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Hyaluronic acid gels with tunable properties by conjugating with a synthetic copolymer
Accepted Manuscript Title: Hyaluronic acid gels with tunable properties by conjugating with a synthetic copolymer Authors: A. Diaconu, L.E. Nita, M. Bercea, A.P. Chiriac, A.G. Rusu, D. Rusu PII: DOI: Reference:
S1369-703X(17)30154-7 http://dx.doi.org/doi:10.1016/j.bej.2017.06.003 BEJ 6725
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
24-2-2017 31-5-2017 6-6-2017
Please cite this article as: A.Diaconu, L.E.Nita, M.Bercea, A.P.Chiriac, A.G.Rusu, D.Rusu, Hyaluronic acid gels with tunable properties by conjugating with a synthetic copolymer, Biochemical Engineering Journalhttp://dx.doi.org/10.1016/j.bej.2017.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Hyaluronic acid gels with tunable properties by conjugating with a synthetic copolymer A. Diaconu, L. E. Nita, M. Bercea, A. P. Chiriac1, A. G. Rusu, D. Rusu “Petru Poni” Institute of Macromolecular Chemistry 41 A, Grigore Ghica Voda Alley, RO – 700487 Iasi, Romania
Corresponding author – Aurica P. Chiriac; Telephone number: +40232217454; Fax number: +40232211299; e-mail address: [email protected]. 1
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Graphical abstract
Highlights
1. Hybrid gels with tunable characteristics were prepared using hyaluronic acid and a
copolymer. 2. The compounds were realized by a grafting-to strategy without using other chemical
additives. 3. Rheological properties, swelling behavior, and network parameters were investigated.
Abstract: Recently, injectable hydrogels became particularly attractive as biomaterials for tissue engineering and drug delivery applications. The intrinsic drug-loading capability of polysaccharide-based hydrogels and their potential use as drug delivery systems can be combined with more sophisticated techniques in order to develop effective approaches for controlled release of bioactive agents. The objective of this study was to design and to test hydrogels based on conjugated hyaluronic acid with a new synthetic copolymer, in order to further development their potential for injectable drug delivery applications. The synthesized materials, obtained by using a grafting-to strategy, were analyzed in terms of rheological
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properties, swelling behavior, contact angle and thermal properties, and the effects of their chemical composition on various properties were assessed. A good structure recovery property and a shear-thinning thixotropic behavior were identified by the rheological tests. The developed hydrogels showed potential as promising injectable biomaterials.
Keywords: hyaluronic acid, poly(itaconic anhydride-co-3,9-divinyl- 2,4,8,10- tetraoxaspiro [5.5] undecane), hydrogels, viscoelasticity.
1. INTRODUCTION
In recent years, tremendous advancements in polymer science have given rise to new opportunities to overcome some of the most fundamental limitations facing pharmacology. There is a clear demand for developing biocompatible systems that can bypass the need for systemic drug administration and are capable of delivering pharmaceutical agents at a controlled rate. Among various kinds of polymeric systems, hydrogels have attracted particular interest in terms of addressing these pharmaceutical challenges as they are a unique class of three – dimensional, polymer networks that may contain a large fraction of aqueous content within their structure [1]. Hydrogels, because of their high water content, controllable porosity and mechanical and compositional similarities between them and native soft tissues in the body, are particularly suitable for biomedical applications such as tissue engineering, controlled release of bioactive agents, bioseparation, space-filling and cell encapsulation. While these properties have motivated significant research of such materials, the practical introduction of hydrogels into the human body is a major challenge limiting their routine clinical application [2]. As a result of these limitations, many efforts have been recently invested in the design of injectable hydrogels [3,4]. These types of materials hold promising implications for improved surgery treatment and other therapies requiring defined quantities of a therapeutic payload in a site-specific and/or timecontrolled fashion – from post-surgery pain medications to drugs that require daily injection. Injectability circumvents the need for surgery to administer the hydrogel in vivo, thus reducing pain and minimizing the risks of infection and enables effective molding of the hydrogel shape in situ by the neighboring tissue to fit cavities and/or tissue [5].
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In the past years, hydrogels based on hyaluronic acid (HA) have been especially attractive as injectable biomaterials due to their unique properties, including biodegradability, biocompatibility, nontoxicity, nonadhesivity, high water absorption capacity and nonimmunogenicity [6]. Additionally, HA-based hydrogels may impart biological activity to cells, including stem cell differentiation [7]. HA macromolecule in dilute aqueous solutions adopts a random coil conformation. Long linear HA chains in more concentrated solutions form an entangled network in which there are some topological constraints on each other called entanglements. Under shear flow, these entangled coiled chains disentangle and align to the flow direction and a shear thinning behavior is registered. In such conditions, even high concentrated HA solutions are easily injected into the human body. However, due to the high water solubility, it quickly disperses when injected into fluid-filled cavities [8]. HA is also subjected to various degradation processes due to hydrolysis and enzymatic hydrolysis by naturally occurring hyaluronidase [9]. Consequently, several chemical modifications of native hyaluronan with synthetic polymers have been developed in order to control and to improve mechanical properties, degradation rate, and clearance such as crosslinking or conjugation, to obtain a more stable material maintaining at the same time its fundamental properties [10,11]. In the case of crosslinking, HA reacts with a crosslinking agent that is capable of creating covalent bonds between HA chains, whereas compounds grafted on HA chains are referred to as conjugates [12]. Biobased materials have attracted considerable attention due to the ever–growing environmental problems, thus, it is considered very important to do research on biomaterials synthesis using renewable resources. Itaconic anhydride (ITA), an unsaturated cyclic anhydride, is regarded as one of key platform chemicals derived from biomass. Also, it is biocompatible, has a bioactive nature and the inclusion of ITA into macromolecular chain structure will induce partly biodegradable character for the prepared compounds. ITA passes to nontoxic degradation products under physiological conditions, when it initially hydrolyzes to itaconic acid followed by oxidation to acetate, lactate, and carbon dioxide. In light of the above, ITA represents an excellent starting material to obtain suitable structures for biomedical applications [13]. In a previous article, we reported the synthesis of a new copolymer based on ITA and an orthoester comonomer named 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5] undecane (U), obtaining a
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new class of copolymers - P(ITAU) - which are dual sensitive to pH and temperature [14]. Furthermore, we extended our research and we developed a new hybrid hydrogel structure based on HA and P(ITAU) by using a grafting-to strategy. By opening the anhydride cycle of the copolymer with hydroxyl groups of hyaluronic acid we created chemically crosslinked hydrogels [15,16]. Comprehensive research on these systems demonstrated that they possess excellent biocompatibility as confirmed by their performance in in vivo studies and sustained drug delivery ability. In the present paper, we extend considerably our earlier studies in order to increase our overall understanding of this type of materials and to optimize a design system in order to meet the requirements for injectable systems. Using the grafting-to approach, one property can be selectively modified while keeping other properties constant, providing a highly adaptable method of engineering injectable, rapidly-gelling hydrogels for potential biomedical applications. The concentration of the polymer is a vital design parameter that affects the hydrogel formation and structure and also the syringeability profile. By modifying the polymer concentration, the hardness or stiffness of the gel can increase or decrease; so it was necessary to vary conveniently this parameter in order to obtain hydrogels with specific requirements [12].
2. EXPERIMENTAL PART
2.1. Materials All chemicals used were reagent grade and used as purchased without further purification. Hyaluronic acid sodium salt (HA) used in this work was produced by SigmaAldrich, with a purity of 99% from Streptococcus equi bacterial glycosaminoglycan polysaccharide (Mw = 1.5×106 Da – 1.8×106 Da). Itaconic anhydride (ITA) (purity 98%), 3, 9divinyl-2,4,8,10-tetraoxaspiro[5.5]
undecane
(U)
(purity
98%),
2,2′-azobis(2-
methylpropionitrile) (AIBN) (purity 98%) and the solvents – 1,4-dioxane (≥ 99.0%) and diethyl ether – were also purchased from Sigma-Aldrich. The water used in the experiments was purified using an Ultra Clear TWF UV System. 2.2. Synthesis of P(ITAU) copolymers and preparation of the gels based on P(ITAU) – grafted onto hyaluronic acid
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Poly(itaconic anhydride-co-3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5] undecane) (P(ITAU)) copolymer was synthesized by radical polymerization in solution using AIBN as initiator and 1,4-dioxane as solvent. Detailed aspects about synthesis, and structural characterization of the copolymer (by FTIR and NMR spectroscopy and other methods) were previously described [14]. Briefly, the copolymer was synthesized through a continuous radical process of polymerization with 0.05:0.07 molar ratio between ITA / U comonomers and 3.7x10-5 mmol 2,2′-Azobis(2methylpropionitrile) radical initiator content for the mentioned comonomer ratio, in a 1,4dioxane solution of 20% concentration. The synthesis was conducted under nitrogen atmosphere, at 75°C, in a constant temperature bath, with a stirring rate of 250 rpm for 17 h. Then the copolymer was separated after solution precipitation in diethyl ether, washed repeatedly with diethyl ether, and dried for 24 h in a vacuum oven at 600 mm HG. We studied the influence of the copolymer content on the development of the new hybrid hydrogels. In this context three variants of copolymer solution, with the same ratio between the comonomers and having respectively 10 wt%, 20 wt% and 40 wt% copolymer content, were obtained and tested. HA was dissolved in deionized water at 1 wt% concentration at room temperature. Grafting P(ITAU) copolymer onto HA was achieved in soft conditions by mixing the copolymer solution in 1,4-dioxane with HA water solution. The gravimetric ratio between HA and the synthetic copolymer was maintained constant at 1:2 value. The sample codes for the prepared hydrogels were chosen in accordance with the copolymer content namely P(ITAU)10_HA, P(ITAU)20_HA and P(ITAU)40_HA. The gels were purified by dialysis (cellulose dialysis membrane with molecular weight cut-off of 14,200 Da) against distilled water for 4 days changing the water twice a day to remove unreacted compounds and 1,4-dioxane. To ensure a better purification the pH and conductivity of the dialyzed water were regularly measured until they reach the values of distilled water (protonated dioxane is slightly more acidic than protonated water [17]. 2.3. P(ITAU)_HA gels characterization 2.3.1 Rheological measurements The rheological properties were assessed with a MCR 302 Anton-Paar rheometer equipped with Peltier device for temperature control and plane-plane geometry (the upper plate having the diameter of 50 mm) with a gap of 500 µm. The water evaporation was limited by using an anti-evaporation device which created a saturated atmosphere near the sample. The
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viscoelastic properties of the gels were tested at 37C by small-amplitude oscillatory shear experiments with oscillation frequency ranging from 0.1 rad/s up to 100 rad/s. Creep tests were also carried out for different shear stress values and, after the stress removal, the recovery behavior was observed. 2.3.2. Swelling measurements The swelling degree was determined in buffer solutions at a physiological temperature of 37oC and pH 7.4, 0.2 M. Each sample was immersed in 5 mL phosphate buffer. At predetermined time intervals, the samples were carefully withdrawn from the swelling medium, weighted after superficially blotting with filter paper and placed again in the same swelling medium. Measurements were continued until a constant weight was obtained for each sample. The amount of absorbed buffer solution was monitored gravimetrically. The swelling degree of a sample was calculated as follows: SD = (Wt − Wd )/Wd
(1)
where Wd and Wt are the weights of dry and wet samples at the time t. 2.3.3 Contact angle The static contact angle of the films was determined by the sessile drop method, at room temperature and controlled humidity, within 10 s, after placing 1 μL drop of water on the film surface, using a CAM-200 instrument from KSV-Finland. To obtain reproducible results for contact angle measurements, several conditions have to be fulfilled, such as: constant temperature during determinations; the same volume of solvent drops; evaluation of the contact angles in different points of the studied surface, the final result being the average of the obtained values (contact angle being measured at least 5 times on different sites of the surface). 2.3.4 Thermal analysis Thermal properties were determined in order to investigate the influence of the synthetic copolymer concentration on the resulted compounds. Thermal analysis was performed using a Jupiter STA 449 F1 (Netzsch) instrument. The samples were previously maintained in a controlled humidity atmosphere, respectively, in the presence of CaCl2 inorganic salt. 7.5–8 mg of samples were heated in an open Al2O3 crucible, under 50 mL/min nitrogen flow rate. Runs were performed in dynamic mode from room temperature up to 600 °C at a heating rate of 10 °C/min. 2.3.5. X-Ray Diffraction Analysis
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X-ray diffraction was studied on dried powder of samples using the Bruker AXS D8 Advance X-ray diffractometer (Bruker AXS, Madison, USA) by applying 36 kV at 25 mA, Cu anode, k1 =1.5406, interval 2 = 270 and 4÷40, time/step = 0.5 sec/step and scanning step size = 0.02.
3. RESULTS AND DISCUSSION
Various bioconjugation techniques are used in order to produce biomaterials and generally they imply ‚grafting – from’ or ‚grafting – to’ aproaches. The ‚grafting – to’ method supposes that the biomolecules are immobilized by reactive coupling reactions. P(ITAU) copolymer was conjugated with HA by using a grafting to strategy, for further ensuring new intramolecular strategies for coupling of various bioactive compounds. By opening the anhydride cycle of the copolymer with hydroxyl groups of HA we created chemically crosslinked hydrogels (Fig. 1). The conjugation between HA and the syntetic copolymer proceeds with fast kinetic via addition reactions, in the absence of any external intervention or small molecules to facilitate gel formation, the process being feasible in mild conditions and make gels prepared using the chemistries discussed herein less invasive and easier for use in multiple applications [15]. Fig. 1. Schematic presentation of the P(ITAU)_HA hydrogels.
3.1 Rheological behavior 3.1.1 Viscoelastic characteristics Frequency sweep tests were carried out in the linear domain of viscoelasticity and the rheological parameters, such as the elastic (G’) and viscous (G”) moduli, as well as the loss tangent (tan δ = G′′/G′), allow to appreciate the network structure of the sample. Figure 2a shows the evolution of the elastic and viscous moduli as a function of the oscillation frequency () for the P(ITAU)_HA and HA samples. As it can be seen, P(ITAU) concentration influences significantly the rheological behaviour. The results indicate a strong structuration for P(ITAU)10_HA sample, the viscoelastic moduli are independent of , G’ >> G” and the loss tangent is around 0.07 (Figure 2b). Such structure is generally considered as a „strong gel”. The
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low tan δ value of this sample indicates an optimum formulation of the gel, i.e., all network chains support the applied stress. The lowest values of G’ and G” were obtained for the P(ITAU)40_HA sample and strong gel-like structure was depicted for P(ITAU)10_HA. For HA solution, these parameters are dependent on and the overall rheological behaviour can be related both to topological interactions, such as entanglements, among the polymers chains and physical or chemical associations. Owing to its chemical structure, PITAU can be regarded as a crosslinker compound. At an increased concentration, the number of bridges formed between the synthetic polymer chains multiply. In consequence, PITAU macromolecules may obstruct each other, so the probability of new associations between the synthetic polymer and HA is reduced. More than that, at high concentrations of the copolymer in solution, the macromolecular chains are intertwined, which can give rise to physical interactions. Thus, the preformed system of PITAU copolymer is penetrating harder into the HA network and to its functional groups. As result, a number of increasingly smaller of anhydride cycles is capable of reacting with available OH groups of HA or to perform these chain–chain associations, number which is inversely proportional to the copolymer concentration. Therefore, a more relaxed and more elastic network will match for P(ITAU)10_HA, and a denser and less elastic network will correspond to P(ITAU)20_HA and respectively for P(ITAU)40_HA. At the same time, the tan δ values of P(ITAU)40_HA sample indicate the existence of some imperfections in the network structure (e.g. dangling chains, un-cross-linked chains and/or loops that do not support stress) that produce a viscous response to deformation, as it was recently reported for hydrogels obtained by in-situ polymerization of acrylamide in the presence of poly(vinylpyrrolidone) [18]. It is worth underlining that all P(ITAU)_HA samples keep their gel-like behaviour throughout the concentration range investigated; tan < 1 (Fig. 2) indicates that a gel structure is formed in all cases, but the network strength is dependent on the sample chemical composition, as well as on the number of crosslinkes formed between the compounds.
Fig. 2. The viscoelastic parameters as a function of the oscillation frequency for P(ITAU)_HA gels as compared with HA solutions.
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HA solution presents a typical viscous behaviour with the viscoelastic moduli dependent on and G” > G’ (tan > 1) bellow 10 rad/s. At the same time, in case of studied gels, the elastic modulus of samples can be tailored by changing the P(ITAU) content. So, from P(ITAU)40_HA up to P(ITAU)20_HA, G' is increased by about 35 times ranging from about 20 Pa to 700 Pa. The properties of these systems, especially P(ITAU)20_HA (G’ = 60 Pa) are comparable to those of materials that are currently used in biomedical applications such as dermal fillers (G’ = 39 Pa – 863 Pa) [19] or viscosupplementation product (G’ = 80 Pa) and are in the range of the rheological properties of many soft tissues [12]. P(ITAU) samples appear as weak structured samples, G’ is close to G” and present values between 1 Pa and 5 Pa (for a better clarity these curves are not shown in Fig. 2). Complex viscosity (*) of the investigated samples with various synthetic polymer concentrations, as a function of the angular frequency, is shown in Fig. 3. * decreases linearly with the increase in frequency within the tested frequency range and the P(ITAU)_HA samples exhibit a shear thinning effect. This behaviour is a key requirement for injectable hydrogels. The η* values decrease with increasing P(ITAU) content in the gel sample. Above 1 rad/s, all dependences present similar slopes, indicating that the structure of all systems are sensitive in the same way to changes in the oscillatory shear conditions. The HA solution presents lower viscosity as compared with P(ITAU)_HA derivatives and the Newtonian plateau is reached at low frequency of oscillation. The Newtonian viscosity of HA is around 20 Pas, approx. twice as compared with P(ITAU)20. Another observation is the sensitivity of P(ITAU)20 to shear increase, above 0.16 rad/s the flow behaviour is pseudoplastic.
Fig. 3. The complex viscosity as a function of the oscillation frequency for P(ITAU)_HA gels, P(ITAU)20 and HA solution.
3.1.2 Creep-recovery behavior Creep-recovery tests are useful for a better understanding of the long-term viscoelastic behavior of hydrogels. A typical creep-recovery curve is shown in the inset of Fig. 4. During the creep, by applying a shear stress, the viscoelastic material exhibits an instantaneous strain (which is correlated with the elastic property of the sample), then the deformation increases in time. When the shear stress is removed, firstly the instantaneous strain, then the delayed strain are
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recovered, these two parameters give the total recovered elastic deformation (rec). The irreversible component of the strain is not recovered. For this investigation, different constant shear stresses (of about 1, 2, 5, 10 and respectively 15 Pa) were applied for 30 s and then the recovery behaviour was followed. The temperature was maintained at 37C during the experiments to simulate the physiological conditions. Fig. 4 presents the creep and recovery curves obtained for the P(ITAU)20_HA sample. After the shear stress removal, the sample restores its equilibrium in approx. 200 s. A high elastic recovery of the network structure after applying shear stresses is an important factor to ensure the successful application of the compound as injectable hydrogel [20]. At the same time, some bonds can be irreversibly broken during the creep so the initial structure may not be completely recovered. Fig. 5 shows the rec as a function of shear stress obtained for the P(ITAU)_HA samples. Also to note that for shear stress up to approx. 18 Pa, P(ITAU)10_HA and P(ITAU)20_HA samples present high elastic recovery (around 90%), whereas P(ITAU)40_HA sample possesses a smaller degree of elasticity (around 50%). Bellow a shear stress of 20 Pa, P(ITAU)20 presents low rec values (approx. 10-11%) and above this value the elastic behavior of the sample fails (pure viscous behavior is registered). HA network is destroyed for very low values of shear stress. From Figure 6 it appears that the P(ITAU)_HA samples present higher elasticity as compared with their precursors.
Fig. 4. Creep-recovery curves obtained for P(ITAU)20_HA at 37C for different shear stress values.
Fig. 5. The elastic recovery (rec) as a function of shear stress obtained for different samples.
3.1.3 Thixotropic behavior Flow can induce reversible or irreversible structural changes in the polymer systems. For the thixotropic systems, the viscosity and the shear stress depend on the shear history and on the microstructure changes [21–23]. For the designed hydrogels, the thixotropic behavior was investigated at 37°C by monitoring the shear stress and viscosity dependences on the shear rate. The hysteresis area is an indicator for the degree of destructuration during flow, higher values for
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thixotropic area indicating higher thixotropy. Maximum of this parameter was recorded for P(ITAU)10_HA (Table 1), and was attributed to a reduced network density for the structure, with reduced as well physical bonds, which was concretized in higher thixotropy for the gel sample.
Table 1 The thixotropic area determined for the P(ITAU)_HA gels.
3.2 Swelling measurements Hydrogels as polymer networks with elastic cross-linked structures have the essential property of filling the interstitial spaces of the network with water and it is important to know the swelling properties of hydrogels because this process has a direct impact on carrier capacity as well as on drug delivery/release [24, 25]. At the same time, the manner of water diffusion in hydrogels with various diffusion constants reflects the size and shape of the network structure [26]. In this context, the interest in elucidating the behavior of the polymeric networks during swelling is understandable. The high swelling ability of hyaluronan chains is caused by the large charge density and the existence of polar groups on the polymeric constituents, and this can be regulated through networks realized by HA conjugation with synthetic polymers which limit the water uptake. The curves of swelling for the prepared hydrogel samples are shown in Fig. 6a. The percentage of the water retention in each tested sample showed a trend in interdependence with the hydrogels composition. This parameter increases with the increase of the synthetic polymer content in the hydrogel matrix. The time to reach the equilibrium uptake was quite different depending also, on the copolymer content during gels preparation. Thus, the hydrogels with 10% P(ITAU) reached equilibrium swelling faster than the other samples, in approximately 50 minutes. The swelling rate is higher due to the difference in pore size and the entanglement degree associated with the polymer concentration. A higher concentration of P(ITAU) copolymer induces a better swelling capacity with an equilibrium swelling degree reached in 150 minutes for P(ITAU)20_HA and P(ITAU)40_HA sample. As it stands though P(ITAU)10_HA network is more relaxed and elastic the degree of swelling of this system is lower compared to P(ITAU)20_HA and P(ITAU)40_HA. The behavior can be justified by the relaxed network which is shortly invaded by water and right after the time
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to reach the equilibrium uptake is installed. Regarding to P(ITAU)20_HA and P(ITAU)40_HA beyond the fact that they have denser networks that provide a slow swelling the electrostatic repulsions intervened between the functional groups make favorable conditions for a continuous process of swelling, respectively for increasing the content of adsorbed water.
Fig. 6. a) Swelling degree of gels with different P(ITAU) concentration as a function of time; b) equilibrium swelling degree of gels as a function of P(ITAU) of concentration in PBS.
With regard to P(ITAU) concentration, as it was already stated, higher polymer concentration may have led to greater swelling tendency. The swelling capacity of a hydrogel depends on the space within the polymer network available for accommodating solvent molecules and on the rate at which polymer chains relax [27]. For 10% concentration the structure of the gel is more relaxed and this provides multiple channels for the diffusion of water molecules leading to the dispersion of the hydrogen bonds formed between the two polymers. Furthermore, the water uptake decreases with increasing the concentration of P(ITAU) from 20% to 40 %. In the case of P(ITAU)40_HA sample, increased concentration of synthetic copolymer induces steric hindrances thus preventing the swelling of the network. The polymer chains of this sample are densely packed because of the involvement of more molecules in crosslinking and entanglements leading to higher elastic forces opposing swelling and limiting the solute transport throughout the network. The value of equilibrium swelling degree of P(ITAU)10_HA is 476 %, as for the values of equilibrium swelling degree for P(ITAU)20_HA and P(ITAU)40_HA are 744.28 % and 710 % (Fig. 6.b).
3.3 Contact angle Surface wettability is one of the most important properties of all materials since it reflects the real structure and chemical composition at the outermost surface. The surface hydrophilic property plays an important role in many properties such as biocompatibility, cell adhesion, spreading and proliferation on the biomaterials surfaces, lubricity, selective absorption and controlled release of molecules [28]. The wettability of the synthetic and natural polymer and one of the obtained hydrogel P(ITAU )20_HA was studied using static water contact angles. P(ITAU) showed a contact angle
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of about 53.8° (Fig. 7), indicating that the surface is moderately hydrophilic. Pure HA exhibited more hydrophilic properties because is rich in polar functional groups, and exhibited a contact angle of 42.6°. The gel displayed a decreased contact angle of 26.2° which is significantly lower than that of the individual components indicating a surface with improved wettability enriched in polar groups.
Fig. 7. Mean static contact angles determined by sessile drop method for P(ITAU), HA and P(ITAU)20_HA sample. These differences can be explained through the structure of the new bioconjugate compounds. Thus, HA chains in solution have an expanded “slightly stiff” random coil structure, meanwhile the size of HA varies with the environment conditions as expected for a flexible polyelectrolyte. [29, 30] At the same time, the investigation of the HA in the solid state confirm the helical conformation with two types of domains with different mobility, which are dependent on the extended hydrogen-bonded arrays. After HA conjugation with P(ITAU) the new macromolecular chains come along in a wormlike pattern with the functional groups released from the physical bonds and capable now for other interactions, concretized in diminution of the static contact angle. 3.4 Thermal analysis TG and DTG curves concerning thermal behavior and changes in thermal stability of the obtained products are depicted in Fig. 8. These showed gradual sample mass loss with increasing temperature, having four or three distinct degradation regions, influenced by the concentration of the copolymer in the final gel structure. The first step of degradation, similar for all batches, may be associated with dehydration from the compound structure as well as the presence of residual monomers; the other regions levels are related to the breakage of the polymeric chains. The main mass loss percentage (going from 70.94 to 81.22 wt. %) was observed in the temperature range from 150 to 300°C – mainly due to the breakage of functional groups (hydroxyl, carboxyl, carbonyl), which generates gaseous compounds with low molecular weight (CO2, CO, H2O, CH2OH, CH2O etc.) [31]. By increasing the heating temperature above 300oC a new stage of degradation appears for P(ITAU)20_HA and P(ITAU)40_HA with weight losses ranging between 11.90 to 9.56 wt. %. This stage is a result of splitting ties hydrocarbon CC and the releases of saturated or unsaturated aliphatic fragments of higher molecular weights and derivatives
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carbonyl. The remaining residue shows a decreasing trend correlated with the concentration of the polymeric matrix. Parameters describing the thermal behavior of these gels, calculated from TG are displayed in Table 2. From the viewpoint of the degradation process, P(ITAU)40_HA sample is more stable and starts degradation later (at 163ºC) than P(ITAU)20_HA and P(ITAU)10_HA (151ºC and 113ºC). This fact is also supported by analyzing the thermal stability as a function of T10 and T20 temperatures which corresponds to 10 or 20% weight loss. The most stable gel corresponds to the one with a higher concentration of P(ITAU), which has a denser network generated by the synthetic matrix. Comparing degradation profiles of both HA [32] and P(ITAU) [14] we can observe significant differences which can be correlated with the appearance of new moieties enhanced by the presence of synthetic copolymer with the consequent increase of the thermal stability of all samples. Furthermore, based on these results the possibility of heat sterilization can be taken into account since no animal-derived raw HA provides superior heat stability for efficient sterilization processes [33].
Fig. 8. TG (a) and DTG (b) curves of P(ITAU)_ HA hydrogels.
Table 2 Thermal parameters of the P(ITAU)_ HA hydrogels. 3.5 X-Ray Diffraction Analysis X-ray diffraction analysis was performed to evidence the physical state of newly synthesized bioconjugate gel structure. XRD patterns are depicted in Fig. 9. P(ITAU)10_HA, P(ITAU)40_HA, P(ITAU) and HA precursors present diffraction patterns with one broad and extended diffraction peak of the diffusion type centered around 15 - 25
(2θ) typical for
amorphous materials. Instead P(ITAU)20_HA has a lot of peaks with different intensities that can be considered as pieces of crystallites embedded in a matrix of amorphous material resulted from ordered bonds intervened between HA and P(ITAU). These crystallites may have occurred due to the optimum ratio between HA and P(ITAU), and this optimal proportion for conjugation can determines the aligned towards the length of the macromolecular chains and with a preferred orientation.
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Fig. 9. XRD patterns for the studied samples.
4. CONCLUSIONS
The general goal was to investigate the properties of new hydrogels based on a polysaccharide (HA) and a synthetic copolymer synthesized by using a grafting – to strategy. Samples of various compositions with different concentration of P(ITAU) were successfully prepared and can be used as a basis for injectable drug delivery systems that may be able to provide sustained and controlled release of a wide variety of bioactive agents. The P(ITAU)_ HA hydrogels exhibit a rheological behavior typical to strong gels and show improved viscoelastic properties at low synthetic polymer concentration. In the creeprecovery tests, a high elastic recovery of the hydrogel network was observed after removing the applied shear stress and the sample restores its equilibrium in approximate 200 seconds. In addition, the hydrogels had shown shear-thinning thixotropic behavior, indicating suitability of these systems as injectable drug delivery vehicles. It was demonstrated that the new structures have a good swelling capacity and also an improved thermal stability dependent on the sample composition. Together, these results demonstrate that the proposed strategy is a valuable toolbox for fine-tuning the rheological and structural properties of injectable HA hydrogels. The facile delivery of these systems and the absence of any external intervention or small molecules to facilitate gel formation make gels prepared using the chemistries discussed herein less invasive and easier for use. Importantly, because of the remarkable feature given by HA, these hydrogels can be used to deliver drugs, biological molecules or cells during the injection process with applications that can be divided in two main fields: regenerative medicine and local therapy through drug delivery.
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ACKNOWLEDGMENTS This work was financially supported by the grant of the Romanian National Authority for Scientific Research, CNCS-UEFISCDI, project number PNII-RU-TE-2014-4-0294 Novel hydrogels synthesis with defined 3D functionality and biodegradable characteristics for bioapplications. The authors thank and appreciate the colleague Dr. Daniel Timpu in “P.Poni” Institute of Macromolecular Chemistry for his expert assistance in X-ray diffraction analyses.
References
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9. U.B.G. Laurent, R.K. Reed, Turnover of hyaluronan in the tissues, Adv. Drug Deliv. Rev. 7 (1991) 237–256. 10. Y.S. Jung, W. Park, H. Parka, D.K. Leeb, K. Na, Thermo-sensitive injectable hydrogel based on the physical mixing of hyaluronic acid and Pluronic F-127 for sustained NSAID delivery, Carbohydr. Polym. 156 (2017) 403–408. 11. A. Dubbinia, R. Censia, M.E. Butinia, M.G. Sabbietib, D. Agasb, T. Vermondenc, P. Di Martinoa, Injectable hyaluronic acid/PEG-p(HPMAm-lac)-based hydrogels dually crosslinked by thermal gelling and Michael addition, Eur. Polym. J. 72 (2015) 423–437. 12. A. Borzacchiello, L. Russo, B.M. Malle, K. Schwach-Abdellaoui, L. Ambrosio, Hyaluronic acid based hydrogels for regenerative medicine applications, Biomed. Res. Int. 2015 (2015) 871218, 1–12. 13. J. Adler, S. Wang, H.A. Lardy, The metabolism of itaconic acid by liver mitochondria, J. Biol. Chem. 229 (1957) 865–879. 14. A. Diaconu, A.P. Chiriac, L.E. Nita, N. Tudorachi, I. Neamtu, C. Vasile, M. Pinteala, Design and synthesis of a new polymer network containing pendant spiroacetal moieties, Des. Monomers. Polym. 18 (2015) 780–788. 15. A.P. Chiriac, L.E. Nita, A. Diaconu, M. Bercea, N. Tudorachi, D. Pamfil, L. MititeluTartau, Hybrid gels by conjugation of hyaluronic acid with poly(itaconic anhydride-co3,9-divinyl-2,4,8,10-tetraoxaspiro (5.5)undecane) copolymers, Int. J. Biol. Macromol. 98 (2017) 407–418. 16. A. Diaconu, L.E. Nita, A.P. Chiriac, L. Mititelu Tartau, F. Doroftei, C. Vasile, M. Pinteala, Self-linked polymer gels [based on hyaluronic acid and poly (itaconic anhydride-co-3, 9-divinyl-2, 4, 8, 10-tetraoxaspiro [5.5] undecane)] as potential drug delivery networks, in Proceedings of the 5th IEEE International Conference on E-Health and Bioengineering – EHB, Romania, 1–4 (2015). 17. M. Mescher, A. Brinkman, D. Bosma, J. Klootwijk, E. Sudhölter, L. Smet, influence of conductivity and dielectric constant of water–dioxane mixtures on the electrical response of SiNW-Based FETs, Sensors 14 (2014) 2350–2361. 18. G. Song, Z. Zhao, X. Peng, C. He, R.A. Weiss, H. Wang, Rheological behavior of tough PVP-in situ-PAAm hydrogels physically cross-linked by cooperative hydrogen bonding, Macromolecules 49 (2016) 8265–8273.
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19. H. Sundaram, D. Cassuto, Biophysical characteristics of hyaluronic acid soft-tissue fillers and their relevance to aesthetic applications, Plast. Reconstr. Surg. 132 (2013) 5S–21S. 20. J. Hu, M. Zhang, J. Hea, P. Ni, Injectable hydrogels by inclusion complexation between a three-armed star copolymer (mPEG-acetal-PCL-acetal-)3 and α-cyclodextrin for pHtriggered drug delivery, RSC Adv. 6 (2016) 40858–40868. 21. J. Mewis, N.J. Wagner, Thixotropy, Adv. Colloid Interface Sci. 147–148 (2009) 214– 227. 22. L.E. Nita, A.P. Chiriac, M. Bercea, B.A. Wolf, Synergistic behavior of poly(aspartic acid) and Pluronic F127 in aqueous solution as studied by viscometry and dynamic light scattering, Colloid Surf. B-Biointerfaces 103 (2013) 544–549. 23. I. Neamtu, A.P. Chiriac, L.E. Nita, M. Bercea, A. Stoleriu, Investigation of poly(aspartic acid)/vinylic polymer interpolymer complex , J. Optoelectron. Adv. Mater. 9 (2007) 981– 984. 24. A. Martínez-Ruvalcaba1, J.C. Sánchez-Díaz, F. Becerra, L.E. Cruz-Barba, A. GonzálezÁlvarez, Swelling characterization and drug delivery kinetics of polyacrylamide-coitaconic acid/chitosan hydrogels, Express Polym Lett. 3 (2009) 25–32. 25. A. Ström, A. Larsson, O. Okay, Preparation and physical properties of hyaluronic acidbased cryogels, J. Appl. Polym. Sci. 132 (2015) 42194, 1–11. 26. Y. Osada, R. Kawamura, K.-I. Sano, Hydrogels of Cytoskeletal Proteins: Preparation, Structure, and Emergent Functions; Springer International Publishing Switzerland 2016. 27. P. Matricardi, F. Alhaique, T. Coviell, Polysaccharide Hydrogels: Characterization and Biomedical Applications, CRC Press, Pan Stanford, 2016. 28. S. Kalia, Y. Haldorai, Organic-inorganic hybrid nanomaterials, Adv. Polym. Sci. 267, (2014) 222–223. 29. Lubomır Lapcık, Jr., Lubomır Lapcık; Hyaluronan: Preparation, Structure, Properties, and Applications, Chemical Reviews 98(8) (1998) 2663-2684. 30. R. L. Cleland, Ionic polysaccharides. II. Comparison of polyelectrolyte behavior of hyaluronatc with that of carboxymethyl cellulose, Biopolymers 6 (1968) 1519 –1529. 31. L.E. Nita, A.P. Chiriac, A. Diaconu, N. Tudorachi, L. Mititelu-Tartau, Multifunctional nanogels with dual temperature and pH responsiveness, Int. J. Pharm. 30 (2016) 165– 175.
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32. K. Benešová, M. Peka, L. Lapík, J. Kuerík, Stability evaluation of n-alkyl hyaluronic acid derivates by DSC and TG measurement, J. Therm. Anal. 83 (2006) 341–348. 33. A.G. Rusu, M.I. Popa, G. Lisa, L. Verestiuc, Thermal behavior of hydrophobically modified hydrogels using TGA/FTIR/MS analysis technique, Thermochim. Acta 613 (2015) 28–40.
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FIGURES
Fig. 1. Schematic presentation of the P(ITAU)_HA hydrogels.
22
Fig. 2. The viscoelastic parameters as a function of the oscillation frequency for P(ITAU)_HA gels as compared with HA solutions.
23
Fig. 3. The complex viscosity as a function of the oscillation frequency for P(ITAU)_HA gels, P(ITAU)20 and HA solution.
24
Fig. 4. Creep-recovery curves obtained for P(ITAU)20_HA at 37C for different shear stress values.
25
Fig. 5. The elastic recovery (rec) as a function of shear stress obtained for different samples.
26
Fig. 6. a) Swelling degree of gels with different P(ITAU) concentration as a function of time; b) equilibrium swelling degree of gels as a function of P(ITAU) of concentration in PBS.
27
Fig. 7. Mean static contact angles determined by sessile drop method for P(ITAU), HA and P(ITAU)20_HA sample.
28
Fig. 8. TG (a) and DTG (b) curves of P(ITAU)_ HA hydrogels.
Intensity (u.a.)
29
HA P(ITAU)10_HA P(ITAU)20_HA P(ITAU)40_HA P(ITAU)
5
10
15
20
25
30
35
(degree)
Fig. 9. XRD patterns for the studied samples.
40
30
Tables
Table 1. The thixotropic area determined for the P(ITAU)_HA gels Sample
P(ITAU)10
Hysteresis area from shear stress curve (Pas-1) 96.92
Hysteresis area from viscosity curve (Pas-1) 227.85
P(ITAU)20
11.59
35.93
P(ITAU)40
18.15
66.40
Table 2. Thermal parameters of the P(ITAU)_ HA hydrogels Sample
P(ITAU)40_HA
Degradation stage I
Tonset (C) 163
Tpeak (C) 217
W (%) 16.44
II
227
250
18.33
III
294
328
36.17
IV
424
432
11.90
residue
P(ITAU)20_HA
T10 (C) 201
T20 (C) 240
175
212
17.16
I
151
217
41.09
II
287
305
31.23
III
419
426
9.56
31
residue
P(ITAU)10_HA
18.12
I
113
-
21.68
II
200
216
26.64
III
296
364
32.90
residue
153
185
18.78
Tonset – the temperature at which the thermal decomposition begins; Tpeak – the temperature at which the degradation rate is maximum; T10 and T20 – the temperature corresponding to 10% and 20% weight loss, respectively; W – the weight loss.
S1369-703X(17)30154-7 http://dx.doi.org/doi:10.1016/j.bej.2017.06.003 BEJ 6725
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
24-2-2017 31-5-2017 6-6-2017
Please cite this article as: A.Diaconu, L.E.Nita, M.Bercea, A.P.Chiriac, A.G.Rusu, D.Rusu, Hyaluronic acid gels with tunable properties by conjugating with a synthetic copolymer, Biochemical Engineering Journalhttp://dx.doi.org/10.1016/j.bej.2017.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Hyaluronic acid gels with tunable properties by conjugating with a synthetic copolymer A. Diaconu, L. E. Nita, M. Bercea, A. P. Chiriac1, A. G. Rusu, D. Rusu “Petru Poni” Institute of Macromolecular Chemistry 41 A, Grigore Ghica Voda Alley, RO – 700487 Iasi, Romania
Corresponding author – Aurica P. Chiriac; Telephone number: +40232217454; Fax number: +40232211299; e-mail address: [email protected]. 1
2
Graphical abstract
Highlights
1. Hybrid gels with tunable characteristics were prepared using hyaluronic acid and a
copolymer. 2. The compounds were realized by a grafting-to strategy without using other chemical
additives. 3. Rheological properties, swelling behavior, and network parameters were investigated.
Abstract: Recently, injectable hydrogels became particularly attractive as biomaterials for tissue engineering and drug delivery applications. The intrinsic drug-loading capability of polysaccharide-based hydrogels and their potential use as drug delivery systems can be combined with more sophisticated techniques in order to develop effective approaches for controlled release of bioactive agents. The objective of this study was to design and to test hydrogels based on conjugated hyaluronic acid with a new synthetic copolymer, in order to further development their potential for injectable drug delivery applications. The synthesized materials, obtained by using a grafting-to strategy, were analyzed in terms of rheological
3
properties, swelling behavior, contact angle and thermal properties, and the effects of their chemical composition on various properties were assessed. A good structure recovery property and a shear-thinning thixotropic behavior were identified by the rheological tests. The developed hydrogels showed potential as promising injectable biomaterials.
Keywords: hyaluronic acid, poly(itaconic anhydride-co-3,9-divinyl- 2,4,8,10- tetraoxaspiro [5.5] undecane), hydrogels, viscoelasticity.
1. INTRODUCTION
In recent years, tremendous advancements in polymer science have given rise to new opportunities to overcome some of the most fundamental limitations facing pharmacology. There is a clear demand for developing biocompatible systems that can bypass the need for systemic drug administration and are capable of delivering pharmaceutical agents at a controlled rate. Among various kinds of polymeric systems, hydrogels have attracted particular interest in terms of addressing these pharmaceutical challenges as they are a unique class of three – dimensional, polymer networks that may contain a large fraction of aqueous content within their structure [1]. Hydrogels, because of their high water content, controllable porosity and mechanical and compositional similarities between them and native soft tissues in the body, are particularly suitable for biomedical applications such as tissue engineering, controlled release of bioactive agents, bioseparation, space-filling and cell encapsulation. While these properties have motivated significant research of such materials, the practical introduction of hydrogels into the human body is a major challenge limiting their routine clinical application [2]. As a result of these limitations, many efforts have been recently invested in the design of injectable hydrogels [3,4]. These types of materials hold promising implications for improved surgery treatment and other therapies requiring defined quantities of a therapeutic payload in a site-specific and/or timecontrolled fashion – from post-surgery pain medications to drugs that require daily injection. Injectability circumvents the need for surgery to administer the hydrogel in vivo, thus reducing pain and minimizing the risks of infection and enables effective molding of the hydrogel shape in situ by the neighboring tissue to fit cavities and/or tissue [5].
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In the past years, hydrogels based on hyaluronic acid (HA) have been especially attractive as injectable biomaterials due to their unique properties, including biodegradability, biocompatibility, nontoxicity, nonadhesivity, high water absorption capacity and nonimmunogenicity [6]. Additionally, HA-based hydrogels may impart biological activity to cells, including stem cell differentiation [7]. HA macromolecule in dilute aqueous solutions adopts a random coil conformation. Long linear HA chains in more concentrated solutions form an entangled network in which there are some topological constraints on each other called entanglements. Under shear flow, these entangled coiled chains disentangle and align to the flow direction and a shear thinning behavior is registered. In such conditions, even high concentrated HA solutions are easily injected into the human body. However, due to the high water solubility, it quickly disperses when injected into fluid-filled cavities [8]. HA is also subjected to various degradation processes due to hydrolysis and enzymatic hydrolysis by naturally occurring hyaluronidase [9]. Consequently, several chemical modifications of native hyaluronan with synthetic polymers have been developed in order to control and to improve mechanical properties, degradation rate, and clearance such as crosslinking or conjugation, to obtain a more stable material maintaining at the same time its fundamental properties [10,11]. In the case of crosslinking, HA reacts with a crosslinking agent that is capable of creating covalent bonds between HA chains, whereas compounds grafted on HA chains are referred to as conjugates [12]. Biobased materials have attracted considerable attention due to the ever–growing environmental problems, thus, it is considered very important to do research on biomaterials synthesis using renewable resources. Itaconic anhydride (ITA), an unsaturated cyclic anhydride, is regarded as one of key platform chemicals derived from biomass. Also, it is biocompatible, has a bioactive nature and the inclusion of ITA into macromolecular chain structure will induce partly biodegradable character for the prepared compounds. ITA passes to nontoxic degradation products under physiological conditions, when it initially hydrolyzes to itaconic acid followed by oxidation to acetate, lactate, and carbon dioxide. In light of the above, ITA represents an excellent starting material to obtain suitable structures for biomedical applications [13]. In a previous article, we reported the synthesis of a new copolymer based on ITA and an orthoester comonomer named 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5] undecane (U), obtaining a
5
new class of copolymers - P(ITAU) - which are dual sensitive to pH and temperature [14]. Furthermore, we extended our research and we developed a new hybrid hydrogel structure based on HA and P(ITAU) by using a grafting-to strategy. By opening the anhydride cycle of the copolymer with hydroxyl groups of hyaluronic acid we created chemically crosslinked hydrogels [15,16]. Comprehensive research on these systems demonstrated that they possess excellent biocompatibility as confirmed by their performance in in vivo studies and sustained drug delivery ability. In the present paper, we extend considerably our earlier studies in order to increase our overall understanding of this type of materials and to optimize a design system in order to meet the requirements for injectable systems. Using the grafting-to approach, one property can be selectively modified while keeping other properties constant, providing a highly adaptable method of engineering injectable, rapidly-gelling hydrogels for potential biomedical applications. The concentration of the polymer is a vital design parameter that affects the hydrogel formation and structure and also the syringeability profile. By modifying the polymer concentration, the hardness or stiffness of the gel can increase or decrease; so it was necessary to vary conveniently this parameter in order to obtain hydrogels with specific requirements [12].
2. EXPERIMENTAL PART
2.1. Materials All chemicals used were reagent grade and used as purchased without further purification. Hyaluronic acid sodium salt (HA) used in this work was produced by SigmaAldrich, with a purity of 99% from Streptococcus equi bacterial glycosaminoglycan polysaccharide (Mw = 1.5×106 Da – 1.8×106 Da). Itaconic anhydride (ITA) (purity 98%), 3, 9divinyl-2,4,8,10-tetraoxaspiro[5.5]
undecane
(U)
(purity
98%),
2,2′-azobis(2-
methylpropionitrile) (AIBN) (purity 98%) and the solvents – 1,4-dioxane (≥ 99.0%) and diethyl ether – were also purchased from Sigma-Aldrich. The water used in the experiments was purified using an Ultra Clear TWF UV System. 2.2. Synthesis of P(ITAU) copolymers and preparation of the gels based on P(ITAU) – grafted onto hyaluronic acid
6
Poly(itaconic anhydride-co-3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5] undecane) (P(ITAU)) copolymer was synthesized by radical polymerization in solution using AIBN as initiator and 1,4-dioxane as solvent. Detailed aspects about synthesis, and structural characterization of the copolymer (by FTIR and NMR spectroscopy and other methods) were previously described [14]. Briefly, the copolymer was synthesized through a continuous radical process of polymerization with 0.05:0.07 molar ratio between ITA / U comonomers and 3.7x10-5 mmol 2,2′-Azobis(2methylpropionitrile) radical initiator content for the mentioned comonomer ratio, in a 1,4dioxane solution of 20% concentration. The synthesis was conducted under nitrogen atmosphere, at 75°C, in a constant temperature bath, with a stirring rate of 250 rpm for 17 h. Then the copolymer was separated after solution precipitation in diethyl ether, washed repeatedly with diethyl ether, and dried for 24 h in a vacuum oven at 600 mm HG. We studied the influence of the copolymer content on the development of the new hybrid hydrogels. In this context three variants of copolymer solution, with the same ratio between the comonomers and having respectively 10 wt%, 20 wt% and 40 wt% copolymer content, were obtained and tested. HA was dissolved in deionized water at 1 wt% concentration at room temperature. Grafting P(ITAU) copolymer onto HA was achieved in soft conditions by mixing the copolymer solution in 1,4-dioxane with HA water solution. The gravimetric ratio between HA and the synthetic copolymer was maintained constant at 1:2 value. The sample codes for the prepared hydrogels were chosen in accordance with the copolymer content namely P(ITAU)10_HA, P(ITAU)20_HA and P(ITAU)40_HA. The gels were purified by dialysis (cellulose dialysis membrane with molecular weight cut-off of 14,200 Da) against distilled water for 4 days changing the water twice a day to remove unreacted compounds and 1,4-dioxane. To ensure a better purification the pH and conductivity of the dialyzed water were regularly measured until they reach the values of distilled water (protonated dioxane is slightly more acidic than protonated water [17]. 2.3. P(ITAU)_HA gels characterization 2.3.1 Rheological measurements The rheological properties were assessed with a MCR 302 Anton-Paar rheometer equipped with Peltier device for temperature control and plane-plane geometry (the upper plate having the diameter of 50 mm) with a gap of 500 µm. The water evaporation was limited by using an anti-evaporation device which created a saturated atmosphere near the sample. The
7
viscoelastic properties of the gels were tested at 37C by small-amplitude oscillatory shear experiments with oscillation frequency ranging from 0.1 rad/s up to 100 rad/s. Creep tests were also carried out for different shear stress values and, after the stress removal, the recovery behavior was observed. 2.3.2. Swelling measurements The swelling degree was determined in buffer solutions at a physiological temperature of 37oC and pH 7.4, 0.2 M. Each sample was immersed in 5 mL phosphate buffer. At predetermined time intervals, the samples were carefully withdrawn from the swelling medium, weighted after superficially blotting with filter paper and placed again in the same swelling medium. Measurements were continued until a constant weight was obtained for each sample. The amount of absorbed buffer solution was monitored gravimetrically. The swelling degree of a sample was calculated as follows: SD = (Wt − Wd )/Wd
(1)
where Wd and Wt are the weights of dry and wet samples at the time t. 2.3.3 Contact angle The static contact angle of the films was determined by the sessile drop method, at room temperature and controlled humidity, within 10 s, after placing 1 μL drop of water on the film surface, using a CAM-200 instrument from KSV-Finland. To obtain reproducible results for contact angle measurements, several conditions have to be fulfilled, such as: constant temperature during determinations; the same volume of solvent drops; evaluation of the contact angles in different points of the studied surface, the final result being the average of the obtained values (contact angle being measured at least 5 times on different sites of the surface). 2.3.4 Thermal analysis Thermal properties were determined in order to investigate the influence of the synthetic copolymer concentration on the resulted compounds. Thermal analysis was performed using a Jupiter STA 449 F1 (Netzsch) instrument. The samples were previously maintained in a controlled humidity atmosphere, respectively, in the presence of CaCl2 inorganic salt. 7.5–8 mg of samples were heated in an open Al2O3 crucible, under 50 mL/min nitrogen flow rate. Runs were performed in dynamic mode from room temperature up to 600 °C at a heating rate of 10 °C/min. 2.3.5. X-Ray Diffraction Analysis
8
X-ray diffraction was studied on dried powder of samples using the Bruker AXS D8 Advance X-ray diffractometer (Bruker AXS, Madison, USA) by applying 36 kV at 25 mA, Cu anode, k1 =1.5406, interval 2 = 270 and 4÷40, time/step = 0.5 sec/step and scanning step size = 0.02.
3. RESULTS AND DISCUSSION
Various bioconjugation techniques are used in order to produce biomaterials and generally they imply ‚grafting – from’ or ‚grafting – to’ aproaches. The ‚grafting – to’ method supposes that the biomolecules are immobilized by reactive coupling reactions. P(ITAU) copolymer was conjugated with HA by using a grafting to strategy, for further ensuring new intramolecular strategies for coupling of various bioactive compounds. By opening the anhydride cycle of the copolymer with hydroxyl groups of HA we created chemically crosslinked hydrogels (Fig. 1). The conjugation between HA and the syntetic copolymer proceeds with fast kinetic via addition reactions, in the absence of any external intervention or small molecules to facilitate gel formation, the process being feasible in mild conditions and make gels prepared using the chemistries discussed herein less invasive and easier for use in multiple applications [15]. Fig. 1. Schematic presentation of the P(ITAU)_HA hydrogels.
3.1 Rheological behavior 3.1.1 Viscoelastic characteristics Frequency sweep tests were carried out in the linear domain of viscoelasticity and the rheological parameters, such as the elastic (G’) and viscous (G”) moduli, as well as the loss tangent (tan δ = G′′/G′), allow to appreciate the network structure of the sample. Figure 2a shows the evolution of the elastic and viscous moduli as a function of the oscillation frequency () for the P(ITAU)_HA and HA samples. As it can be seen, P(ITAU) concentration influences significantly the rheological behaviour. The results indicate a strong structuration for P(ITAU)10_HA sample, the viscoelastic moduli are independent of , G’ >> G” and the loss tangent is around 0.07 (Figure 2b). Such structure is generally considered as a „strong gel”. The
9
low tan δ value of this sample indicates an optimum formulation of the gel, i.e., all network chains support the applied stress. The lowest values of G’ and G” were obtained for the P(ITAU)40_HA sample and strong gel-like structure was depicted for P(ITAU)10_HA. For HA solution, these parameters are dependent on and the overall rheological behaviour can be related both to topological interactions, such as entanglements, among the polymers chains and physical or chemical associations. Owing to its chemical structure, PITAU can be regarded as a crosslinker compound. At an increased concentration, the number of bridges formed between the synthetic polymer chains multiply. In consequence, PITAU macromolecules may obstruct each other, so the probability of new associations between the synthetic polymer and HA is reduced. More than that, at high concentrations of the copolymer in solution, the macromolecular chains are intertwined, which can give rise to physical interactions. Thus, the preformed system of PITAU copolymer is penetrating harder into the HA network and to its functional groups. As result, a number of increasingly smaller of anhydride cycles is capable of reacting with available OH groups of HA or to perform these chain–chain associations, number which is inversely proportional to the copolymer concentration. Therefore, a more relaxed and more elastic network will match for P(ITAU)10_HA, and a denser and less elastic network will correspond to P(ITAU)20_HA and respectively for P(ITAU)40_HA. At the same time, the tan δ values of P(ITAU)40_HA sample indicate the existence of some imperfections in the network structure (e.g. dangling chains, un-cross-linked chains and/or loops that do not support stress) that produce a viscous response to deformation, as it was recently reported for hydrogels obtained by in-situ polymerization of acrylamide in the presence of poly(vinylpyrrolidone) [18]. It is worth underlining that all P(ITAU)_HA samples keep their gel-like behaviour throughout the concentration range investigated; tan < 1 (Fig. 2) indicates that a gel structure is formed in all cases, but the network strength is dependent on the sample chemical composition, as well as on the number of crosslinkes formed between the compounds.
Fig. 2. The viscoelastic parameters as a function of the oscillation frequency for P(ITAU)_HA gels as compared with HA solutions.
10
HA solution presents a typical viscous behaviour with the viscoelastic moduli dependent on and G” > G’ (tan > 1) bellow 10 rad/s. At the same time, in case of studied gels, the elastic modulus of samples can be tailored by changing the P(ITAU) content. So, from P(ITAU)40_HA up to P(ITAU)20_HA, G' is increased by about 35 times ranging from about 20 Pa to 700 Pa. The properties of these systems, especially P(ITAU)20_HA (G’ = 60 Pa) are comparable to those of materials that are currently used in biomedical applications such as dermal fillers (G’ = 39 Pa – 863 Pa) [19] or viscosupplementation product (G’ = 80 Pa) and are in the range of the rheological properties of many soft tissues [12]. P(ITAU) samples appear as weak structured samples, G’ is close to G” and present values between 1 Pa and 5 Pa (for a better clarity these curves are not shown in Fig. 2). Complex viscosity (*) of the investigated samples with various synthetic polymer concentrations, as a function of the angular frequency, is shown in Fig. 3. * decreases linearly with the increase in frequency within the tested frequency range and the P(ITAU)_HA samples exhibit a shear thinning effect. This behaviour is a key requirement for injectable hydrogels. The η* values decrease with increasing P(ITAU) content in the gel sample. Above 1 rad/s, all dependences present similar slopes, indicating that the structure of all systems are sensitive in the same way to changes in the oscillatory shear conditions. The HA solution presents lower viscosity as compared with P(ITAU)_HA derivatives and the Newtonian plateau is reached at low frequency of oscillation. The Newtonian viscosity of HA is around 20 Pas, approx. twice as compared with P(ITAU)20. Another observation is the sensitivity of P(ITAU)20 to shear increase, above 0.16 rad/s the flow behaviour is pseudoplastic.
Fig. 3. The complex viscosity as a function of the oscillation frequency for P(ITAU)_HA gels, P(ITAU)20 and HA solution.
3.1.2 Creep-recovery behavior Creep-recovery tests are useful for a better understanding of the long-term viscoelastic behavior of hydrogels. A typical creep-recovery curve is shown in the inset of Fig. 4. During the creep, by applying a shear stress, the viscoelastic material exhibits an instantaneous strain (which is correlated with the elastic property of the sample), then the deformation increases in time. When the shear stress is removed, firstly the instantaneous strain, then the delayed strain are
11
recovered, these two parameters give the total recovered elastic deformation (rec). The irreversible component of the strain is not recovered. For this investigation, different constant shear stresses (of about 1, 2, 5, 10 and respectively 15 Pa) were applied for 30 s and then the recovery behaviour was followed. The temperature was maintained at 37C during the experiments to simulate the physiological conditions. Fig. 4 presents the creep and recovery curves obtained for the P(ITAU)20_HA sample. After the shear stress removal, the sample restores its equilibrium in approx. 200 s. A high elastic recovery of the network structure after applying shear stresses is an important factor to ensure the successful application of the compound as injectable hydrogel [20]. At the same time, some bonds can be irreversibly broken during the creep so the initial structure may not be completely recovered. Fig. 5 shows the rec as a function of shear stress obtained for the P(ITAU)_HA samples. Also to note that for shear stress up to approx. 18 Pa, P(ITAU)10_HA and P(ITAU)20_HA samples present high elastic recovery (around 90%), whereas P(ITAU)40_HA sample possesses a smaller degree of elasticity (around 50%). Bellow a shear stress of 20 Pa, P(ITAU)20 presents low rec values (approx. 10-11%) and above this value the elastic behavior of the sample fails (pure viscous behavior is registered). HA network is destroyed for very low values of shear stress. From Figure 6 it appears that the P(ITAU)_HA samples present higher elasticity as compared with their precursors.
Fig. 4. Creep-recovery curves obtained for P(ITAU)20_HA at 37C for different shear stress values.
Fig. 5. The elastic recovery (rec) as a function of shear stress obtained for different samples.
3.1.3 Thixotropic behavior Flow can induce reversible or irreversible structural changes in the polymer systems. For the thixotropic systems, the viscosity and the shear stress depend on the shear history and on the microstructure changes [21–23]. For the designed hydrogels, the thixotropic behavior was investigated at 37°C by monitoring the shear stress and viscosity dependences on the shear rate. The hysteresis area is an indicator for the degree of destructuration during flow, higher values for
12
thixotropic area indicating higher thixotropy. Maximum of this parameter was recorded for P(ITAU)10_HA (Table 1), and was attributed to a reduced network density for the structure, with reduced as well physical bonds, which was concretized in higher thixotropy for the gel sample.
Table 1 The thixotropic area determined for the P(ITAU)_HA gels.
3.2 Swelling measurements Hydrogels as polymer networks with elastic cross-linked structures have the essential property of filling the interstitial spaces of the network with water and it is important to know the swelling properties of hydrogels because this process has a direct impact on carrier capacity as well as on drug delivery/release [24, 25]. At the same time, the manner of water diffusion in hydrogels with various diffusion constants reflects the size and shape of the network structure [26]. In this context, the interest in elucidating the behavior of the polymeric networks during swelling is understandable. The high swelling ability of hyaluronan chains is caused by the large charge density and the existence of polar groups on the polymeric constituents, and this can be regulated through networks realized by HA conjugation with synthetic polymers which limit the water uptake. The curves of swelling for the prepared hydrogel samples are shown in Fig. 6a. The percentage of the water retention in each tested sample showed a trend in interdependence with the hydrogels composition. This parameter increases with the increase of the synthetic polymer content in the hydrogel matrix. The time to reach the equilibrium uptake was quite different depending also, on the copolymer content during gels preparation. Thus, the hydrogels with 10% P(ITAU) reached equilibrium swelling faster than the other samples, in approximately 50 minutes. The swelling rate is higher due to the difference in pore size and the entanglement degree associated with the polymer concentration. A higher concentration of P(ITAU) copolymer induces a better swelling capacity with an equilibrium swelling degree reached in 150 minutes for P(ITAU)20_HA and P(ITAU)40_HA sample. As it stands though P(ITAU)10_HA network is more relaxed and elastic the degree of swelling of this system is lower compared to P(ITAU)20_HA and P(ITAU)40_HA. The behavior can be justified by the relaxed network which is shortly invaded by water and right after the time
13
to reach the equilibrium uptake is installed. Regarding to P(ITAU)20_HA and P(ITAU)40_HA beyond the fact that they have denser networks that provide a slow swelling the electrostatic repulsions intervened between the functional groups make favorable conditions for a continuous process of swelling, respectively for increasing the content of adsorbed water.
Fig. 6. a) Swelling degree of gels with different P(ITAU) concentration as a function of time; b) equilibrium swelling degree of gels as a function of P(ITAU) of concentration in PBS.
With regard to P(ITAU) concentration, as it was already stated, higher polymer concentration may have led to greater swelling tendency. The swelling capacity of a hydrogel depends on the space within the polymer network available for accommodating solvent molecules and on the rate at which polymer chains relax [27]. For 10% concentration the structure of the gel is more relaxed and this provides multiple channels for the diffusion of water molecules leading to the dispersion of the hydrogen bonds formed between the two polymers. Furthermore, the water uptake decreases with increasing the concentration of P(ITAU) from 20% to 40 %. In the case of P(ITAU)40_HA sample, increased concentration of synthetic copolymer induces steric hindrances thus preventing the swelling of the network. The polymer chains of this sample are densely packed because of the involvement of more molecules in crosslinking and entanglements leading to higher elastic forces opposing swelling and limiting the solute transport throughout the network. The value of equilibrium swelling degree of P(ITAU)10_HA is 476 %, as for the values of equilibrium swelling degree for P(ITAU)20_HA and P(ITAU)40_HA are 744.28 % and 710 % (Fig. 6.b).
3.3 Contact angle Surface wettability is one of the most important properties of all materials since it reflects the real structure and chemical composition at the outermost surface. The surface hydrophilic property plays an important role in many properties such as biocompatibility, cell adhesion, spreading and proliferation on the biomaterials surfaces, lubricity, selective absorption and controlled release of molecules [28]. The wettability of the synthetic and natural polymer and one of the obtained hydrogel P(ITAU )20_HA was studied using static water contact angles. P(ITAU) showed a contact angle
14
of about 53.8° (Fig. 7), indicating that the surface is moderately hydrophilic. Pure HA exhibited more hydrophilic properties because is rich in polar functional groups, and exhibited a contact angle of 42.6°. The gel displayed a decreased contact angle of 26.2° which is significantly lower than that of the individual components indicating a surface with improved wettability enriched in polar groups.
Fig. 7. Mean static contact angles determined by sessile drop method for P(ITAU), HA and P(ITAU)20_HA sample. These differences can be explained through the structure of the new bioconjugate compounds. Thus, HA chains in solution have an expanded “slightly stiff” random coil structure, meanwhile the size of HA varies with the environment conditions as expected for a flexible polyelectrolyte. [29, 30] At the same time, the investigation of the HA in the solid state confirm the helical conformation with two types of domains with different mobility, which are dependent on the extended hydrogen-bonded arrays. After HA conjugation with P(ITAU) the new macromolecular chains come along in a wormlike pattern with the functional groups released from the physical bonds and capable now for other interactions, concretized in diminution of the static contact angle. 3.4 Thermal analysis TG and DTG curves concerning thermal behavior and changes in thermal stability of the obtained products are depicted in Fig. 8. These showed gradual sample mass loss with increasing temperature, having four or three distinct degradation regions, influenced by the concentration of the copolymer in the final gel structure. The first step of degradation, similar for all batches, may be associated with dehydration from the compound structure as well as the presence of residual monomers; the other regions levels are related to the breakage of the polymeric chains. The main mass loss percentage (going from 70.94 to 81.22 wt. %) was observed in the temperature range from 150 to 300°C – mainly due to the breakage of functional groups (hydroxyl, carboxyl, carbonyl), which generates gaseous compounds with low molecular weight (CO2, CO, H2O, CH2OH, CH2O etc.) [31]. By increasing the heating temperature above 300oC a new stage of degradation appears for P(ITAU)20_HA and P(ITAU)40_HA with weight losses ranging between 11.90 to 9.56 wt. %. This stage is a result of splitting ties hydrocarbon CC and the releases of saturated or unsaturated aliphatic fragments of higher molecular weights and derivatives
15
carbonyl. The remaining residue shows a decreasing trend correlated with the concentration of the polymeric matrix. Parameters describing the thermal behavior of these gels, calculated from TG are displayed in Table 2. From the viewpoint of the degradation process, P(ITAU)40_HA sample is more stable and starts degradation later (at 163ºC) than P(ITAU)20_HA and P(ITAU)10_HA (151ºC and 113ºC). This fact is also supported by analyzing the thermal stability as a function of T10 and T20 temperatures which corresponds to 10 or 20% weight loss. The most stable gel corresponds to the one with a higher concentration of P(ITAU), which has a denser network generated by the synthetic matrix. Comparing degradation profiles of both HA [32] and P(ITAU) [14] we can observe significant differences which can be correlated with the appearance of new moieties enhanced by the presence of synthetic copolymer with the consequent increase of the thermal stability of all samples. Furthermore, based on these results the possibility of heat sterilization can be taken into account since no animal-derived raw HA provides superior heat stability for efficient sterilization processes [33].
Fig. 8. TG (a) and DTG (b) curves of P(ITAU)_ HA hydrogels.
Table 2 Thermal parameters of the P(ITAU)_ HA hydrogels. 3.5 X-Ray Diffraction Analysis X-ray diffraction analysis was performed to evidence the physical state of newly synthesized bioconjugate gel structure. XRD patterns are depicted in Fig. 9. P(ITAU)10_HA, P(ITAU)40_HA, P(ITAU) and HA precursors present diffraction patterns with one broad and extended diffraction peak of the diffusion type centered around 15 - 25
(2θ) typical for
amorphous materials. Instead P(ITAU)20_HA has a lot of peaks with different intensities that can be considered as pieces of crystallites embedded in a matrix of amorphous material resulted from ordered bonds intervened between HA and P(ITAU). These crystallites may have occurred due to the optimum ratio between HA and P(ITAU), and this optimal proportion for conjugation can determines the aligned towards the length of the macromolecular chains and with a preferred orientation.
16
Fig. 9. XRD patterns for the studied samples.
4. CONCLUSIONS
The general goal was to investigate the properties of new hydrogels based on a polysaccharide (HA) and a synthetic copolymer synthesized by using a grafting – to strategy. Samples of various compositions with different concentration of P(ITAU) were successfully prepared and can be used as a basis for injectable drug delivery systems that may be able to provide sustained and controlled release of a wide variety of bioactive agents. The P(ITAU)_ HA hydrogels exhibit a rheological behavior typical to strong gels and show improved viscoelastic properties at low synthetic polymer concentration. In the creeprecovery tests, a high elastic recovery of the hydrogel network was observed after removing the applied shear stress and the sample restores its equilibrium in approximate 200 seconds. In addition, the hydrogels had shown shear-thinning thixotropic behavior, indicating suitability of these systems as injectable drug delivery vehicles. It was demonstrated that the new structures have a good swelling capacity and also an improved thermal stability dependent on the sample composition. Together, these results demonstrate that the proposed strategy is a valuable toolbox for fine-tuning the rheological and structural properties of injectable HA hydrogels. The facile delivery of these systems and the absence of any external intervention or small molecules to facilitate gel formation make gels prepared using the chemistries discussed herein less invasive and easier for use. Importantly, because of the remarkable feature given by HA, these hydrogels can be used to deliver drugs, biological molecules or cells during the injection process with applications that can be divided in two main fields: regenerative medicine and local therapy through drug delivery.
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ACKNOWLEDGMENTS This work was financially supported by the grant of the Romanian National Authority for Scientific Research, CNCS-UEFISCDI, project number PNII-RU-TE-2014-4-0294 Novel hydrogels synthesis with defined 3D functionality and biodegradable characteristics for bioapplications. The authors thank and appreciate the colleague Dr. Daniel Timpu in “P.Poni” Institute of Macromolecular Chemistry for his expert assistance in X-ray diffraction analyses.
References
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FIGURES
Fig. 1. Schematic presentation of the P(ITAU)_HA hydrogels.
22
Fig. 2. The viscoelastic parameters as a function of the oscillation frequency for P(ITAU)_HA gels as compared with HA solutions.
23
Fig. 3. The complex viscosity as a function of the oscillation frequency for P(ITAU)_HA gels, P(ITAU)20 and HA solution.
24
Fig. 4. Creep-recovery curves obtained for P(ITAU)20_HA at 37C for different shear stress values.
25
Fig. 5. The elastic recovery (rec) as a function of shear stress obtained for different samples.
26
Fig. 6. a) Swelling degree of gels with different P(ITAU) concentration as a function of time; b) equilibrium swelling degree of gels as a function of P(ITAU) of concentration in PBS.
27
Fig. 7. Mean static contact angles determined by sessile drop method for P(ITAU), HA and P(ITAU)20_HA sample.
28
Fig. 8. TG (a) and DTG (b) curves of P(ITAU)_ HA hydrogels.
Intensity (u.a.)
29
HA P(ITAU)10_HA P(ITAU)20_HA P(ITAU)40_HA P(ITAU)
5
10
15
20
25
30
35
(degree)
Fig. 9. XRD patterns for the studied samples.
40
30
Tables
Table 1. The thixotropic area determined for the P(ITAU)_HA gels Sample
P(ITAU)10
Hysteresis area from shear stress curve (Pas-1) 96.92
Hysteresis area from viscosity curve (Pas-1) 227.85
P(ITAU)20
11.59
35.93
P(ITAU)40
18.15
66.40
Table 2. Thermal parameters of the P(ITAU)_ HA hydrogels Sample
P(ITAU)40_HA
Degradation stage I
Tonset (C) 163
Tpeak (C) 217
W (%) 16.44
II
227
250
18.33
III
294
328
36.17
IV
424
432
11.90
residue
P(ITAU)20_HA
T10 (C) 201
T20 (C) 240
175
212
17.16
I
151
217
41.09
II
287
305
31.23
III
419
426
9.56
31
residue
P(ITAU)10_HA
18.12
I
113
-
21.68
II
200
216
26.64
III
296
364
32.90
residue
153
185
18.78
Tonset – the temperature at which the thermal decomposition begins; Tpeak – the temperature at which the degradation rate is maximum; T10 and T20 – the temperature corresponding to 10% and 20% weight loss, respectively; W – the weight loss.