinos Voniatis

1Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary 3Department of Surgical Research and Techniques, Semmelweis University, Budapest, Hungary Formal analysis, Investigation, Methodology, Visualization, Writing &#; original draft Find articles by Constantinos Voniatis 1,3, Daniella Feher

Daniella Feher

3Department of Surgical Research and Techniques, Semmelweis University, Budapest, Hungary Methodology, Validation Find articles by Daniella Feher 3, Gyorgyi Szabo

Gyorgyi Szabo

3Department of Surgical Research and Techniques, Semmelweis University, Budapest, Hungary Validation Find articles by Gyorgyi Szabo 3, Rita Varga

Rita Varga

1Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary Data curation Find articles by Rita Varga 1, Lilla Reiniger

Lilla Reiniger

41st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary Validation Find articles by Lilla Reiniger 4, David Juriga

David Juriga

1Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary Methodology, Validation Find articles by David Juriga 1, Zoltan Kiss

Zoltan Kiss

5Department of Polymer Engineering, Faculty of Mechanical Engineering, Budapest University of Technology and Economics, Budapest, Hungary 6Biomechanical Research Center, Faculty of Mechanical Engineering, Budapest University of Technology and Economics, Budapest, Hungary Formal analysis, Validation Find articles by Zoltan Kiss 5,6, Eniko Krisch

Eniko Krisch

2Department of Food, Agricultural and Biological Engineering, College of Food, Agricultural, and Environmental Sciences, The Ohio State University, Wooster, OH, United States of America Formal analysis, Writing &#; original draft Find articles by Eniko Krisch 2, Gyorgy Weber

Gyorgy Weber

3Department of Surgical Research and Techniques, Semmelweis University, Budapest, Hungary Methodology, Supervision Find articles by Gyorgy Weber 3, Andrea Ferencz

Andrea Ferencz

3Department of Surgical Research and Techniques, Semmelweis University, Budapest, Hungary Supervision Find articles by Andrea Ferencz 3, Gabor Varga

Gabor Varga

7Department of Oral Biology, Semmelweis University, Budapest, Hungary Supervision Find articles by Gabor Varga 7, Miklos Zrinyi

Miklos Zrinyi

1Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary Supervision Find articles by Miklos Zrinyi 1, Krisztina S Nagy

Krisztina S Nagy

1Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary 7Department of Oral Biology, Semmelweis University, Budapest, Hungary Formal analysis, Methodology, Supervision, Writing &#; original draft Find articles by Krisztina S Nagy 1,7, Angela Jedlovszky-Hajdu

Angela Jedlovszky-Hajdu

1Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary Conceptualization, Funding acquisition, Investigation, Supervision, Visualization, Writing &#; original draft Find articles by Angela Jedlovszky-Hajdu 1,* Editor: Wenguo Cui8

1Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary 2Department of Food, Agricultural and Biological Engineering, College of Food, Agricultural, and Environmental Sciences, The Ohio State University, Wooster, OH, United States of America 3Department of Surgical Research and Techniques, Semmelweis University, Budapest, Hungary 41st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary 5Department of Polymer Engineering, Faculty of Mechanical Engineering, Budapest University of Technology and Economics, Budapest, Hungary 6Biomechanical Research Center, Faculty of Mechanical Engineering, Budapest University of Technology and Economics, Budapest, Hungary 7Department of Oral Biology, Semmelweis University, Budapest, Hungary 8Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital, CHINA

Roles

Kristof Molnar: Conceptualization, Formal analysis, Visualization, Writing &#; original draft Constantinos Voniatis: Formal analysis, Investigation, Methodology, Visualization, Writing &#; original draft Daniella Feher: Methodology, Validation Gyorgyi Szabo: Validation Rita Varga: Data curation Lilla Reiniger: Validation David Juriga: Methodology, Validation Zoltan Kiss: Formal analysis, Validation Eniko Krisch: Formal analysis, Writing &#; original draft Gyorgy Weber: Methodology, Supervision Andrea Ferencz: Supervision Gabor Varga: Supervision Miklos Zrinyi: Supervision Krisztina S Nagy: Formal analysis, Methodology, Supervision, Writing &#; original draft Angela Jedlovszky-Hajdu: Conceptualization, Funding acquisition, Investigation, Supervision, Visualization, Writing &#; original draft Wenguo Cui: Editor

Received May 7; Accepted Jul 4; Collection date .

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Abstract

In this work two types of biodegradable polysuccinimide-based, electrospun fibrous membranes are presented. One contains disulfide bonds exhibiting a shorter (3 days) in vivo biodegradation time, while the other one has alkyl crosslinks and a longer biodegradation time (more than 7 days). According to the mechanical measurements, the tensile strength of the membranes is comparable to those of soft the connective tissues and visceral tissues. Furthermore, the suture retention test suggests, that the membranes would withstand surgical handling and in vivo fixation. The in vivo biocompatibility study demonstrates how membranes undergo in vivo hydrolysis and by the 3rd day they become poly(aspartic acid) fibrous membranes, which can be then enzymatically degraded. After one week, the disulfide crosslinked membranes almost completely degrade, while the alkyl-chain crosslinked ones mildly lose their integrity as the surrounding tissue invades them. Histopathology revealed mild acute inflammation, which diminished to a minimal level after seven days.

1. Introduction

Polymer hydrogels are three-dimensional polymer networks that contain a large amount of aqueous solution. Hydrogels possess the properties of both solid and liquid materials: they can maintain their shape as solids do, yet small molecules for example drugs, can migrate through them by diffusion (as they would do in fluids). Furthermore, due to their high water content, they resemble the mammalian soft tissues, therefore, hydrogels are commonly used in clinical practice and experimental medicine in a wide range of applications including diagnostics, drug delivery, regenerative medicine [1&#;5]. However, hydrogels are typically fragile, brittle resulting in breaking upon bending, suturing or other basic handling maneuvers during surgeries, severely limiting their applicability as implants [6, 7].

Fibrous hydrogel membranes are soft materials composed of sub-micron diameter hydrogel fibers bundled together that create a loose and flexible sheet. While still being considered as hydrogels, fibrous hydrogel membranes have additional advantages. Not only these membranes possess the favorable features of hydrogels, but their structure is similar to the extracellular matrix found ubiquitously around almost every human cell. Therefore, these membranes are exceptional candidates for cell cultivation or tissue regeneration applications. Furthermore, being composed of fibers, the material should be more flexible, but also more resistant to the damage caused by the sutures due to the free movement and bending of their fibers.

Fibrous membranes have been extensively researched for a wide variety of fields [8] including biomaterials [9, 10]. One method for polymer fiber preparation is electrospinning, where polymer fibers are created under the effect of a strong electrical field [11]. By introducing a crosslinking reaction to electrospinning, the aforementioned gel fibers can be obtained, which, without chemical or enzymatic degradation, will not dissolve after being immersed in a solution but only absorb the surrounding fluid [12]. It is important to note here that in gel fibers crosslinks are between polymer chains inside the fibers and not between fibers. Typically, one of the two following implementation methods is utilized for the synthesis of crosslinked fibrous membranes (gel fibers). In post-electrospinning methods, the fibrous membrane (having the crosslinking agent already in the polymer solution) is electrospun first and the crosslinks are formed subsequently in a chemical reaction [13&#;15]. By utilizing this method, however, the number of crosslinks cannot be properly regulated. On the other hand, in reactive electrospinning, the chemical crosslinking reaction takes place during the fiber formation. Typically, a UV active reagent is added to the polymer that under the effect of UV light will auto-crosslink the polymer chains inside the fibers. Although by utilizing this method the number of crosslinks can be regulated, most of these UV active reagents are toxic [16, 17].

Poly(aspartic acid) (PASP) is a synthetic biocompatible polymer composed of aspartic acid molecules interconnected by peptide bonds and thus exhibits a peptide-like chemical structure, which ensures its biodegradability [18&#;24]. PASP based hydrogels are promising materials for tissue engineering and cell cultivation. Juriga et al. displayed how MG-63 cells could not just attach and proliferate on PASP based hydrogels, but were also able to grow inside the gels and establish 3D colonies [25]. Nevertheless, PASP based hydrogels are simply too fragile for conventional implantation. In addition, although the fabrication these membranes was proven feasible, [26, 27] as promising as these materials are, no information is available regarding any implantation attempts, in vivo biocompatibility or a biodegradability profile.

PASP can be prepared from its anhydride, polysuccinimide (PSI) via hydrolysis under mild alkaline conditions (Fig 1) [28]. Unlike PASP, PSI is a reactive polymer, therefore, it can be easily modified at room temperature by nucleophilic reagents such as primary amines, giving PSI a major advantage compared to other synthetic biocompatible and biodegradable polymers. This enables us to synthesize a large variety of PSI derivatives with adjustable properties for different applications (Fig 1), which can be later on hydrolyzed to the corresponding PASP derivatives either in vitro or in vivo [19&#;24, 29&#;31]. By utilizing multifunctional amines for crosslinking, advanced functional PSI gels or with their hydrolysis PASP hydrogels can be created [28].

In this work we present the fabrication and characterization two PSI based crosslinked fibrous membranes (referred to as fibrous membranes from now on) which can be implanted by conventional surgical techniques while also retaining their hydrolytic and enzymatic degradability. The two types of membranes were fabricated having different chemical compositions: a disulfide crosslinked one intended for fast biodegradation and an alkyl-chain crosslinked one with possibly longer biodegradation time, both intended for tissue engineering. These samples were implanted under the skin in PSI form to see if they can hydrolyze and form PASP based samples, how fast the hydrolysis occurs, how the surrounding tissue reacts to these changes and to see the short-term biodegradability of the membranes. The biocompatibility/biodegradability was comprehensively investigated.

2. Experimental

2.1. Materials

L-aspartic acid (Sigma-Aldrich, UK), cysteamine (CYSE) (Sigma-Aldrich, UK), dimethylformamide (DMF) (VWR International, USA), dimethylsulfoxide (DMSO) (Sigma-Aldrich), o-phosphoric acid (VWR), 1,4-diaminobutane (DAB) (99%, Aldrich), imidazole (ACS reagent, &#;99%, Sigma-Aldrich), citric-acid*H2O (ACS reagent, &#;99.9%, VWR), sodium chloride (99&#;100.5%, Sigma-Aldrich), phosphate buffer saline (PBS) (Tablet, Sigma), sodium hydroxide (VWR International, USA) were of analytical grade and were used as received. For the aqueous solutions, ultrapure water (Human Corporation ZeneerPower I Water Purification System) was used. For 1 L of imidazole buffer imidazole (pH 8: 12.988 g), citrate (pH 8: 1.728 g), sodium chloride (pH 8: 11.466 g) and ultrapure water were used. In all cases the exact pH was adjusted by the addition of hydrochloric acid and followed by digital pH meter (Thermo Scientific&#; Orion&#; 4-Star Plus pH/ISE Benchtop Multiparameter Meter).

For the in vitro experiments MG-63 human osteosarcoma cell line (Sigma-Aldrich, USA), Minimum Essential Medium (Gibco, USA), fetal bovine serum (Gibco, USA), non-essential amino acids (Gibco, USA), L-glutamine (Gibco, USA), penicillin and streptomycin (Gibco, USA), commercially available cell proliferation reagent (WST-1, Roche, Switzerland), Vybrant DiD vital dye (Molecular Probes, USA) were used.

2.2. Preparation of polysuccinimide and cysteamine modified polysuccinimide

PSI was prepared by thermal polycondensation of L-aspartic acid in the presence of o-phosporic acid catalyst, under vacuum at 180°C as described in our previous paper (Fig 1A) [18]. For the modification of PSI with cysteamine (CYSE), first 0.04 g of CYSE was dissolved in a mixture of 0.7 g DMF and 0.4 g DMSO in a glass reactor, then 2 g of PSI dissolved in DMF (25 w/w%) was added and mixed vigorously under Argon atmosphere at room temperature for 45 minutes (Fig 1C). After the synthesis, the final polymer concentration was 15 w/w% and theoretically, every 10th repeating unit was modified with cysteamine (PSICYSE). Details of the synthesis and chemical characterization can be found in our previous paper.29

2.3. Preparation of electrospun cystamin crosslinked polysuccinimide fibers

Cystamine crosslinked PSI fibers were prepared by a homemade apparatus consisting of a Genvolt P high voltage power supply, a KD Scientific KDS100 syringe pump, a Fortuna Optima 7.140 33 glass syringe with Luer-lock, a blunt G18 needle made by Hamilton and a grounded plate collector covered with aluminum foil. The electrospinning parameters were set at 0.8 ml/h flow rate, 15 cm target distance (distance between the tip of the needle and collector) and 12 kV potential. Samples were prepared from 1 ml of 15 w/w% PSICYSE solution. During the electrospinning process, as fibers were expelled from the needle, the thiol side chains of the PSICYSE formed disulfide bonds between the polymer chains upon oxidation by the atmospheric oxygen before reaching the collector (Fig 1D). The crosslinked polymer is denoted as PSICYS. Due to practical reasons and restrictions of our current setup the amount of electrospun material we can synthesize during a single electrospining session is limited. A simple solution to this issue is removing a sample from the aluminum collector, folding it then compressing it resulting in smaller yet thicker membranes. In our case 3 x 4 cm rectangles were prepared (Fig 1G). To reinforce them, they were then compressed by 5 t weight along their whole surface for 5 min using an Atlas Manual 15T Hydraulic Press (GS). Finally, disks of 16 mm diameter (Fig 1G) were cut out and subsequently sterilized by dry heat thermal sterilization in a Memmert SLP 500 at 120 oC for 2 hours [32]. According to thermal gravimetry and differential thermal analysis, there is no physical or chemical change in the samples after applying this sterilization method: further details can be found in our previous paper [26].

2.4. Preparation of 1,4-diaminobuthane crosslinked PSI fibers (PSIDAB)

25 w/w% PSI/DMF solution was electrospun at 15 cm collector distance and 1 ml/h feeding rate (Fig 1E). 1 x 1 cm square samples were then cut and immersed in a 0.5 M DAB/EtOH solution (crosslinker solution) for different time intervals (1 min, 5 min, 10 min, 20 min, 30 min, 60 min, 120 min, 180 min, 1 day). After the reaction, samples were thoroughly immersed in DMF as a simple dissolution test. The reaction between PSI and DAB can be seen on Fig 1F. For the in vivo experiments, the PSIDAB samples of the 1-hour crosslinking time were chosen. Before the crosslinking, (similarly to the PSICYS samples) the PSI membrane was folded and compressed, then disks of 16 mm diameter were cut before immersing them in the crosslinker solution. Finally, samples were thoroughly washed with ultrapure water. Samples were securely sealed and stored in ultrapure water containing a small amount of ClO2 for sterilization (Fig 1F) [33].

2.5. Characterization

2.5.1. Scanning Electron Microscopy (SEM)

For SEM studies, the samples were treated in different ways according to their type: PSI-based membranes: a small part of the membrane was cut out and placed on conductive tape for coating and microscopy; PASP-based membranes: samples were first thoroughly washed with ultrapure water and freeze-dried, then a small portion was placed on conductive tape for coating and microscopy; samples from in vivo experiments: a small portion of membranes was resected from the in vivo samples and was washed in an excessive amount of 100 mM Na-cacodylate (pH 7.2) solution, then stored in a 1 V/V% glutaraldehyde solution in 100 mM Na- cacodylate (pH 7.2). To dehydrate the membranes, the samples were first placed for 5 min in a series of ethanol solutions: 20, 50, 70, 85, 96 V/V% (diluted with water) then in a 1:1 ethanol (96 V/V%) and acetone mixture and finally in a porous container with pure acetone. In a slow process, acetone was replaced with supercritical CO2 and slowly heated until complete evaporation. The dry samples were then placed on conductive tape for coating and microscopy. Micrographs were taken using a ZEISS EVO 40 XVP scanning electron microscope equipped with an Oxford INCA X-ray spectrometer (EDS). An accelerating voltage of 20 kV was applied. For the measurements, samples were sputter-coated with gold in 20&#;30 nm thickness with a 2SPI Sputter Coating System. Fiber diameters were measured with ImageJ software. In every case where membranes had clear fiber morphology, 50 individual fibers were measured and averages were calculated.

2.5.2. Multiphoton microscopy

Multiphoton microscopy enables the in-depth investigation of samples that either have auto-fluorescent properties or have been labelled with fluorescent dyes prior to the investigation. For the examination of PSI and PASP based membranes, a two-photon microscope (Femto2d, Femtonics, Hungary) with a Spectra Physics Deep See laser was used at an 800 nm wavelength to induce the auto-fluorescence of PSI and PASP [25]. The emitted photons were detected in the green channel. Images were taken with 10x objective by the MES4.4v program. The brightness and contrast of the pictures were enhanced for better visualization.

2.5.3. Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR)

Chemical structures and the success of synthesis were investigated with a Jasco FT/IR- (Able-Jasco, Japan) with DTGS detector. All spectra were collected over the range 400&#; cm-1 at a resolution of 2 cm-1. The background spectra were measured on a clean and dry diamond crystal. The number of scans accumulated was 128.

2.5.4. Synthesis of PASP-based membranes by the hydrolysis of the PSI membranes

The hydrolytic stability of PSI and PASP based samples were investigated in vitro. PASP samples were prepared by mild alkaline hydrolysis of the electrospun PSI membranes (PSICYS or PSIDAB) in an imidazole-based buffer solution of pH 8 (I = 250mM). The chemical reaction of PSI turning into PASP is depicted in Fig 1B. A consequence of the hydrolysis is the swelling of the membranes [28]. 100% conversion is achieved when the membranes reach their equilibrium size. Based on our previous study, PSIDAB spheres with a diameter of 5.5 mm obtain their equilibrium size in 5 hours [28]. Furthermore, the smaller the spheres were, the faster they obtained their equilibrium size during hydrolysis. Although the fiber in the membranes are much smaller, to ensure complete hydrolysis samples were kept in the buffer for 24 hours. The swelling investigation was performed as it is crucial regarding technical surgical aspects but also to compare any in vitro and in vivo differences. The diameter of 3 PSI disks was measured before and after hydrolysis with a caliper. Changes in thickness were considered negligible therefore the degree of swelling was calculated as diameter PASPDAB/PSIDAB*100. The average fiber diameter was calculated with standard error (confidence of 95%) using standard procedure.

After the hydrolysis, samples were washed with ultrapure water to get rid of the salts and were subsequently freeze-dried for SEM and ATR-FTIR. PASP membranes are denoted by changing the PSI part in the original sample name to PASP (PASPCYS, PASPDAB).

2.5.5. Mechanical analysis

Mechanical analysis of the PSICYS and PSIDAB membranes was performed to assess their loading capacity but also investigate whether they can be reliably used during a standard surgical procedure (suturability). Therefore, the methods we followed aimed to assess both the properties of the materials themselves, as well as the membrane-suture interactions. All samples were immersed in saline to replicate the in vivo environment where the samples would inevitably swell. For the measurements, a ZWICK Z005 tensile testing machine (Ulm, Germany) was used with standard clamps holding the samples, while registering the force and displacement of the crosshead at a constant pulling rate of 10 mm/min. The initial sample size was always 2 cm long and 1 cm wide. Sample thickness was measured with a caliper. From the recorded data, the maximum bearing load was obtained. The ultimate tensile strength was calculated by dividing the maximum bearing load by the initial cross-section area. From each sample type at least 4 parallel samples were measured. In the case of suture-sample interaction investigation (Suture Retention Test), on one side of the previously mentioned samples, a simple interrupted suture was placed in the center 0.5 cm from the top and 0.5 cm from each side. The suture was pulled out from the fixed sample at a constant speed of 10 mm/min. This is in line with the recommendation of Pensalfini et al. for the measurement of suture retention strength and is in line with AAMI/ISO/ANSI Standard () [34]. In their work they found that sample width of 1 cm and suture placed 0.5 cm from the edge of the sample are necessary to exclude any effect of sample geometry on the suture retention.

2.6. In vitro tests

2.6.1. Cell culture

A human osteosarcoma cell line, MG-63 was cultured as a subconfluent monolayer under standard conditions (100% humidity, 37°C and 5% CO2) in a humidified incubator (Nuaire, USA). These osteoblast-like cells were cultured in Minimum Essential Medium, supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 2 mM L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin.

2.6.2. Cell viability assay

Disks of an average diameter of 6 mm were cut from the electrospun membranes. To minimize the probability of a bacterial or fungal infection, the samples were stored in sterile-filtered PBS containing sodium azide. Before introducing the disks to the cells, the membrane samples were sterilized in a 300 ppm chlorine-dioxide solution (in PBS) for 10 min and they were incubated in the completed medium for 1 hour. First, the gel disks were placed into the wells of low cell binding 96 well microplates (flat bottom, Nunc, Denmark). After that, the MG-63 cells were seeded onto the gel disks at a concentration of 20 000 cells/well in 200 μl medium/well and incubated for 24 or 72 hours at 37°C.

Cell viability was evaluated by a colorimetric assay using a commercially available cell proliferation reagent (WST-1). The WST-1 reagent was diluted with uncompleted MEM solution (lacking phenol red) in a ratio of 1:20. After washing the wells with PBS to remove the non-attached and slightly attached cells, 200 μl of WST-1 solution was added to each well and the cells were incubated at 37°C for 4 h. The absorbance of the supernatant was measured at 450 nm with a reference wavelength of 655 nm using a microplate reader (Model , Bio-Rad Laboratories, Japan).

2.6.3. Multiphoton microscopy of the cells

To visualize the MG-63 cells growing on the surface of the fibrous membranes, they were labelled with the fluorescent vital dye Vybrant DiD before seeding (according to the manufacturer&#;s suggested protocol). Membrane disks of 6 mm in diameter were placed into Lab-tek 8 chamber slides (Nunc, USA) with tissue culture surface treatment, and 40 000 cells were seeded onto each disk. After 24 hours, the samples were fixed in 4% paraformaldehyde (in PBS) at RT and they were stored in PBS at 4°C until investigation under a multiphoton microscope. The red fluorescence shows the cells due to the Vybrant DiD vital staining while the green color indicates the autofluorescence of the PSI or PASP based membranes [25].

2.7. Animal model

Biocompatibility and biodegradability of electrospun PSICYS and PSIDAB samples were investigated on 24 male Wistar rats (250 g) as 12 parallel measurements were carried out for each sample type. Before implantation, the PSICYS and PSIDAB samples were immersed in sterile physiological saline (0.9%) for 10 minutes to reach their equilibrium size. Sedation of animals was performed with a mixture of ketamine and xylazine (~0.8 ml/animal). Samples were implanted at the nape under the skin. After a 1&#;2 cm long incision along the dorsal midline, samples were placed and fixed on the paramedian line via a single simple interrupted suture using an Atramat 2-0 polyglycolic acid absorbable suture material. Skin closure was performed with 3&#;4 simple interrupted stitches using the same suture material. Post-operatively for both PSICYS and PSIDAB, animals were randomly divided into two groups of 6 animals each. Animals were kept in individual cages and observed daily for evidence of wound complications, such as skin dehiscence seroma, hematoma, or infection. The experimental protocol adhered to rules laid down by the Directive of the European Parliament and of the Council on the protection of animals used for scientific purposes and was approved by the Semmelweis University&#;s Institutional Animal Care and Use Committee. The accreditation number of the laboratory is 22.1//3/. We have not used control animals in the experiments for two reasons: firstly, according to /63/EU guideline of the European Union, in animal studies the number of animals used should be reduced as low as reasonably possible; if applicable animals should be replaced with animals of lower hierarchy or other types of studies (in vitro); additionally, the in vivo study should be refined to provide animal care of the highest possible standards. Secondly, for the control animal, the surgical procedure would just involve a simple incision on the skin of the animal then sutured with the same technique and material. This would result in a standard wound healing process that is comprehensively documented in basic pathology books, like Robbins Pathology [35]. Termination and sample retrieval were performed after 3 days (Group A) and 7 days (Group B). Samples were preserved in formaldehyde and then sent for histological evaluation, whereas in the case of PSI-DAB, SEM micrographs were also taken from retrieved and then freeze-dried samples.

2.7.1. Histology

PSIDAB and PSICYS samples were collected on the 3rd and 7th days after implantation as follows: the skin tissue was separated, and the samples were dissected around the suture including the muscle tissue then placed in 4 V/V% formaldehyde solution. After fixation, water was eliminated according to a standardized protocol in a Leica ASP300 enclosed tissue processor, samples were then embedded in paraffin and slices of 4 μm thickness were cut (Leica microtome). After removing the paraffin, slices were stained by standard haematoxylin-eosin staining protocol. All slides were digitalized with a Pannoramic 250 Flash Scanner (3DHISTECH Ltd.). Although no standardized protocol for specifically evaluating foreign body inflammatory reactions exists, some indicators are widely accepted as criteria of grading the inflammatory response and used in other areas of medicine. For quantifying the immune response and inflammatory grade to the implanted samples, a scoring system based on the work of Planck et al. was used as shown in Table 1 [36]. A small piece from the 7th day PSIDAB was collected with a tweezer and investigated by a multiphoton microscope without any staining, and after freeze-drying by SEM to try and visualize leukocytes (macrophages, lymphocytes).

Table 1. Scoring system used to evaluate the histopathology samples [35].

Point Value 0 1 2 3 4 Average Score 0&#;0.49 0.50&#;1.49 1.50&#;2.49 2.50&#;3.50 > 3.50 Inflammation Grade None Mild Moderate Severe Macrophage infiltration None Mild Moderate Severe Lymphocyte Infiltration None Mild Moderate Severe Foreign Body Giant Cell Formation None Mild Moderate Severe Granulation Tissue and Fibrosis thickness None Narrow Band Moderate Band Wide Band Extensive Band

2.8. Statistical analysis

When applicable, the data written in the text is represented by an average followed by the standard error calculated at p = 0.05 confidence level. The same confidence level was used for all calculations. The number of elements in each sample is given in brackets at the corresponding averages and errors. The error bars on figures represent the standard errors. For the hypothesis tests in Section 3.5 double-sided, two-sample Student t-tests were conducted using the averages and standard deviations of the samples. In the in vitro analysis Kruskal-Wallis ANOVA and median test were used for statistical evaluation of the data, applying the STATISTICA 10 software (Statsoft, USA). In case of p < 0.05 was considered a difference as statistically significant.

3. Results and discussion

3.1. Preparation of PSI, PSICYS and PSIDAB membranes

Regarding the synthesis, electrospinning and physicochemical properties of PSI and cysteamine modified polysuccinimide (PSICYSE) can be found in detail in our previous article.29 After electrospinning of both PSI and PSICYS a white sheet of fibrous material was obtained, that was easy to remove from the collector. Small parts of the white fibrous sheets were dipped in N,N-dymethilformamide (DMF) to check dissolution. The PSI sheet dissolved immediately contrary to the PSICYS sheet that only swelled indicating the presence of crosslinks. The reactive electrospinning had no significant effects on the fiber formation as the average diameter of PSICYS fibers (900 ± 70 nm (n = 50), Fig 2B) does not differ significantly from that of the PSI fibers (910 ± 80 nm (n = 50), Fig 2A). According to the SEM images, both the PSI and the PSICYS fibers had a smooth surface without any defects.

The idea for the implementation of post electrospinning crosslinking for the synthesis of PSIDAB was to immerse the fibrous membrane in the solution of a crosslinker to elicit crosslinks between the polymer chains inside the fibers. To check whether this hypothesis was right, previously prepared PSI fiber membranes were immersed in a 0.5 M DAB/EtOH solution for different periods and then washed with DMF to confirm dissolution. DAB is a feasible crosslinker for PSI as proven in some published works [18, 37]. PSI is soluble in N,N-dimethylformamide, dimethyl sulfoxide and partly in N-methylpirolidone. To prevent PSI from dissolving, we chose ethanol as the reaction solvent in which the crosslinking process is a heterogeneous reaction. After a 1- and 5-minute long immersion time in DAB/EtOH, samples dissolved in DMF. Only after a 10-minute immersion time did samples remain intact when immersed in DMF. In other words, the minimal reaction time for crosslinking of this system is 10 minutes. However, to ensure full crosslinking and reproducibility, 1 hour of crosslinking time was chosen at the preparation of PSIDAB for the in vivo experiments as no significant difference was found in either shape or morphology between the 10 minute and 1hour samples (S1 Fig in S1 File). The average fiber diameter of PSIDAB was slightly smaller (800 ± 30 nm, (n = 50)) (Fig 2C), compared to the PSI fibers used in this series (average fiber diameter was 911 ± 41 nm, (n = 50)), since the more crosslinks there are in a hydrogel, the more it shrinks.

3.2. Preparation of PASPCYS and PASPDAB membranes

Our objective was to synthesize fibrous samples that can be easily degraded in vivo (fast biodegradation) and samples whose degradation takes a longer time (slow biodegradation). PSICYS and PSIDAB differ from each other in both synthesis and chemical structure, which leads to different reactions in vivo. Disulfide bridges, such as the ones in PSICYS, are often in the focus of drug delivery research since those are cleavable in redox environments found in vivo [18, 37]. On the contrary, PSIDAB contains crosslinks that are not sensitive to such conditions and thus theoretically cannot be cleaved that easily, resulting in longer in vivo biodegradation times. Juriga et al. demonstrated that PASPCYS bulk hydrogels degrade in a collagenase type I solution but also in minimal essential media used for cell cultivation, which supports the original idea of the fast dissolution of PSICYS membranes and slow degradation of PSIDAB samples [25]. The membranes in the present project were implanted in the animals in PSI forms. However, for biodegradability, the membranes must first undergo hydrolysis after implantation, in other words turn into their respective PASP forms (S2 and S3 Figs in S1 File). Without hydrolysis the biodegradation would be compromised. Typically, pH in the connective tissue is approximately 7.4 while in the skin ranges between 4&#;6. However, in the case of acute inflammation due to surgery or damage to the skin tissue, it can rise to pH 7.4 or in the case of chronic inflammation even pH 8 [38]. Since PSI hydrolyzes to PASP at pH&#;7.4, it is strongly suggested that hydrolysis occurs in vivo for both PSICYS and PSIDAB samples implanted under the skin.

As samples were implanted in PSI based form, it was essential to investigate morphological changes caused by hydrolysis. To imitate these, PSICYS and PSIDAB samples were hydrolyzed in vitro as well. During this process, the PSI based membranes turn into the PASP-based membranes and swell in aqueous medium. Considerable change was observed in the fibrous structure of PSICYS after hydrolysis. During hydrolysis, the PASPCYS fibers fuse creating bundles and bulk parts along the fibers, therefore the fibrous structure was only partially maintained (Fig 2D). This is often observable in gel fiber systems in different degrees. In the case of PSIDAB fibers, although crosslinking by itself did not change the fiber morphology, hydrolysis did. As evidenced by SEM images on washed and freeze-dried PASPDAB samples (with 1 hour crosslinking time), the fusion of fibers occurred in two ways: a. in some cases where fibers happened to be parallel and touched each other they fused into flat sheets (Fig 2E) b. in other instances where fibers touched each other in any degree an interconnected fibrous structure was created at the connection points. The fusion of fibers is not an unprecedented phenomenon and it has been observed in crosslinked networks [39, 40]. Zhang et al. reported a similar method for the post electrospinning crosslinking of PSI fibers with 1,2-diaminoethane in methanol solution, where the fibrous structure was severely damaged due to hydrolysis [41]. Our work demonstrates that PSI fibers crosslinked with DAB proved to be a better option for retaining the fibrous structure of PASPDAB. It caused minimal change in the fiber morphology, which alterations should not have any effect on either the mechanical or in vivo performance of the membranes. An example for the macroscopic changes of a PSIDAB&#;PASPDAB transition can be seen in Fig 3. In this case, the fibrous membranes grew from 16 ± 0 mm to 21 ± 1 mm, which corresponds to 31 ± 7% (n = 3, p = 0.05). Similar behavior was demonstrated for PSI based bulk hydrogels [42] but also other PASP based fibrous membranes with different crosslinkers as well [27]. Further details on the swelling behavior of different size PSIDAB membranes during hydrolysis and PASPDAB membranes in different pH solutions can be found in the supporting document. The detailed chemical analysis of PSI, PSICYS, PASPCYS, PSIDAB and PASPDAB can be found in the supplementary information and in S4 Fig in S1 File.

3.3. Mechanical analysis

Assessment of the mechanical properties of an implant is crucial before commencing in vivo experiments. While different methods are available to measure the stiffness, we chose uniaxial stress-strain measurements so we could obtain real-world practical results about the membranes that could be compared with in vivo tissues or market available implants and biomaterials [43]. Measurements were performed on wet PSI, PSICYS and PSIDAB membranes (all of the membranes were immersed in saline solution to mimic the environment of the in vivo experiments). In this regard, tensile strength is generally documented in one of two units: a. In Force/area (N/m2) for tissue or materials with well-defined dimensions or b. In Force/length (N/cm) for more complex materials with hard to define dimensions e.g., surgical meshes and specialized dressings. We present results as applicable.

In the first set of experiments, the maximum load-bearing capacity and the ultimate tensile strength of the raw membranes were investigated (Fig 4A and S5 Fig in S1 File). All samples exhibited similar initial behavior as their stress-strain curves are very steep, representing the high rigidity of the materials. The ultimate tensile strength of the membranes PSI was ± kPa or 12.32 ± 0.94 N/cm (n = 8, p = 0.05) of PSICYS it was ± kPa or 3.8 ± 0.9 N/cm (n = 5, p = 0.05) and of PSIDAB it was ± 390 kPa or 6.0 ± 1.0 N/cm (n = 6, p = 0.05) (Fig 4C and 4D). Since the standard errors in the measurements were quite high, we used two-sample t-test to see if there was a significant difference between the membrane types. According to the analysis, there was no significant difference between the maximum bearing loads of PSICYS and PSIDAB, however, they were both significantly different from the PSI membranes. In terms of performance the membranes are not suitable as bone, cartilage, or tendon implants or surgical meshes for hernia repair as their tensile strength is simply not high enough. Nevertheless, they are suitable as soft tissue implants. The tensile strength of the membranes is comparable to the tensile strengths of soft connective tissues and visceral tissues [43&#;45].

In the second set of experiments, a simple interrupted suture was placed on the samples (Fig 4F), which was then torn out by the mechanical tester while the force and displacement of the crosshead were registered (Suture Retention Test). Similarly, the assessed properties were the ultimate tensile strength and the maximum load-bearing capacity. Typical force-displacement curves of PSI, PSICYS and PSIDAB can be seen in Fig 4B whereas all the measured curves can be found in S6 Fig in S1 File.

In every case, the maximum bearing load of the suture retention test was considerably smaller than compared to the raw sample measurements. Ultimate tensile strength would be in theory calculated by dividing the maximum sustained load with the thickness of the suture material. However, the results would be highly overestimating the strength of the membranes. A relative tensile strength was calculated by dividing the maximum bearing loads by the thicknesses of the samples. For PSI membranes it was 91 ± 6 N/cm (n = 4, p = 0.05), for PSICYS membranes it was 87 ± 11 N/cm (n = 6, p = 0.05) while for PSIDAB membranes it was 30 ± 10 N/cm (n = 6, p = 0.05) (Fig 4E). According to two-sample t-tests, the PSIDAB once again was significantly different from the PSI and PSICYS samples that exhibited the same behavior. The load-bearing capacity of the sample may seem exceedingly small, but it is worth mentioning that no standardized criteria regarding suture retention tests are available. The measurement was rather performed to assess whether the membranes would withstand the surgical handling and in vivo suture fixation. Apart from instrument-based evaluations, during a manual surgical maneuver and suture fixation evaluation it was apparent (Fig 4G) that the membranes will indeed resist the intervention and fixation in the animals. Additionally, it is noteworthy that increasing the thickness of the fabricated membranes, will consequently result in an increase of their tensile strength as well. On the contrary, bulk hydrogels will still tear under the effect of sutures as their susceptibility to suturing is not in correlation with their thickness but with their texture.

3.4. In vitro experiments

Initially, both PASPCYS, PSIDAB and PASPDAB membranes were planned to be investigated. Although PASPCYS was stable in PBS for several days, it slowly degraded and lost its integrity completely during 8 days in the MEM solution (S7 Fig in S1 File). In contrast to this, PASPDAB could maintain its physical properties. This is in line with the expected biodegradability of the two materials. PSIDAB underwent hydrolysis and turned into PASPDAB, while shifting the pH of MEM toward the acidic region indicated by the MEM turning yellow.

By applying fluorescent pre-labelling, the cells could be visualized by multiphoton microscopy (Fig 5). On the plastic surface of tissue culture wells (Fig 5A), many healthy cells showing normal, star-like morphology can be seen. However, we could not find any cells on the PSIDAB membranes (Fig 5B), supposedly in this case the cells were only able to loosely attach to the membranes and all of them were removed during the fixation process including several washing steps. In addition, PSI based membranes shifted the pH value of the cell culture medium into the acidic range (its color always turned to yellow) indicating that these membranes do not support the survival of the cells under in vitro conditions. Nevertheless, large number of cells with healthy star-like morphology were observed on the PASPDAB membranes (Fig 5C and 5D). Fig 5E shows the control PASPDAB sample to demonstrate how the fibrous structure would look like without the cells.

The results of the viability assays (Fig 5F) show that the viability of MG-63 cells seeded onto PSIDAB and PASPDAB were similar to the control 1 day after the seeding. PASPDAB membranes showed no signs of cytotoxicity as the MG-63 osteosarcoma cells were able to attach to their surface and proliferate. These results are in agreement with our previous study, where biocompatibility of PASPDAB based bulk hydrogels was proven using the same cell line [25]. On the contrary the viability on PSIDAB decreased by day 3 (Fig 5F). As it was mentioned earlier, the MEM turned yellow due to the hydrolysis of PSIDAB marking a shift in pH toward the acidic range. MG-63 cells are not viable in acidic MEM which possibly caused the cell death, and a documented low viability. Therefore, based on this experiment it cannot be decided whether PSIDAB is cytotoxic as cell death most probably was caused by the pH shift. Nevertheless, this phenomenon is amplified in an in vitro setting as no fluid exchange is available. In contrast, in an in vivo environment, the body can readily compensate the lower pH. In this regard the results of the in vivo investigation should give further insight whether the shift is pH tolerable and whether meshes are biocompatible.

3.5. In vivo experiment

The purpose of the animal experiments was to investigate the surgical applicability of PSICYS and PSIDAB based membranes as suturable implants as well as to evaluate biocompatibility and biodegradability. The most important results of the in vivo studies are summarized in Table 2. The intra-operative surgical handling was easy for both materials and implantation took place without any difficulties. Samples were very flexible, and they could be rolled up or twisted in various degrees therefore their application is not limited to open surgical procedures but could be easily used for laparoscopic interventions as well (Fig 4D). Fixation with sutures was successful without any considerable damage to the implant. During the fixation suturability of the membranes was excellent and neither the sutures were torn out, nor the membranes were damaged. During the 3-day (Group A) and 7-day (Group B) observation no visible irritations, animal misbehavior or other macroscopically observable complications were detected. The animals behaved just as they did before surgery: normal food intake and bowel movement, mobility, and behavior with the caretakers were observed.

Table 2. Collection of results of in vivo experiments, where at the biocompatibility rows the determined scores can be seen inside the brackets.

PSICYS PSIDAB IMPLANTATION Handling and surgery Easy handling, good suturability, no difficulties AFTER 3 DAY Macroscopic results Hydrolysis and swelling by approximately 40% in size Biocompatibility Moderate acute inflammation (1.83) Moderate acute inflammation (2.17) Narrow band of granulation tissue (1.33) Narrow band of granulation tissue (1.00) Biodegradation No observable degradation Tissue invasion No tissue invasion according to histopathology No tissue invasion according to histopathology
Attachment of cells to the surface of the samples AFTER 7 DAY Macroscopic results Samples either not found or completely covered and incorporated by new tissue Samples lost their integrity and strength Easy truncation by tweezers Biocompatibility Mild acute inflammation (1.17) Mild acute inflammation (1.33) Moderate band of granulation tissue (1.50) Narrow band of granulation tissue (1.33) Biodegradation Although degradation was observable macroscopically, histopathology showed the remnants of fibrous samples at implantation area Degradation was not observable, however the loss of mechanical strength and integrity indicates it Tissue invasion Not according to histopathology Cells invaded the samples for several millimeters Incorporation of samples into the granulation tissue

3.5.1. 3-day results

Three days after implantation (Group A) animals were terminated, implantation area was investigated, and the samples were resected. Both PSICYS (Fig 6A and S8 Fig in S1 File) and PSIDAB (Fig 7A) samples were found in their respective animals with physiological wound healing without any observable complications. There were no visible differences between the samples in different groups. However, both sample types grew by approximately 40% in diameter (from 16 mm to ~ 22 mm) and turned from the implanted white paper-like membranes to soft swollen gels (Figs 6A, 7A and 7B). Similar size change (16 mm to ~21 mm) due to hydrolysis was observed in vitro for both samples (Fig 3A and 3B, PSICYS not shown). This strongly suggests that hydrolysis of PSI based systems occurs in vivo, which is the first step in the biodegradation of PSI based materials. PSICYS and PSIDAB did not cause any irritation in the animals macroscopically nor created excessive skin tissue.

When a foreign object is implanted into a body, a natural response, the so-called foreign body type reaction is elicited [46]. Usually, it is a mild inflammation with granulocytes and histiocytes trying to restore the damaged area to its natural state for example by attacking or segregating the foreign object in a capsule (fibrosis) [47]. Although there is always some degree of foreign body type reaction and mild inflammation after any operation it is highly desirable to keep it minimal. Histopathological investigation as expected, revealed an acute inflammation in the tissue surrounding both types of samples further proving a physiological response of the animals to the implants. However, in neither case was this inflammation considered as a strong reaction. According to the scoring of Planck et al. [36] this inflammatory response is considered mild or moderate in the case of the PSIDAB samples (Table 2). PASPCYS samples were easily found on the slides surrounded by a rim of inflammatory cells predominantly comprising neutrophil granulocytes (Fig 6C and S9 Fig in S1 File), yet the cells did not invade the membrane. In the case of the PSIDAB samples the inflammatory reaction of the body was stronger, with a mixture of neutrophil granulocytes, lymphocytes and histiocytes surrounding and invading the outer part of the membrane, making it difficult to distinguish the tissue and the implant (Fig 7D and S10 Fig in S1 File, where the black arrows indicate the supposed boundary between the sample and the tissue). Around both membranes a narrow (Table 2). fibroblastic reaction and granulation tissue are visible (Figs 6 and 7), which will become new connective tissue. It is worth emphasizing, that no giant foreign body cells were observed in any of the samples for either membrane.

3.5.2. 7-day results

Although similar results to the 3-day group were expected after 7 days group as well, significant differences were found between PSICYS and PSIDAB samples. In the case of PSICYS membranes (or in this state PASPCYS, because it was evidenced by the 3-day experiment that hydrolysis takes place) the samples were either not found in the animals (2 cases) or a soft new tissue was found in their place (4 cases) (Fig 6B). The soft granulation tissue grew around the suture, but it was easily deformed and removed. Upon cutting it half, there was no evidence for the presence of any PASPCYS remnants observable with the naked eye leading to the conclusion that these samples degraded and dissolved in 7 days. Furthermore, no signs of irritation, infection, or foreign body reaction neither on nor around the PASPCYS samples was found. Although macroscopically PASPCYS samples were impossible to find in the implantation area after 7 days, microscopically a fibrous substance (supposedly the PASPCYS matrix) was found with a moderate band of granulation tissue surrounding it (Fig 6D and S11 Fig in S1 File). In this state, the previously moderate inflammation declined to mild inflammation, while neovascularization (Table 2) was found in the granulation tissue. The new vessels formation (neovascularization) combined with the fact that no giant foreign body cells were found, highly indicates a healthy granulation tissue progressing to the proliferative phase of physiological wound healing.

In comparison, PASPDAB samples were found in a swollen, softer and more fragile state than their 3-day counterparts (Fig 7C). Therefore, hydrolysis has indeed occurred turning PSIDAB into PASPDAB. These samples were easily torn and dissected into separate layers or truncated by tweezers suggesting a high reduction in physical strength and consistency (S12 Fig in S1 File). Due to the deformation of the samples and incorporation of the surrounding tissue, it was hard to distinguish the samples from the granulation tissue macroscopically. Just as in the case of PSICYS, there were no macroscopic signs of irritation, infection, or foreign body type reaction neither on nor around the PASPDAB samples. Histopathology revealed the resolution of the acute inflammation observed on the 3rd day post-implantation to a mild level (Table 2). Furthermore, the incorporation of the granulation tissue into the sample evolved into a stage where the boundary between the sample and granulation tissue was almost impossible to mark exactly (Fig 7E and S13 Fig in S1 File). The living tissue being able to invade the membrane is an exceptional result as it suggests a proper tissue integration. When examining biomaterials, a very frequent phenomenon is the encapsulation and segregation of the implant via a fibrous capsule which is not considered as true tissue integration [48]. Additionally, giant foreign body cells were not found in the granulation tissue surrounding these samples either, further supporting the results. We found that the granulation tissue band was still narrow even when not considering the part which grew into the samples. However, the thickness of granulation tissue surrounding the samples may have been just inaccurately determined. To determine the exact composition of the PSIDAB matrix after 7 days, a small sample was gathered with a tweezer and investigated by multiphoton- and scanning electron microscope without any treatment (Fig 8). Both the fibrous sample and the cells surrounding it were visible under the microscope glowing in green with similar intensities, and thus it was not possible to properly distinguish the two by digital subtraction. Nonetheless, the retained fibrous structure of the DAB crosslinked sample was still observable. As a reference, the PASPDAB fibrous microstructure can be found as prepared in vitro (without the cells) under the multiphoton microscope in Fig 5F.

This phenomenon was also observed on the scanning electron microscopy investigations of the PASPDAB samples resected 7 days post-implantation (Fig 8B and 8C). The depicted cells are impossible to recognize and classified as they were possibly flattened due to the preparation process [46]. It is also observable in the figure that under the cellular shell the fibrous structure was intact without any observable degradation or morphological change. This was also similar to the fibrous structure of the PASPDAB membranes prepared in vitro (Fig 5F). Furthermore, many spheres were found with 6&#;8 μm in diameter suggesting the presence of either granulocytes, lymphocytes or red blood cells inside the matrix close to the surface, which is understandable given that histopathology showed vascularization around these samples. Therefore, these are the most probable corpuscular elements (based on the literature) with comparable size with the ones found on the SEM images (Fig 8D) [49, 50].

When comparing the two membranes we can clearly see a difference in in vivo behavior. The shorter and longer biodegradation times of PSICYS and PSIDAB accordingly are evident. This verifies our hypothesis that both PSIDAB and PSICYS systems hydrolyze and swell in vivo turning into the corresponding PASP based systems. The exact degradation mechanism of PSICYS has yet to be clarified, while PSIDAB maintains its physical form after 7 days implanted under the skin showing reduction in physical strength and consistency, thus showing signs of degradation.

4. Conclusion

Poly(aspartic acid) (PASP) hydrogel systems possess advantageous properties such as biodegradability, biocompatibility and easy functionalization. In this work we have presented the fabrication and characterization of electrospun crosslinked fibrous polysuccinimide (PSI) membranes. These membranes can readily hydrolyze into PASP fibrous hydrogels. These provide a reliable system during the implantation and a template even closer to the body&#;s innate ECM. Two types of PSI fibrous membranes were prepared: one based on disulfide crosslinks exhibiting fast biodegradation and another based on alkyl-chain crosslinks showing longer biodegradation time. According to mechanical assessments, both membranes showed adequate mechanical properties for suturing and surgery. In vitro tests showed that the disulfide crosslinked membrane dissolves in cell culture conditions in 8 days whereas the alkyl-chain crosslinked one was still stable. Furthermore, the membranes exhibited no cytotoxic side effects as MG-63 osteosarcoma cells could attach to their surface and freely proliferate. Implantability was tested in vivo on small animals. Membranes were implanted in Wistar rats, under the skin at the backside of the neck. The membranes changed into PASP based membranes in 3 days and after 7 days most of the disulfide crosslinked membranes disappeared, proving biodegradation. Histopathologic examinations in both cases showed only a mild to moderate acute inflammation, which diminished after 7 days. Moreover, excessive tissue invasion was also observed proving the biocompatibility and tissue integration capabilities of these systems. These results coupled with the easily modifiable chemical structure of the poly(amino acid) based system makes them ideal for advanced functionalized materials and platforms for biomedical research.

Supporting information

Data Availability

All relevant data are within the manuscript and its S1 File and S1 Graphical abstract files.

Funding Statement

This work was supported by the National Research, Development and Innovation Office (NKFIH FK ), the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (JHA) and by the new national excellence program of the Ministry for Innovation and Technology (ÚNKP-20-5-SE-9). The research was further financed by the Higher Education Institutional Excellence Programme of the Ministry for Innovation and Technology in Hungary, within the framework of the Therapeutic Development thematic programme of the Semmelweis University and by EFOP-3.6.3-VEKOP-16--.

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. Nov 12;7:755. doi: 10./fchem..

Hydrogels Based on Poly(aspartic acid): Synthesis and Applications

Hossein Adelnia

Hossein Adelnia

1Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia 2School of Pharmacy, University of Queensland, Woolloongabba, QLD, Australia Find articles by Hossein Adelnia 1,2, Idriss Blakey

Idriss Blakey

1Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia Find articles by Idriss Blakey 1, Peter J Little

Peter J Little

2School of Pharmacy, University of Queensland, Woolloongabba, QLD, Australia 3Department of Pharmacy, Xinhua College of Sun Yat-sen University, Guangzhou, China Find articles by Peter J Little 2,3, Hang T Ta

Hang T Ta

1Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia 2School of Pharmacy, University of Queensland, Woolloongabba, QLD, Australia Find articles by Hang T Ta 1,2,*

1Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia 2School of Pharmacy, University of Queensland, Woolloongabba, QLD, Australia 3Department of Pharmacy, Xinhua College of Sun Yat-sen University, Guangzhou, China

Received Aug 21; Accepted Oct 22; Collection date .

Copyright © Adelnia, Blakey, Little and Ta.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

PMCID: PMC  PMID:

Abstract

This review presents an overview on the recent progress in the synthesis, crosslinking, interpenetrating networks, and applications of poly(aspartic acid) (PASP)-based hydrogels. PASP is a synthetic acidic polypeptide that has drawn a great deal of attention in diverse applications due particularly to its biocompatibility and biodegradability. Facile modification of its precursor, poly(succinimide) (PSI), by primary amines has opened a wide window for the design of state-of-the-art hydrogels. Apart from pH-sensitivity, PASP hydrogels can be modified with suitable species in order to respond to the other desired stimuli such as temperature and reducing/oxidizing media as well. Strategies for fabrication of nanostructured PASP-based hydrogels in the form of particle and fiber are also discussed. Different cross-linking agents for PSI/PASP such as diamines, dopamine, cysteamine, and aminosilanes are also introduced. Finally, applications of PASP-based hydrogels in diverse areas particularly in biomedical are reviewed.

Keywords: hydrogels, poly(aspartic acid), poly(succinimide), crosslinking, nanoparticles, interpenetrating polymer networks (IPNs)

Introduction

Hydrogels are 3-D networks composed of water-soluble polymer chains linked together by chemical or physical bonds. They are employed as carriers for delivery of bioactive agents, as wound healing films, bio-sensing materials, implants, and scaffolds in tissue engineering, etc. (Ullah et al., ; Wang L. et al., ; Al Harthi et al., ). In the presence of water, instead of dissolution, hydrogels are swollen from a few to several times of their own dry weight depending on the crosslinking degree. Similar to water-soluble polymers, based on their chemical structures, hydrogels can be divided into anionic (Ullah et al., ), cationic (Qi et al., ), non-ionic (Golabdar et al., ), and zwitterionic (Vatankhah-Varnosfaderani et al., ).

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Anionic polymers, which are essentially poly(acid)s, show globule to coil transition upon pH increment and/or reduction of ionic strength (Abu-Thabit and Hamdy, ; Meka et al., ). This behavior is reflected in hydrogels as swelling when the polymer is cross-linked (Varaprasad et al., ). Aside from polysaccharide-based anionic polymers, most of anionic hydrogels are not biodegradable, thereby posing environmental problems and creating pollution challenges in the long-term (Guilherme et al., ; Pakdel and Peighambardoust, ). Therefore, seeking a suitable substitute that is biodegradable and non-toxic is of outmost importance.

Poly(aspartic acid) (PASP), a synthetic poly(amino acid) with a protein-like amide bond in its backbone, and a carboxylic acid as a pendant group in each repeating unit, has drawn a great deal of attention and so the demand for its production has significantly grown. The former bond provides PASP with degradability (Nakato et al., ; Tabata et al., , ), while the latter groups gives the polymer acidic properties and negative charge (Yang et al., ; Sattari et al., ). Various enzymes such as trypsin (Zhang C. et al., ), chymotrypsin (Wei et al., ), dispase and collagenase I (Juriga et al., ), as well as different media such as activated sludge (Alford et al., ) and river water (Tabata et al., ) have been examined for biodegradation of PASP-based hydrogels and polymers. Depending on the condition (e.g., enzyme concentration and temperature), complete degradation varies from a few days to one month.

PASP hydrogels typically exhibit the same response to pH and ionic strength as other anionic ones, such that higher swelling ratio can be achieved by increasing pH or lowering ionic strength (Zhao et al., ; Sharma et al., ). This property, which is called polyelectrolyte effect, stems from ionization of carboxylic groups. Ionization (or de-protonation) creates negative charges along the chain/network, causing extended chain conformation and globule to coil transition. On the other hand, high ion concentration shields the network charge and lowers swelling (Yang et al., ). Additionally, by changing crosslinking density, mechanical properties and swelling could be tuned (Vatankhah-Varnoosfaderani et al., ). More importantly, PASP hydrogels can be readily modified with a wide variety of species to meet the need of any given application. This feature arises from highly reactive imide rings in the intermediate poly(succinimide) (PSI), which allow grafting of different molecules bearing primary amine group under mild conditions without using any catalyst. Therefore, considering facile modification, biocompatibility, and biodegradability, PASP-based hydrogels offers potential advantages over conventional anionic hydrogels [e.g., poly(acrylic acid)] and can be considered as a promising choice for hydrogel preparation in diverse applications.

In the light of the aforementioned features, in this paper we provide a review on the synthesis, gelation process, cross-linking agents, and recent applications of hydrogels based on PASP.

Synthesis of Polyaspartic Acid

PASP homopolymer is generally synthesized through poly-condensation of aspartic acid (ASP) monomer or polymerization of maleamic acid which is produced from maleic anhydride and a nitrogen source like ammonia or urea as shown in Figure 1. Whatever the method is, the reaction yields the intermediate poly(anhydroaspartic acid), i.e., poly(succinimide) (PSI). The subsequent alkaline hydrolysis of PSI leads to imide ring opening through either carbonyl groups, resulting in a mixture of α and β. Also, as mentioned, PSI can easily undergo a nucleophilic reaction with primary and secondary amines without catalyst even at room temperature to yield poly(aspartamide) derivatives, allowing one to tailor-make PASP to be exploited as a versatile and multi-functional hydrogels (Feng et al., ; Nayunigari et al., ; Zhang S. et al., ).

Poly-Condensation of Aspartic Acid

Thermal poly-condensation of ASP at elevated temperatures (typically higher than 160°C) can either be conducted in bulk (Nakato et al., ; Zrinyi et al., ) or in solution (Low et al., ; Tomida et al., a) in the presence or absence of a catalyst. The reaction by-product, i.e., water, should be eliminated during the course of polymerization. The most effective solvent and catalyst have been found to be the mixture of mesitylene/sulfolane (7/3, w/w) and phosphoric acid, respectively (Tomida et al., a). The reactions catalyzed by phosphoric acid yield linear chain whereas uncatalyzed reactions lead to branching (Wolk et al., ). High temperature, high catalyst, and aspartic acid monomer concentration can significantly increase molecular weight (Mw) (Jalalvandi and Shavandi, ; Yavvari et al., ). Recent studies have also shown when phosphoric acid is utilized as both catalyst and polymerization media (aspartic acid monomer: phosphoric acid, 1:1), PASP with high Mw and reaction yield is achieved (Zakharchenko et al., ; Moon et al., ; Szilágyi et al., ). It is noteworthy to mention that the use of solvent though improves heat transfer, it may reduce the reaction rate, as the availability of functional groups (NH2 and COOH) is reduced (Stevens, ). Additionally, solvent should be removed after the reaction by washing polymer. Therefore, bulk reaction under batch and continuous (through extruder) conditions is preferable in industry (Kokufuta et al., ; Nakato et al., ; Zrinyi et al., ).

Polymerization of Maleamic Acid/Ammonium Salt of Maleic Acid

The second method involves polymerization of maleamic acid without catalyst for 6&#;8 h at high temperature (>160°C), during which period water is removed by distillation (shown in Figure 1) (Koskan and Meah, ; Wood, ; Boehmke and Schmitz, ; Ni et al., ; Shi et al., ). Maleamic acid is prepared by reacting maleic anhydride (MA) (or maleic acid) with anhydrous ammonia or urea as a nitrogen source or heating the monoammonium salt of maleic acid. This method was first introduced as a patent by Boehmke, where PASP was synthesized with a relatively low degree of polymerization 15&#;20%, using ammonia (AN) and MA which was heated in water (at 75°C) to change to maleic acid (Boehmke, ). The reaction is typically carried out without solvent in a reactor, oven (Freeman et al., ), or under microwave irradiation (Huang et al., ). Although this method employs industrially inexpensive and available raw materials such as maleic anhydride and ammonia, it gives low yields and low molecular weight (Boehmke, ; Koskan and Meah, ; Wood, ; Boehmke and Schmitz, ; Freeman et al., ; Ni et al., ; Huang et al., ; Shi et al., ).

Crosslinking

Commonly, hydrogels based on PASP are prepared either via crosslinking of PSI followed by alkali hydrolysis, or by crosslinking of PASP itself. Various types of crosslinking agents can be used. Because of the simplicity, the agent is generally introduced by PSI modification or the gelation process itself is carried out on PSI followed by alkali hydrolysis.

Hydrogels Based on Diamines

Simple succinimide ring opening by primary amines allows the use of various diamines for the synthesis of a PASP hydrogel (Jalalvandi and Shavandi, ). This reaction occurs at room temperature without requiring any catalyst (Fang et al., a,b). Gyenes et al. () employed different natural amines and amino acid derivatives such as putrescin, spermine, spermidine, lysine, and cystamine for crosslinking. They indicated that the cystamine-based hydrogels dissolve above pH 8.5 as the disulfide linkage breaks under alkaline media. Gyarmati et al. () reported the synthesis of super-macroporous PASP hydrogels using 1,4-diaminobutane as a cross-linker under cryogenic condition of DMSO. Phase separation was induced by freezing DMSO as the solvent of PSI. As a result, highly porous interconnective hydrogels (pore size 9&#;259 μm) was fabricated, which is useful for in vitro cell seeding with pH-induced detachment of the grown cells. In a similar study, aside from chemical crosslinking with hexamethylenediamine (HMDA), freeze/thaw technique was also applied to induce phase separation and physical crosslinking (Zhao and Tan, ). Swelling behavior was highly affected by changing freeze/thaw cycle number, time, and temperature. Chen et al. () also prepared PASP superabsorbent cross-linked by HMDA in the presence of organic bentonite (OB) with high swelling capacity (491 g/g in water). It was shown that OB can serve as a crosslinker due to its surface amine groups since high OB content (above 3%) led to lower swelling.

Hydrogels Based on Disulfide Bond

Crosslinking through disulfide or thiol containing agents endows an interesting feature to the PASP-based hydrogels. The reaction of thiol to disulfide can be carried out under application of a reducing agent. This reaction can be reversed in the presence of an oxidizing agent. Therefore, PSI is generally modified with thiol groups (cysteamine or cystamine) for the preparation of reducing/oxidizing-responsive PASP hydrogels (Molnar et al., ). In order to maintain structural integrity in different media, a permanent linker such as a diamine can be employed (Figure 3A; Zrinyi et al., ; Krisch et al., ). Recently, such dual cross-linked hydrogels have drawn a great deal of attention due to swelling under reductive state. For instance, Zrinyi et al. () synthesized PASP with diaminobutane (DAB), and cystamine (CYS) as permanent and cleavable crosslinkers, respectively. They showed that disulfide bonds arising from the latter is broken by the addition of a reducing agent, leading to an increase in swelling and a decrease in modulus. Likewise, redox- and pH-responsive PASP hydrogels were prepared by dual crosslinking using cysteamine, and 1,4-diaminobutane which creates reversible and irreversible bonds, respectively (Gyarmati et al., ). It was indicated that swelling degree of hydrogel and elastic modulus can be tuned by reducing/oxidizing agents without hydrogel disintegration/dissolution. Swelling increased as pH increased both under oxidized and reduced states. However, under the latter condition, swelling was higher. The hydrogels maintained their mechanical stability under repeated redox cycles for at least three cycles and the reversibility was shown to be independent of initial redox state of PASP (reduced or oxidized) (Figures 2A,B). Krisch et al. () employed poly(ethylene glycol) diglycidyl ether (PEGDGE) for crosslinking thiolated PASP in order to secure structural integrity of the hydrogels in reducing media. A part of thiol groups were reacted with the former to establish a non-cleavable gel junction while the remaining ones were oxidized into breakable disulfide bonds. It should be noted that the epoxide groups with thiol groups form unbreakable S-C bonds.

Hydrogels Based on Dopamine

Catechol moieties in dopamine exhibit a multifunctional characteristic for the design of mussel-inspired coatings (Ryu et al., , ; Saiz-Poseu et al., ). Complex formation of catechol with boron and/or iron ions (Fe3+) can be employed for hydrogel preparation (Vatankhah-Varnoosfaderani et al., ; Krogsgaard et al., ). Injectable dopamine modified PASP hydrogels with superior adhesive character were synthesized by complexation with Fe3+ ions (gelation time around 1 min; Figure 3B; Gong et al., ). It was suggested that the resulting crosslinking are composed of both Fe3+ coordination as well as covalent quinone-quinone bonds. Boric acid was also shown to crosslink dopamine-modified PASP and yield hydrogels due to boron&#;catechol coordination (Wang B. et al., ). The prepared hydrogels had autonomous self-healing feature due to such a coordination.

Other Hydrogels

Apart from diamine and disulfide crosslinking, other strategies have also been developed for fabrication of PASP hydrogels. The reaction of hydrazine and aldehyde, radical polymerization of pendant double bond, sol-gel reaction of aminosilane, and application of gamma-irradiation are some examples that will be discussed in this section for preparation of PASP hydrogels.

Lu et al. () prepared injectable PASP-based hydrogel through introduction of hydrazine and aldehyde to PSI backbone by hydrazine hydrate and 3-amino-1,2-propanediol, respectively. Therefore, hydrazine and aldehyde modified PASPs were used as two gel precursors as shown in Figure 3C. The same strategy was used for crosslinking of oxidized alginate and polyaspartamide conjugated with RGD peptide (Jang and Cha, ).

Conventional radical polymerization of allyl amine monomer grafted onto PSI can also lead to crosslinking (Figure 3D; Umeda et al., ; Némethy et al., ). Minimum value of allyl amine content was found to be 5% for gel formation (Umeda et al., ).

Gamma-irradiation can be typically utilized for crosslinking of polymers as it delivers high amount of energy and is capable of forming free radical on polymer backbone. Using such radiation (dosage of 32&#;100 kGy), Tomida et al. (b) prepared PASP hydrogel (Figure 3E). It was shown that the reaction should be conducted under N2 atmosphere as oxygen scavenges free radicals. It was also found that low polymer concentration, as well as low Mw does not lead to gelation and also acidic conditions destabilize the generated radicals.

γ-aminopropyltriethoxysilane (APTS) (an aminosilane) is generally used for attachment of organic/inorganic materials, and surface modification (Adelnia et al., ; Bidsorkhi et al., ). Its amine and hydroxyl groups make it an excellent candidate as a linker. Meng et al. () introduced APTS on PSI backbone and used it as a crosslinker for PASP gel formation (Figure 3F).

Ethylene glycol diglycidyl ether (EGDGE) can also react with PASP to yield hydrogels (at 180°C for 30 min, dry state, pH before drying 5&#;6.5) (Chang and Swift, ). As the degree of ionization of PASP as well as the protonation of epoxide ring is highly dependent on pH, crosslinking occurs at optimum pH of 5&#;6.5. Acidic media hydrolyse epoxide group whereas alkaline media reduce the protonated acid group concentration required for nucleophilic attack on the epoxide ring. Meng et al. () however, utilized EGDGE for PSI crosslinking and compared it with hydrazine as a diamine. The produced bonds of the former and the latter are ester and amide, respectively. The PASP hydrogels with the latter had faster swelling kinetic, while lower stability in terms of maintaining the absorbed water.

PASP Hydrogel Nanostructures

PASP Hydrogel Particles (i.e., Nanogels and Microgels)

Hydrogel nanoparticles (i.e., nanogels) can find much more applications compared to their own bulk counterpart especially as a carrier for delivery of bioactive agents (Cuggino et al., ; Molaei et al., ). Preparation of such structures is generally carried out via inverse type emulsion techniques where aqueous phase containing hydrophilic polymer is dispersed in an organic solvent (typically hydrocarbons such as hexane) containing emulsifier (e.g., span-80; Krisch et al., ). Formation of small droplets/particles requires high shear stress which can be applied through high speed homogenizer or sonication. Schematic representation of typical emulsification is drawn in Figure 4A. Network formation should be conducted after particle formation as premature crosslinking leads to bulk gelation and does not allow emulsification. For example, Krisch et al. () prepared nanogels of thiolated PASP by inverse miniemulsion (water in n-hexane). After particle formation, the thiolated groups of PASP were oxidized by sodium bromate NaBrO3, giving rise to gel formation. To maintain structural integrity of nanogels in reducing media, the same group utilized poly(ethylene glycol) diglycidyl ether for crosslinking of a part of S-H groups. The nanogels diameter increased in reducing media due to disulfide bond breakage and thus swelling while maintaining nanogel integrity (Figure 4B; Krisch et al., ).

Inverse emulsion method, though effective in preparation of small size and uniform particles, is accompanied with several problems such as using toxic organic solvent, relatively high amount of emulsifier, and the need for medium substitution to water. Therefore, alternative methods need to be adopted. Since PSI is hydrophobic, its particles can be formed in aqueous media (Hill et al., ). PASP-g-PEG hydrogel nanoparticles were fabricated via self-association of hydrophobic PSI units in water (i.e., micelle formation), followed by their hydrolysis (Park et al., ). The particles were crosslinked with HMDA or cystamine. PASP particles can also be formed via self-assembly with cationic polymers such as chitosan, as it is an anionic polyelectrolyte. Such an electrostatic self-assembly (also referred to as ionic gelatification) can yield composite nanoparticles in water through polyelectrolyte complexation (i.e., electrostatic charge attraction of the two polymer; Zheng et al., ; Zhang et al., ; Wei Wang et al., ). PASP/chitosan particles were prepared by drop-wise addition of chitosan to PASP solution. When chitosan/PASP ratio increased from 0.75 to 2.5, the size increased from 84 to 1,364 nm (Hong et al., ).

PASP Hydrogel Nanofibers

Regarding the fiber formation, since gels cannot flow, network formation should be conducted after fiber formation similar to the emulsification mentioned above. A typical PASP-based fiber preparation is exhibited in Figure 4C. In this method, PSI is dissolved in its solvent (commonly DMF or DMSO) and electrospun into nanofibers. The resulting PSI nanofibers are cross-linked in this step, employing a suitable agent (e.g., ethylenediamine) and converted to PASP by alkali hydrolysis (Zhang et al., ; Zhang C. et al., ). Interestingly, Zhang C. et al. () found that inter-fiber crosslinking can also occur, resulting in hydrogels with interconnected microporous structure. This causes higher deformation, swelling kinetic, and swelling ratio compared to hydrogel films. Another study revealed that crosslinking can also be carried out during electrospinning for cysteamine-grafted PASP (diameter 80&#;500 nm; Molnar et al., ). However, relatively lower polymer concentration (15 wt.%) compared to conventional electrospinning process should be used to avoid premature gelation.

Interpenetrating Polymer Network (IPN) Based on PASP

Interpenetrating polymer networks (IPN) are a class of materials composed of two chemically distinct, but highly compatible polymers that are uniformly mixed in each other in microscopic scales without any phase separation. IPNs are divided into semi-IPNs and full IPNs, in which one or both components are cross-linked, respectively (Roland, ). IPNs are generally fabricated to take advantage of the features of both components.

For instance, poly(N-isopropylacrylamide) [poly(NIPAAm)] which is a well-known polymer with LCST at around physiological temperature, can be introduced for providing the IPN with temperature sensitivity. Liu et al. () prepared NIPAAm/PASP IPN hydrogels that show response both to pH and temperature. They first cross-linked PSI with a diamine, followed by its hydrolysis to PASP hydrogel. The hydrogel was then swelled with NIPAAm monomer/N,N&#;-methylene bisacrylamide (MBA) crosslinker followed by their polymerization. Némethy et al. () synthesized NIPAAm/PASP co-network hydrogels by grafting allyl amine monomer onto PSI backbone followed by its radical polymerization with NIPAAm, and PSI hydrolysis. Nistor et al. () evaluated swelling degree of PASP/PNIPAAm semi-IPN as a function of pH, temperature and NIPAAm content (Figures 5A,B). Other polymers including poly(vinyl alcohol), poly(acrylic acid), and poly(acrylamide) have also been employed for PASP-based IPN preparation as summarized in Table 1. Zhao et al. () introduced PAA to PASP-based semi-IPN hydrogels by polymerization of acrylic acid and MBA as a cross-linker in the PASP solution. It was found that the swelling ratio increases with increasing PASP content as well as temperature (range of 40&#;60°C). The incorporation of high Mw PASP also inhibited gel formation due to steric hindrance. Jv et al. () indicated that semi-IPNs based on PASP/PAA possess excellent ability for removal of methylene blue and neutral red with maximum adsorption of 357.14 and 370.37 mg/g, respectively (Figure 5C). Magnetic nanoparticles of Fe3O4, were incorporated into the hydrogels for facile separation of the dye-containing solid. Lee et al. () exhibited that PASP improves mechanical properties of brittle PAAm significantly. They suggested that the addition of multivalent cations such as Fe3+, Al3+, Pb2+, Cu2+ results in ionic coordination, and thus creation of second network. Iron cation (Fe3+) had the highest impact on improving mechanical properties (Figure 5D).

Table 1.

Polymer 2 X-linker1 X-linker2 Type Characteristic References 1 N-isopropylacrylamide 1,4-diaminobutane (DAB) N,N&#;-methylene bisacrylamide (MBA) IPN Excellent pH-responsiveness Némethy et al., Hexamethylenediamine (HMDA) MBA IPN Dual pH- and temperature- sensitive hydrogel. Large porous structure; fast shrinking and re-swelling Liu et al., Diethylene Glycol diacrylate (DEGDA) Semi-IPN Dual pH- and temperature-sensitive hydrogel Nistor et al., 2 Acrylic acid MBA Semi-IPN The freezing temperature resulted in a more porous hydrogel and faster swelling/deswelling rates Lim et al., MBA Semi-IPN Improving responsive behavior of the hydrogel to alternating changes in inorganic salt, pH, and temperature Zhao et al., MBA Semi-IPN The hydrogels had dye pollutant removal ability. Magnetic NPs were added for separation of the solid after dye removal Jv et al., MBA Semi-IPN Polygorskite clay caused higher swelling rate than that of pure hydrogels of PAA/PASP Ma et al., MBA and Ethylene glycol dimethylacrylate (EGDMA) Semi-IPN MBA and EGDMA resulted in higher swelling behavior in acidic and basic medium, respectively Sharma et al., 3 Acrylamide MBA Semi-IPN Metal cations (Fe3+, Al3+, Pb2+, Cu2+) led to creation of second network, and increased mechanical strength and decreased swelling ratio of the gel Lee et al., 4 poly(vinyl alcohol) γ-aminopropyltriethoxysilane (APTS) Semi-IPN and IPN Interpenetrating PASP/PVA hydrogel resulted in higher, faster swelling ratio, and higher drug releasing Lu et al.,

PASP Hydrogel Applications

Apart from conventional and common applications of hydrogels such as hygiene, and agricultural products, PASP hydrogels can be utilized in a wide variety of biomedical engineering areas such as development of scaffolds for tissue engineering, and carriers for sustained or targeted drug delivery systems (DDS). This is mainly due to its biocompatibility, biodegradability, as well as stimuli-responsive characteristic. Regarding the latter, in particular pH- and redox/oxidation-sensitivity of PASP has been exploited for DDS (Horvát et al., ; Sim et al., ). In this section, some recent studies in these regards are presented.

PASP/PNIPAAm co-network hydrogels loaded with sodium diclofenac (DFS) showed pH sensitivity such that the release of DFS increased when the gel is delivered from stomach (pH 1.2) into the bowels (pH 7.6) (Némethy et al., ). Such a conventional pH sensitivity feature can protect both the stomach from the side effects of DFS and the drug itself from acidity of stomach. However, in another study, unusual pH-response was observed in PASP hydrogels cross-linked with hydrazine and aldehyde (Lu et al., ). The release rate of DOX was accelerated by decreasing pH from 7 to a weak acidic condition (ca. pH 5). This behavior was attributed to instability of the hydrazone bond in acidic media, resulting in loosening of gel network. DFS was also employed as an ocular drugs and loaded in in-situ gelling thiolated PASP for its sustainable delivery (Horvát et al., ). The polymer due to its negative charge showed strong mucoadhesion, as well as high resistance against lachrymation of the eye. This is attributed to mucin glycoproteins role for crosslinking (i.e., disulfide linkage). The drug release showed a burst-like profile in the first hour followed by sustained release up to 24 h. In another work, fluorescent dextran (FTIC-Dx) was loaded into thiolated PASP nanogels prepared by inverse emulsion (Krisch et al., ). Disulfide bonds were cleaved by a reducing agent for gel disintegration, and release of the loaded drug. As seen in Figure 6A, the release profile dramatically increased by the addition of DTT as a reducing agent. The same redox-response and DOX release was seen in thiolated PASP-g-PEG nanogels (Park et al., ). Under reductive intracellular conditions, the prepared nanogels were shown to have the ability to release DOX and efficiently translocated to the nucleus of cancer cells (Figure 6B). Epigallocatechin Gallate (EGCG) which is the main bioactive element of green tea and is unstable in vitro was encapsulated in PASP/chitosan particles (Hong et al., ). The release of EGCG was investigated by simulation of food ingestion pH condition. It was demonstrated that EGCG is much more effective against rabbit atherosclerosis when encapsulated into PASP/chitosan.

Jang and Cha () incorporated RGD peptide to PSI for improving 3T3 fibroblast cell adhesion. PSI was further modified with hydrazide, and subsequently reacted with oxidized alginate, bearing aldehyde groups. The Schiff reaction (i.e., aldehyde-hydrazide, yielding hydrazone bond) leads to in-situ gelation of poly(aspartamide)/alginate. As shown in Figure 6C, RGD-modified hydrogels possessed much better cell viability, adhesion and proliferation compared to un-modified hydrogels. Juriga et al. () also modified thiolated PASP hydrogels with RGD and utilized them as scaffolds for MG-63 osteoblast-like cells. It was shown that RGD introduction leads to compacted cluster formation of the cells. The prepared scaffolds provided the osteoblast-like cells with excellent condition for adhesion, viability, and proliferation.

Zakharchenko et al. () fabricated polymer tubes and encapsulated yeast cells within them. The tubes composed of bilayer cross-linked films of PSI/polycaprolactone. Upon the hydrolysis of PSI in physiological buffer environment, and conversion into PASP gels, the films self-rolled due to the produced internal stress as a result of swelling of the lower layer, i.e., PASP (Figure 6D). Such a self-rolling was exploited for cell encapsulation. Hydrolysis of PSI was shown to be step like process and initiates after nearly 8 h in PBS buffer.

PASP due to its negative charge can endow electrostatic stability to colloidal systems. For example, iron oxide (Fe3O4) nanoparticles coated with a thin layer of PASP hydrogels had improved colloidal stability (Vega-Chacón et al., ). The composite magnetic particles did not show any adverse effect on cell viability of L929 fibroblast. Also, particles exhibited response to pH, presenting them as promising candidate for magnetic drug delivery. Iron oxide nanoparticles as negative contrast agents for magnetic resonance imaging (MRI) have been employed widely for detection of diseases (Ta et al., , a,b, ; Gaston et al., ; Wu et al., ; Yusof et al., ; Zhang et al., ). Multifunctional PASP nanoparticles containing iron oxide nanocrystals and doxorubicin was also developed for simultaneous diagnosis and treatment of cancer by Yang et al. (). Iron oxide nanocrystals were loaded in PASP nanoparticles through an emulsion method using octadecyl grafted PASP, then doxorubicin (DOX), was incorporated in the magnetic PASP nanoparticles. It was shown that the DOX loaded nanoparticles exhibited high T2 relaxivity and strong cytotoxicity for cancer cells.

Due to its strong ability for chelation, PAPS nanofiber hydrogels were utilized as chemosensor for Cu2+ ions detection (Zhang et al., ). The hydrogels showed high sensitivity and selectivity to Cu2+ ions compared with other ions such as Ag+, and Ca2+ where no color change was observed (Figures 6E,F). The detection limit of as low as 0.01 mg/L was reported.

Because of its bio-degradation and water uptake, PASP hydrogels could be regarded as a promising candidate for ecological restoration and plant survival especially in arid area. Wei et al. () employed PASP hydrogel to transplant Xanthoceras sorbifolia seedlings. The survival rate and the leaf water content were improved in soils containing PASP hydrogels.

Conclusion and Prospects

Although synthesis of PASP-based hydrogels is relatively more complex than other anionic-based hydrogels such as PAA-based ones, its biocompatibility and biodegradability make it attractive particularly in biomedical applications. Though pH-responsive, PASP is further modified with other moieties to provide sensitivity to the desired stimuli as well including temperature and reducing/oxidizing media. Incorporation of other water-soluble polymers into the PASP network may also provide the final hydrogel with superior properties. Scrutinizing the literature, it is found that PASP hydrogels has not yet been employed for inhibition of scale formation in which PASP solution has exhibited promising results (Hasson et al., ). Moreover, PASP hydrogel fibers may potentially be a good candidate as scaffold for cell culture as well as tissue engineering. Additionally, due to its anionic nature, PASP-based hydrogels can be used for preparation of electrically-responsive materials (Murdan, ).

Author Contributions

HA wrote and revised the manuscript. IB and PL supervised the work. HT supervised and revised the manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Glossary

Abbreviations

APTS

γ-aminopropyltriethoxysilane

AN

ammonia

ASP

Aspartic acid

CYS

cystamine

DAB

1,4-diaminobutane

DEGDA

diethylene glycol diacrylate

DTT

dithiothreitol

DDS

drug delivery system

EGDGE

ethylene glycol diglycidyl ether

EGDMA

ethylene glycol dimethylacrylate

FTIC-Dx

fluorescent dextran

HMDA

hexamethylenediamine

MA

maleic anhydride

MBA

N,N&#;-methylene bisacrylamide

MB

methylene blue

NR

neutral red

PAAm

poly(acrylamide)

PASP

poly(aspartic acid)

PEGDGE

poly(ethylene glycol) diglycidyl ether

poly(NIPAAm)

poly(N-isopropylacrylamide)

PSI

poly(succinimde)

TMEDA

tetramethylenediamine (TMEDA).

Footnotes

Funding. This work was funded by National Health and Medical Research Council (HT: APP).

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