FIELD OF THE INVENTION

The present invention discloses and claims a novel initiator-free synthesis method for the preparation of a nontoxic and biocompatible photo-crosslinked hydrogels from natural and synthetic polymers.

BACKGROUND

Hydrogel is a three-dimensional, crosslinked polymer network which consists of hydrophilic moieties (-COOH, -OH, -NH2 etc.). These hydrophilic groups are mainly responsible for retaining high amount of water within the structure. The main reason for the importance of hydrogels in biomedical applications is mainly due to its extraordinary permeability for nutrients, oxygen and other water-soluble metabolites. Hydrogels are made from natural (collagen, silk, alginate, chitosan, hyaluronic acid etc.) and synthetic (PEG, PLGA, PVA etc.) sources.

Hydrogel fabrication can be categorized into two main types depending on crosslinking mechanism involved. In physically crosslinked hydrogels, polymer chains interact through hydrogen bonding, hydrophobic interactions, Van der Waals forces and ionic interactions. While in chemically crosslinked hydrogels, covalent bonding results in superior mechanical properties as compared to physical ones. Different kinds of reactions and chemistries are involved in establishing covalent bonds between polymer chains such as; addition, condensation reaction, Michael addition, click chemistry (alkyne-azide reaction), free radical polymerization etc. All these reactions require photo- initiator/coimtiator or catalyst. However, the use of these toxic photo- ini tiator/coinitiator or catalyst, specifically injectable hydrogels, is serious concern for the biocompatibility of these gels for biomedical applications.

Previous studies and inventions require the use of photo-initiator/coinitiator or a catalyst for hydrogel synthesis under specific conditions. These conditions can be harsh or soft depending on the reaction type. For example, in traditional addition reaction, successively addition of alkene monomers occurs at high temperature, pressure and in the presence of toxic catalyst. In normal photo-polymerization reactions, some photo-initiators or co-initiators (Eosin- Y, Irgacure, LAP, NVP, TEA etc.) are used to promote the formation of radicals which lead to branching and crosslink but are commonly toxic to living cells.

In a typical alkyne-azide chemistry (Copper(I)-catalyzed [3+2] azidealkyne cycloaddition, CuAAC), copper is used as a catalyst which is toxic to cells. However, in a modified version of this chemistry ([3+2] cycloaddition of azide and strain-promoted alkyne, SPAAC), alkyne was replaced by dibenzocyclooctyne (DBCO) and reaction can be proceeded in the absence of a catalyst and at room temperature as well [1], Currently, this chemistry is the solely state-of-the-art. In other examples, hydrogel formation with high molecular weight PEO (Mw=* 106Da) under electron and y-irradiation was reported which involved the synthesis of hydrogel through the radiolysis of water-generating hydroxyl radicals that attack the polymer chain [2, 3],

In the current state of the art (SPAAC), there are some problems that limit their applications. One of these limitations involves the use of complex reactions and expensive chemicals in producing Azide and DBCO functionalities in polymer [4], Secondly, the color of the product formed is yellowish due to inherent yellow color of DBCO and consequently, hydrogel becomes opaque in nature, which restricts it applications where optical transparency is required. Furthermore, in other examples, where, electron and y-irradiation are involved in hydrogel formation, is applicable on very high molecular weight polymers only and use of such harmful radiations make the system impractical. We have solved these limitations associated with the present methods in our invention presented here.

In one study, fabrication and characterization of poly(vinyl alcohol)/chitosan hydrogel thin films via UV irradiation was explained. It was mentioned to prepare a series of poly (vinyl alcohol) / chitosan (PVA / CTS) hydrogel thin films by ultraviolet scattering with the addition of acrylic acid (AA) monomer as a crosslinker without the addition of any photo-initiator. In this study, thin films were prepared from PVA and chitosan in the presence of acrylic acid. In the prepared film, PVA and chitosan chains were physically entrapped between chemically crosslinked structure of polyacrylic acid and chains were merely having physical interactions like hydrogen bonding [5],

In another recent study, a method was mentioned which comprising microfluidic formation of monodispersed spherical microgels composed of triple-network crosslinking. In the scope of the study, the triple-mesh (3XN) type hydrogel system contains minimally modified natural GRAS materials, partially oxidized dextran (Odex), Teleostane and N-carboxyethyl chitosan (CEC), and the use of crosslinkers or photo-initiators is not required. Thus, it has proven to be a novel biodegradable and mechanically powerful in-situ gelling hydrogel system. In this study, authors have used partially oxidized dextran, teleostean and N- carboxyethyl chitosan for making microgel by using microfluidics technique. Prerequisite modification of polymers was carried out to achieve crosslinking through Schiff base reaction. These kinds of modifications are not easy to do for all other polymers making this procedure less versatile [6],

A further study mentioned about flexible oligomer spacers as the key to solid-state photopolymerization of hydrogel precursors. The prepolymers obtained within the scope of the study show good solid-state photoreactivity even in the absence of a photo-initiator. In this reported study, authors first synthesized model precursor (PEG-OEOAcr) through complex chemical reactions. First, they reacted PEG with diisocyanate in the presence of a toxic catalyst at elevated temperature followed by reaction with oligo-PEG-acrylate. Then this precursor was used to make hydrogel. Their system contains complex procedures to be carried out in the presence of toxic catalyst and ultimately their system was not capable enough to produce strong enough hydrogel in the absence of initiator (few pascals). Therefore, they used Irgacure (a photo-initiator) to enhance their mechanical properties [7], Whereas we are reporting very high modulus of our hydrogel via very simple and facile method (in our case, PEG based hydrogel is = 51kPa).

In the European patent document EP2585497, the production of hydrophilic gels from polyurethane-based photo-initiators was mentioned. Self-initiating photoinitiator functionalities are disclosed within the scope of the invention. Upon UV or visible light stimulation, these photo-initiators are separated and crosslinked predominantly by a Norrish type I mechanism, without furthermore a conventional photo-initiator. In this patent, polymer based crosslinker is used for making gels. Different diisocyanates can be used for making a crosslinker and this diisocyanate further reacts with different functional groups. Basically, this method is not useful for making hydrogel, especially cells containing hydrogels, because all the diisicyanates are inherently toxic to cells. So, these can’t be used in prepolymer solutions. Further, diisocyanate reacts with different functional groups at certain conditions, either require catalyst or high temperature. Most importantly, such polymerization can’t be proceeded in water or aqueous medium because of many side reactions. It requires organic solvent if the reaction is to proceed in solution form. For example, isocyanate functional group is not stable in the presence of water and converts to -NH2 and CO2 becomes in active towards reaction proceeding. Conclusively, this reported invention is beyond the scope of biomedical applications where cells are used in aqueous medium to encapsulate them in polymeric network.

Conclusively, the present invention is advantageous over all these studies/inventions or any other reported one. In the present invention, potential of hydrogel formation of polymers (PEG, chitosan, alginate, gelatin) is shown under UV and Visible light in the absence of any photo-initiator. Further, any polymer, either natural or synthetic, can be used to form hydrogel through our reported procedure. It just needs one single step like acrylation of any polymer through - NH2 or -OH or both functional groups and then exposing its solution to UV or Visible light. These needs and other needs are satisfied by the present invention. In this invention, we addressed this urgent need through the design of anthracene- incorporated alginate gel network.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a novel initiator-free synthesis method for the preparation of a nontoxic and biocompatible photocrosslinked hydrogels from natural and synthetic polymers.

The other aspect of the present invention is to provide a novel initiator-free synthesis method to eliminate the use of toxic initiators during photopolymerization of natural as well as synthetic polymers.

Another aspect of the present invention is to provide a novel initiator-free synthesis method via a simple and facile process to synthesize acrylated form of any polymer and thus acrylated/methacrylated form of any polymer can be directly used. The Polymer mentioned here can be either functionalized through acrylation as we reported before [8, 9] or acrylated form of any commercially available polymer can also be used directly.

A further aspect of the present invention is to provide a novel initiator-free synthesis method to obtain transparent hydrogels in nature.

This present invention provides a novel initiator-free synthesis method which uses the capability of different wavelengths of light to carry out the crosslinking reaction step. In the present invention, the reaction step is performed under commonly used UV (k=365nm) and visible light (k=430 nm), which is more compatible with cells. This will enable us to encapsulate cells in polymer solution prior to gelation. All the gels made were mechanically robust and ready to use for end use biomedical applications. All these reactions were carried out in the absence of any photo-initiator/co-initiator or catalyst.

Accordingly, a broad embodiment of the invention is directed to a synthesis method which can be used for biomedical applications.

This object and other objects of this invention become apparent from the detailed discussion of the invention that follows.

Brief Description of Figures

The present invention is illustrated in the accompanying figures wherein;

Figure 1 is an illustration of the reaction to make hydrogel under UV and Visible light.

Figure 2 is a set of illustrations of scanning electron microscopic (SEM) images of synthesized hydrogels under UV (A) AlgiMA, (B) CSMA, (C) GelMA, (D) PEGDA) and visible light ((E) AlgiMA, (F) CSMA, (G) GelMA).

Figure 3 is a set of illustrations of storage (G’) and loss modulus (G”) profiles of hydrogels under UV light. ((A) AlgiMA, (B) CSMA, (C) GelMA, (D) PEGDA, (E) PEGDA-Azide)

Figure 4 is a set of illustrations of storage (G’) and loss modulus (G”) profiles of hydrogels under visible light. ((A) AlgiMA, (B) CSMA, (C) GelMA)

Figure 5 is an illustration of FTIR analysis of PEG-DA polymer and respective hydrogel.

The parts on the figures are referred and the information corresponding these references are presented below.

PS. Polymer Solution pPS. Prepolymer Solution

RUPP. Rheometer’s upper parallel plate

QP. Quartz plate

R. Rheometer

H. Hydrogel

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a novel initiator-free synthesis method for the preparation of a nontoxic and biocompatible photo-crosslinked hydrogels (H) from natural and synthetic polymers which are crosslinked upon exposure to light of a suitable wavelength.

Unless specified otherwise, the term “initiator” refers to a photo-initiator, a coinitiator and a catalyst, and more particularly refers to a photo-initiator. This invention is suitable for use in a ‘clinically desirable’ approach for the preparation of hydrogels (H) in toxic-free medium, which is critical for biomedical applications and in particular for cells encapsulated within hydrogels (H).

The approach developed here is applicable to different kinds of polymers that are natural or synthetic. Any type of polymer that contain amine (-NH2), hydroxyl (- OH) or both functional groups can be utilized to functionalize the polymers with acrylic groups and can further be used during photo-crosslinking in the absence of a photo-initiator, co-initiator or a catalyst.

An object of the present invention is a method for the preparation of a completely non-toxic and biocompatible hydrogel (H), including an initiator-free and only light activated photo polymerization reaction step which named here as “initiator- free photo-click addition reaction” (IFPCAR).

The present invention relates to a preparation method of hydrogel (H) which is a cross-linked macro molecule network.

According to the invention, surprisingly it is found that using different wavelengths of light to carry out the crosslinking reaction is sufficient to achieve the photopolymerization.

The present invention relates to a method for the preparation of photo crosslinked hydrogel (H). In particular, the preparation method comprises photopolymerization of acrylic groups.

The preparation method of the invention involves, in the first step, the preparation of acrylated and/or methacrylated polymer solution (PS). This step is named also as acrylation and/or metacrylation step. The polymer used in the polymer solution (PS) is selected from the group of any polymer containing acrylate and/or methacrylate groups.

The preparation method of the invention involves, in the second step, photo crosslinking of polymer solution (PS) by photo-polymerization of acrylic groups in the absence of any initiator.

According to the invention, a method for the preparation of a nontoxic and biocompatible photo-crosslinked hydrogels (H) comprising the step of:

- providing an acrylated and/or methacrylated polymer solution (PS),

- exposing the polymer solution (PS) to light of a suitable wavelength to induce crosslinking in the absence of initiator.

According to the present invention, the polymer used in the first step is an acrylated and/or methacrylated form of any polymer containing the group of -NH2 or -OH or both. It just needs one single step like acrylation of any polymer through -NH2 or -OH or both functional groups. In a preferred embodiment the polymer is selected from the group comprising natural polymer such as alginate, chitosan, gelatin and synthetic polymers such as PEG. However, any natural or synthetic polymer that contains hydroxyl (-OH) or amino (-NH2) or both functional groups, can be used to synthesize hydrogel (H) by the procedure proposed in this invention. These functional groups can be used to induce acrylation in the polymer and subsequently this functionalized polymer can be utilized to synthesize hydrogel (H). On the other hand, acrylated/methacrylated form of any polymer can also be directly used in the present invention. Further, polymers (alginate, chitosan, gelatin) were functionalized through methacrylation, where acrylated PEG (PEGDA) was directly used after purchasing. In second step, hydrogel (H) is made by utilizing polymer solution (PS) in the first step. Hydrogel (H) is made through photo-polymerization under UV (X=365nm) or visible light (X=430 nm), in the absence of initiator, where only acrylic groups of polymers took part in reaction.

The other advantage of the technique developed here is the capability of using different wavelengths of light to carry out the crosslinking reaction. The reaction is successfully performed under commonly used UV (k=365nm) and visible light (k=430 nm), which is more compatible with cells. This success will enable to encapsulate cells in polymer solution (PS) prior to gelation. All the gels made were mechanically robust and ready to use for end use applications. All these reactions were carried out in the absence of any photo-initiator/co-initiator or catalyst.

In the present invention, a technique is developed to eliminate the use of such toxic initiators during photo-polymerization of natural as well as synthetic polymers. According to the invention, a simple method is required to synthesize acrylated form of any polymer. Further, acrylated/methacrylated form of any polymer can be directly used. The resultant hydrogels (H) are also transparent in nature The potential of the invention is demonstrated for hydrogels (H) prepared from both natural and synthetic polymers.

These examples are intended to representative of specific embodiments of the invention and are not intended as limiting the scope of the invention.

SPECIFIC EMBODIMENTS

In these embodiments, a preparation procedure was applied to provide the nontoxic and biocompatible hydrogel (H). After obtaining the candidate hydrogel (H) by the preparation method illustrated in figures, experimental studies lead to determine the hydrogel (H) features. Furthermore, characterization of the present invention was studied.

Examples

Example 1 Formation of Hydrogel (H)

In this invention, a facile and versatile procedure is reported to synthesize hydrogel (H) under UV and as well as visible light in the absence of any kind of photo-initiator/co-initiator as illustrated in Figure 1. Polymer will either be functionalized through acrylation as we reported before [8, 9] or acrylated form of any commercially available polymer can also be used directly. We have synthesized hydrogels (H) with the help of light (k=365nm or 430nm) in the absence of any photo-initiator. We showed the potential of this procedure by using natural (alginate, gelatin, chitosan) and as well as synthetic (Polyethylene glycol) diacrylate, PEGDA) polymers. We successfully made hydrogel (H) from all the polymers by using appropriate concentration of each polymer solution (PS). Alginate, chitosan and gelatin were reacted first with methacrylic anhydride (MA) to make respective methacrylated form of each polymer (AlgiMA, CSMA or GelMA). After purification and lyophilization, dried powder was used to make solution while PEGDA was used as purchased. In this simple procedure, methacrylated form of each polymer was dissolved in distilled water (w/w%) at appropriate concentration (AlgiMA, 15%; CSMA, 2%; GelMA, 25%; and PEGDA, 25%). 320 pL of polymer solution (PS) was poured onto a quartz plate (QP) (Dia=20mm) fitted on a Rheometer (R). Rheometer (R) was modified to house two different light sources (Figure 1). Solution was exposed to light (either with 365nm or 430nm) with a power of 150 and 140mW/cm2 respectively.

Example 2 Scanning Electron Microscopic (SEM) Analyses of Hydrogel (H) For obtaining the cross-sectional images, hydrogels (H) were dipped in liquid nitrogen first and then were cut without disturbing the cross-sectional features. Images were acquired by using a ZEISS ULTRA PLUS FIELD EMISSION SCANNING ELECTRON MICROSCOPE and are provided in Figure 2 as a hydrogel (H) synthesized under UV (A; AlgiMA, B; CSMA, C; GelMA, D; PEGDA) and visible light (E; AlgiMA, F; CSMA, G; GelMA). All these images represent the typical porous structure of a hydrogel (H).

Example 3 Measurements and Comparative Studies

Rheological and chemical analysis was performed to confirm the mechanical strength of formed hydrogels (H) and chemistry involved

Storage (G’) and loss modulus (G”) profiles were recorded as a function of time (1 Hz frequency and 5% strain) to monitor the in-situ crosslinking. G’ and G” of hydrogels (H) made under UV and visible light are provided in Figure 3 and Figure 4, respectively. In all hydrogels (H), gel point was achieved where storage modulus crosses the loss modulus, and this crossover is shown with an arrow. We believe that photo-polymerization involved in our reaction classically follows singlet oxygen (102) mechanism. To prove this, we performed a control reaction in the presence of sodium azide (250mM) in prepolymer solution (pPS). Sodium azide is a well-known oxygen quencher and its presence in a system inhibits reaction that involves singlet oxygen. We can see from Figure 3-E, where PEGDA was exposed to 365nm in the presence of sodium azide and modulus didn’t increase even after 4000 seconds. Furthermore, modulus of hydrogel (H) depends on methacrylation% and initial concentration of each polymer solution (PS) and that can be tuned depending on required mechanical properties of hydrogel (H). Chemical analysis of polymer and hydrogel (H) was performed using Fourier transform infrared spectroscopy in attenuated total reflection mode (Thermo- Scientific iS50 ATR-FTIR). The change in intensity of -C=C- band at 1413cm' ¹ in PEGDA polymer and respective dried hydrogel (H) was compared (Figure 5). The decrease in intensity in hydrogel (H) corresponds to addition reaction going on. This result strengthens our hypothesis that during hydrogel (H) formation, photoclick-addition reaction is going on.

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