Patent Application
20100144900
The present invention relates to a stereo photo hydrogel, to a process of making stereo photo hydrogels, to polymers for use in making such hydrogels and a pharmaceutical kit comprising such polymers.
Hydrogels have been widely used for biomedical applications, such as tissue engineering and drug delivery, due to their favorable characteristics.1-3 Hydrogels are water-swollen networks of crosslinked hydrophilic polymers. Their high water content renders them highly biocompatible and also leads to minimal adsorption of proteins. The mechanical properties of hydrogels parallel those of soft tissues, making them particularly interesting for tissue engineering. Hydrogels may be formed in situ, thus allowing easy mixing of cells and bioactive molecules, such as proteins, with the polymer solutions prior to gelation.4-6 Moreover, in situ hydrogel formation enables the preparation of complex shapes and use of minimally invasive surgery. In situ forming hydrogels have been prepared by physical and chemical crosslinking methods. Physically crosslinked hydrogels include those based on hydrophobic interactions and hydrogen bonds between thermosensitive block or graft polymers7-11, stereocomplexation between poly(L-lactide) (PLLA) and poly(D-lactide) (PDLA) graft12 and block copolymers13-15, inclusion complexation using α-dextrin polymers16-20, and ionic interactions between oppositely charged microparticles21 or peptides22. The crosslinking conditions for these gels are generally very mild, thus allowing the entrapment of labile compounds, such as proteins. However, in general they are mechanically weak compared to chemically crosslinked hydrogels and changes in the external environment (e.g. ionic strength, pH, temperature) may give rise to disruption of the network.
Chemically crosslinked hydrogels have been formed in situ by Michael addition between thiols and acrylates or vinyl sulfones23-29, reaction between aldehydes and dihydrazides30 or amines31, reaction between activated esters and amines32 and redox initiated radical chain polymerization of (meth)acrylates33-37. Photopolymerization of (meth)acrylates5 using UV-light38-41 or visible light42-44 has been mostly used for in situ formation of chemically crosslinked hydrogels. Biodegradable hydrogels prepared by photocrosslinking of polyethylene glycol)-poly(lactide) (PEG-PLA) diacrylate derivatives were first reported by the group of Hubbell.42 More recently, this group has prepared degradable hydrogels by the incorporation of plasmin degradable peptide sequences.39,43 When modified with cell-adhesive RGD peptide sequences, these hydrogels supported three-dimensional outgrowth of human fibroblasts embedded as a cluster within the hydrogel. Another type of degradable hydrogel was prepared by copolymerization of a hyaluronic acid methacrylate derivative and PEG diacrylate.44 Fibroblasts adhered and proliferated when cultured on the RGD functionalized hydrogels. The group of Anseth has done much work on degradable hydrogels based on PEG-PLA dimethacrylates.40 It was shown that by using combinations of PEG and PEG-PLA dimethacrylates and/or by changing the PLA block length, the hydrogel degradation rate, compressive modulus and crosslinking density could be tuned to provide suitable scaffolds for cartilage tissue engineering.41 The major advantage of photopolymerization is the spatial and temporal control over the polymerization. However, photopolymerization in vivo is hampered by the absorption of UV-light by the skin (>99%). In clinical applications, fast gelation is desired to prevent diffusion of hydrogel precursors or bioactive molecules to the surrounding tissue. Elisseeff et al. have reported on transdermal photopolymerization of a 20 wt % PEG dimethacrylate aqueous solution injected subcutaneously into nude mice by UV-irradiation for 3 min at 2 mW/cm2 incident light intensity.45 In this study, high molecular weight PEG (100,000) was used as an additive to prevent rapid diffusion of the gel precursors after injection and to increase the mechanical properties of the photopolymerized hydrogel. A drawback is that it is very difficult to excrete high molecular weight PEG by the kidneys.46 Elisseeff et al. have studied the UV-light attenuation by the skin using swine skin as a model.47 The incident light intensity of 100 mW/cm2 was attenuated by the skin to ca. 0.05 mW/cm2. After 3 min of UV-irradiation of a 20 wt % PEG dimethacrylate aqueous solution with 0.04 wt % photoinitiator concentration, a conversion of ca. 10% was reached. The remaining unsaturated bonds may cause toxicity problems and incomplete conversion may result in hydrogels with weak mechanical properties.48 The polymerization rate may be increased by increasing the photoinitiator concentration or the intensity of the incident light. However, due to their toxicity photoinitiators can only be used at low concentrations (ca. 0.01-0.05 wt %)49 and the intensity of the UV-light is limited to ca. 5-10 mW/cm2 to prevent cell damage. Visible light is less attenuated by the skin, but efficient initiators with less cytotoxicity are required.49,50 Another problem of photopolymerization is that fast polymerization is generally accompanied by substantial heat effects.48 The resulting temperature rise may cause local cell morbidity and tissue necrosis surrounding the implant.
The present invention has for its object to provide a hydrogel which is fast in situ forming and is robust. The present invention is based on the insight that by the combined use of stereo complexation and photo polymerization gelation will be fast and photo polymerization will be facilitated. After rapid formation of the stereo complexed hydrogel, subsequent photo polymerization can be carried out at a relatively low polymerization rate to provide gels with the desired mechanical properties.
Furthermore, the local temperature rise by heat generated by the photo polymerization will be limited. In addition, the photo polymerization may take place at lower initiator concentrations and at lower irradiation intensity as compared to gel formation without using preformed stereo complexes.
Under particular circumstances, the stereo photo hydrogel formed will have increased mechanical properties and if desired prolonged degradation times. Finally, the stereo complexation aids in the photo polymerization of the photo cross-linkable component. The stereo photo hydrogels formed will have increased storage moduli and if desired improved degradation times in comparison to photo hydrogels formed by photo polymerization only. Accordingly, the present invention provides stereo photo hydrogels formed by stereo complexed and photo cross-linked polymers, which polymers comprise at least two types of polymers having at least one hydrophilic component, at least one hydrophobic mutually stereo complexing component, and at least one of the types comprises at least one photo cross-linkable component. Accordingly, this stereo photo hydrogel is formed by stereo complexed and photo cross-linked polymers. These polymers comprise at least two types of polymers. The polymers of both types have at least one hydrophilic component required for forming the hydrogel. The polymers further comprise at least one hydrophobic stereo complexing component which will mutually stereo complex thereby forming the stereo hydrogel. At least one of the two types of polymers comprises at least one photo cross-linkable component. Accordingly, after formation of the stereo hydrogel the polymers of at least one of the two types are cross-linked by photo polymerization thereby forming the stereo photo hydrogel.
According to a preferred embodiment the two types of polymers comprise at least one mutually photo cross-linkable component. Accordingly, both types of polymers are mutually photo cross-linked such that both types of polymers are covalently bound thereby forming a very robust stereo-photo hydrogel.
Examples of the hydrophilic component for each of the two types of polymers are PEG, dextran, hyaluronic acid, pullulan, chondroitin sulfate, poly (vinyl alcohol), poly(hydroxyethyl methacrylate), poly(aspartic acid), poly (glutamic acid), poly(acrylic acid), and poly((C1-C6)alkyloxazoline), such as poly(methyl- or ethyl-oxazoline). Preferred is PEG having a number average molecular weight of for instance 10,000-50,000, such as 20-30,000.
An example for the hydrophobic stereo complexing component comprises poly (L-lactide) or poly (D-lactide). In general each poly(L-lactide) or poly (D-lactide) could comprise 3-30 lactyl units per poly (L- or D-lactide). More preferably the number of lactyl units per poly (L- or D-lactide) is 10-20, more preferably 12-16. This depends on the hydrophobic character required for the stereo complexing component relative to the hydrophilic character of the hydrophilic component and the character of the photo cross-linkable component.
The photo cross-linkable component may comprise acrylate, methacrylate, acrylamide and fumarate. Preferred as a photo cross-linkable components are acrylate and methacrylate.
The photo cross-linkable component may be cross-linked using visible or ultraviolet irradiation, depending on the use. For the in-vivo formation of the stereo-photo hydrogel it is preferred to use long wavelength ultraviolet irradiation. When using long wavelength ultraviolet irradiation the intensity of the UV irradiation may be relatively low. For reasons, that due to the prior formation of the stereo hydrogel, photo crosslinking may be carried out at lower rates. The UV irradiation may be as low as 0.05-20 mW/cm2 (when there is a tissue barrier (such as intact skin) or from 2-20 mW/cm2 when there is no tissue barrier. When using visible light, the intensity of the visible light is preferred from 30-100 mW/cm2.
The two types of polymers may have the same or mutually different structures. According to one structure the stereo complexing component and the photo cross-linkable component are both directly linked to the hydrophilic component. According to another structure the photo cross-linkable component is linked to the stereo complexing component which in turn is linked to the hydrophilic component.
With both type of structures hydrogels may be formed, in which the constituting polymers have the form of a triblock or the form of a multi arm structure. When having a multi arm structure, the number of arms is preferably between 3-12, more preferably between 8-10 arm structures.
It is noted that various type of structures (of a different composition than the polymers according to the present invention) are disclosed in US 2003/0087985 in FIGS. 1A-1J.
The hydrogels according to the invention have advantageous properties. One of the advantageous properties of the hydrogel is a storage modules G′ larger than 1 kPa. Storage moduli G′ up to 150 kPa may be obtained. Accordingly, the hydrogels may have a storage modules G′ within the range of about 1-150 kPa, preferably within the range of 1-100 kPa.
Another aspect of the present invention relates to the process of making the stereo-photo hydrogels according to the invention. Based on the insight of the present invention the stereo-photo hydrogel is formed by first stereo complexing two types of polymers (as described above). Subsequently, the formed stereo complexed hydrogel is subjected to photo cross-linking thereby forming the stereo-photo hydrogel.
Accordingly, the present invention provides a process of making stereo photo hydrogel comprising the steps of
Accordingly, the two types of polymers are mixed which will, dependent on the circumstances, result in the stereo complexing of both types of polymers within a given time period. In this time period the stereo complexed hydrogel is formed. Subsequently, the stereo complexed hydrogel is subjected to photo cross-linking using irradiation thereby forming the stereo-photo hydrogel. The irradiation may be (low intensity) UV irradiation or visible light.
As indicated above it is preferred that the two types of polymers both comprise a photo cross-linkable component. Accordingly, both types of polymers are photo cross-linked resulting in a robust stereo photo hydrogel. If desired, the photo cross-linking may be carried out in the presence of a photo initiator. When the photo cross-linking is to take place within the animal or human body it is preferred to use an in situ compatible photo initiator (preferably in an amount of 0.001 to 0.05 wt %). Suitable examples are 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651), 1-hydroxycyclohexyl phenyl ketone (Irgacure 184), 2-methyl-1-[4-(methylthio) phenyl]-2-(4-morpholinyl)-1-propanone (Irgacure 907), and 2-hydroxy-1-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Darocur 2959), camphorquinone (CQ) with ethyl 4-N,N-dimethylaminobenzoate (4EDMAB) and triethanolamine (TEA) and the photosensitizer isopropyl thioxanthone.
The mixture of the two types of polymers may comprise the polymers in any suitable concentration for subsequent stereo complexing. In general the polymer concentration is within the range of 5-30 wt-v %. Preferably, the polymer concentration is within the range of 10-20 wt-v %. Dependent on the type of polymers, their structure and the concentration the stereo complexing time is within the range of 1 minute-3 days. Accordingly, it is possible in relation to the objective use of the photo stereo hydrogels to select the time of stereo complexing as required. Particularly, short or relatively short times for stereo complexing may be selected when it is desired to include within the stereo complexed hydrogel and subsequently within the stereo-photo hydrogel a component which may leach out or may be diffused out of the stereo photo hydrogel over an extended period of time and not during the stage of forming the stereo complex and the formation of the stereo-photo hydrogel. Then relatively short times for stereo complexing may be selected. Preferably, the time for stereo complexing is within 2 hours to 1 day, and more preferably within 4-10 hours.
The stereo-photo hydrogels will be subject to hydrolysis and thereby degradation of the hydrogel. Hydrolysis preferably takes place by hydrolysis of the hydrophobic component and more preferably by hydrolysis of the poly-lactide chain. When the stereo-photo hydrogel is formed of polymers having structures in which the photo cross-linkable component is at the end of some or all hydrophobic components, then degradation will result in a clear solution within a period of time of about 1 day-7 weeks, such as 1-3 weeks.
If the stereo-photo hydrogel is formed by structures in which the hydrophobic component and the photo cross-linkable component are both directly linked to the hydrophilic component, then degradation by hydrolysis of the hydrophobic component will result in a swollen hydrogel which will subsequently degrade by hydrolysis of the photo cross-linkable component and then form a clear solution. Degradation from the swollen hydrogel into the clear solution will take 2-30 weeks, such as 5-25 weeks, more preferably 7-21 weeks. Clearly, the swollen hydrogel has different properties than the stereo-photo hydrogels.
The hydrogels may comprise a pharmaceutically active agent or a moiety that binds a pharmaceutically active agent. Pharmaceutically active agents may be any pharmaceutically active compound used for therapy, diagnosis or prophylaxis of a human or animal body. The pharmaceutically active agent may comprise cells and biologically active molecules such as proteins, antibodies and the like.
Another aspect of the present invention relates to the polymers of both types as discussed above. Both types of polymers are suitable for use in making a hydrogel which may comprise a pharmaceutical active agent or moiety that binds a pharmaceutically active agent. Such hydrogel may have the form of a medicament for the treatment of the human or animal body. The treatment is related to the biological activity of the pharmaceutically active agent as discussed above.
Finally, the present invention relates to a pharmaceutical kit which may be used for in-situ or in-vitro forming of a stereo-photo hydrogel according to the present invention. Such pharmaceutical kit comprises two containers each comprising one of two types of polymers having at least one hydrophilic component, at least one hydrophobic mutually stereo complexing component, and at least one of the types comprises at least one photo cross-linkable component. When the stereo-photo hydrogel is to comprise the pharmaceutically active agent or a moiety binding a pharmaceutically active agent, then it is preferred that the pharmaceutically active agent is contained in one or both containers for each of the two types of polymers. If possible or needed, the pharmaceutically active agent or the moiety that binds a pharmaceutically active agent is present in a separate container. Prior to the formation of the stereo hydrogel and the stereo-photo hydrogel, the content of one or more containers is mixed.
Mentioned and other features and characteristics of the stereo hydrogel, stereo-photo hydrogel and the process of making these hydrogels will be further illustrated by the following examples which are given for information purposes only and are not intended to restrict to any extend the present invention. In these examples reference will be made to the figures wherein:
L-lactide and D-lactide were obtained from Purac and recrystallized from dry toluene. Eight-arm star PEG (Mn, NMR=21,800) was supplied by Nektar and used as received. The single site Zn-complex catalyst (Zn(Et)[OC6H4(CH2N(Me)2)-2, Me-4]) was kindly provided by Professor G. van Koten of the University of Utrecht (The Netherlands). Methacrylic anhydride was purchased from Merck and Irgacure 2959 from Ciba Specialty Chemicals. Both were used as received. Dichloromethane (DCM) and triethylamine (TEA) were dried over calcium hydride and potassium hydroxide, respectively, and distilled prior to use. Eight-arm poly(ethylene glycol)-poly(L-lactide) and poly(ethylene glycol)-poly(D-lactide) star block copolymers with 12 lactyl units per poly(lactide) (PLA) block (PEG-PLLA12 and PEG-PDLA12, respectively) were prepared as reported previously (Mn, PEG=21,800).53
PEG-PLLA12-MA and PEG-PDLA12-MA were synthesized by partial methacrylation of the hydroxyl groups of PEG-PLLA12 and PEG-PDLA12, respectively, according to the procedure reported by Lin-Gibson et al.54 Typically, PEG-PLLA12 (5.0 g, 0.174 mmol, dried overnight under vacuum over phosphorous pentoxide) was dissolved in 18 ml of DCM. A solution of TEA (0.171 g, 1.690 mmol) in 1 ml of DCM was added and the reaction mixture was cooled in an ice bath. Subsequently, a solution of methacrylic anhydride (0.244 g, 1.583 mmol) in 2 ml of DCM was added dropwise. The reaction mixture was stirred for 2 days at 30° C. and the product was recovered by precipitation in a mixture of cold diethyl ether/hexane/methanol (10/1/1 v/v). Degree of methacrylation: 40%, yield: 88%. 1H NMR (CDCl3): δ 1.4 (m, CH(CH3)OH end group PLA), 1.5 (m, CHCH3), 1.9 (s, C(CH3)═CH2), 3.6 (m, PEG methylene protons), 4.2-4.3 (m, CH2OCO, linking unit PEG-PLA), 4.3-4.4 (q, CH(CH3)OH end group PLA), 5.1 (m, CHCH3), 5.6 and 6.2 (C(CH3)═CH2)
PEG-MA/PLLA and PEG-MA/PDLA, in which both MA and PLA blocks are directly linked to PEG, were synthesized by ring opening polymerization of lactide using partially methacrylate functionalized eight-arm star PEG (PEG-MA). For the synthesis of PEG-MA, typically, PEG (16.0 g, 0.734 mmol) was dissolved in 33 ml of DCM. A solution of TEA (0.442 g, 4.368 mmol) in 1 ml of DCM was added and the reaction mixture was cooled in an ice bath. Subsequently, a solution of methacrylic anhydride (0.654 g, 4.242 mmol) in 2 ml of DCM was added dropwise. The reaction mixture was stirred for 2 days at 30° C. and the product was recovered by precipitation in a mixture of cold diethyl ether/hexane/methanol (10/1/1 v/v). Degree of methacrylation: 42%, yield: 90%. 1H NMR (CDCl3): δ 1.9 (s, C(CH3)═CH2), 3.6 (m, PEG methylene protons), 4.2 (m, CH2OCO, linking unit PEG-MA), 5.6 and 6.2 (C(CH3)═CH2)
PEG-MA/PLLA and PEG-MA/PDLA were synthesized by ring opening polymerization of L-lactide and D-lactide, respectively, in DCM at room temperature, initiated by the remaining hydroxyl groups of PEG-MA (dried overnight under vacuum over phosphorous pentoxide). The single site Zn-complex Zn(Et)[OC6H3(CH2Me2)-2-Me-4] was used as a catalyst. Typically, PEG-MA (3.0 g, 0.136 mmol) (degree of methacrylation 42%) and L-lactide (0.532 g, 3.694 mmol) were dissolved in 14 ml of DCM ([LA]0=0.25 M). A solution of single site Zn-complex catalyst (0.064 g, 0.247 mmol) was added in 1 ml of DCM and the reaction mixture was stirred for 1 h. The polymerization was terminated by the addition of an excess of glacial acetic acid and the polymer was precipitated in a mixture of cold diethyl ether/methanol (20/1 v/v). Lactide conversion: 95%, yield: 85%. 1H NMR (CDCl3): δ 1.4 (m, CH(CH3)OH end group PLA), 1.5 (m, CHCH3), 1.9 (s, C(CH3)═CH2), 3.6 (m, PEG methylene protons), 4.2 (m, CH2OCO, linking unit PEG-MA), 4.2-4.3 (m, CH2OCO, linking unit PEG-PLA), 4.3-4.4 (q, CH(CH3)OH end group PLA), 5.1 (m, CHCH3), 5.6 and 6.2 (C(CH3)═CH2)
Characterization. 1H NMR spectra (CDCl3) were recorded on a Varian (nova Spectrometer (Varian, Palo, Alto, USA) operating at 300 MHz. The number of lactyl units per PLA block was calculated based on the methyl protons of lactyl units (δ 1.4-1.5) and the methylene protons of PEG (δ 3.6). The number of methacrylate groups per PEG molecule was determined based on the methylene protons of PEG (δ 3.6) and the methylene protons of the methacrylate group (δ 5.6 and 6.2).
Critical gel concentrations (CGCs) were determined as described before.53 Briefly, polymer solutions were prepared by dissolving the polymers in deionized water overnight. Subsequently, polymer solutions of equimolar amounts of PEG-PLLA-MA and PEG-PDLA-MA, or PEG-MA/PLLA and PEG-MA/PDLA star block copolymers were mixed and equilibrated overnight. The CGCs were determined at room temperature by inverting the vials. When the sample showed no flow within 20 s, it was regarded as a gel.
Rheology experiments were performed on a US 200 Rheometer (Anton Paar), as described previously.53 Briefly, a flat plate measuring geometry (25 mm diameter, gap 0.5 mm), a frequency of 1 Hz and a strain of 1% were used. Polymer solutions in HEPES buffered saline (pH 7.0, 100 mM, adjusted to 300 mOsm with NaCl) containing equimolar amounts of PEG-PLLA-MA and PEG-PDLA-MA, or PEG-MA/PLLA and PEG-MA/PDLA star block copolymers were mixed, homogenized, quickly applied to the rheometer and measured at 37° C.
Combined UV-irradiation and rheology experiments were performed on a US 200 Rheometer (Anton Paar) equipped with a UV-light source (350-400 nm, 16 mW/cm2). The samples were irradiated from above. A flat plate measuring geometry (10 mm diameter, gap 0.1 mm), a frequency of 1 Hz and strains of 1% or 5% were used. Both strains are within the linear viscoelastic region. Both PEG-PLA-MA or PEG-MA/PLA stereocomplexed hydrogels (stereo hydrogels) and solutions of PEG-PLLA-MA or PEG-MA/PLLA single enantiomers in HEPES buffered saline were UV-irradiated and at the same time measured at 37° C. Irgacure 2959 was used as photoinitiator at a concentration of 1-5 mol % relative to the methacrylate groups. The stereo hydrogels were measured 10 min after mixing the enantiomeric solutions, unless mentioned otherwise.
Hydrogels for scanning electron microscopy (SEM) experiments and swelling/degradation tests were prepared similarly in a 96 wells plate with sample volumes of 125 μl, resulting in cylinders of ca. 4 mm in height and 6 mm in diameter. PEG-PLA12-MA or PEG-PLA16/MA stereo-photo hydrogels were prepared by UVA-irradiation (250 mW/cm2) for 10 min of the stereo hydrogels (equilibrated for ca. 15 min after mixing of the enantiomeric solutions) with 8 mol % initiator concentration (with respect to the methacrylate groups) prepared in HEPES buffered saline. Photo hydrogels were formed similarly by UVA-irradiation of PEG-PLLA12-MA or PEG-MA16/PLLA single enantiomer solutions in HEPES buffered saline.
SEM experiments were performed on freeze-dried hydrogels using a LEO Gemini 1550 FEG-SEM, fitted with a field Emission Gun, and a voltage of 2 kV. Freeze-dried hydrogels were prepared by freezing in liquid nitrogen and subsequent freeze-drying at −50° C. and 5×10−7 bar overnight.
For the swelling/degradation tests, the hydrogel cylinders were placed in vials and after addition of 1 ml of HEPES buffered saline the hydrogels were allowed to swell at 37° C. The swelling experiment was performed in duplicate or triplicate. The swollen hydrogels were weighed at regular intervals after removal of the buffer. After each weighing the buffer was refreshed. The swelling ratio of the hydrogels was calculated from the initial hydrogel weight after hydrogel preparation (W0) and the swollen hydrogel weight after exposure to buffer (Wt):
Two types of methacrylate functionalized poly(ethylene glycol)-poly(lactide) (PEG-PLA) star block copolymers, PEG-poly(L-lactide)-methacrylate (PEG-PLLA-MA) and PEG-poly(D-lactide)-methacrylate (PEG-PDLA-MA) (
PEG-MA/PLA copolymers were prepared by a two-step synthesis procedure. First ca. 40% of the hydroxyl end groups of an eight-arm star PEG (Mn=21,800) were methacrylated. Subsequently, the ring opening polymerization of L-lactide or D-lactide was initiated by the remaining hydroxyl groups of methacrylate functionalized PEG, using the single-site Zn-complex as a catalyst and dichloromethane (DCM) as a solvent at room temperature. PEG-MA/PLLA and PEG-MA/PDLA copolymers were obtained by precipitation in a diethyl ether/methanol mixture (20/1 v/v). PEG-MA/PLA copolymers with 12 and 16 lactyl units per PLA block were prepared by varying the feeding ratio of lactide to PEG (Table 1, entry 5-8). The use of the single site Zn-catalyst allowed excellent control over the degree of polymerization of the PLA blocks and the methacrylation reaction was reproducible, giving similar degrees of methacrylation (Table 1).
1H NMR
1H NMR
a)The ring opening polymerization of lactide was performed in DCM for 1 h at RT using PEG or partially methacrylate functionalized PEG as an initiator and the single site Zn-complex Zn(Et)[OC6H3(CH2Me2)-2-Me-4] as a catalyst, ([LA]0 = 0.25 M, PEG hydroxyl groups:Zn catalyst = 2:1). The methacrylation was performed in DCM for 2 days at 30° C. ([OH]0 ≈5 mM, MA:OH:TEA = 1:1.5:1.1)
b)Number of lactyl units per PLA block.
c)Based on feed composition and conversion.
The influence of the methacrylate groups and the PLA block length on stereocomplex hydrogel (denoted as stereo hydrogel) formation was studied at room temperature. Aqueous solutions of equimolar amounts of PEG-PLLA-MA and PEG-PDLA-MA, or PEG-MA/PLLA and PEG-MA/PDLA star block copolymers were mixed and after equilibration it was tested whether the sample had turned into a gel by the vial tilting method. Table 2 shows that the critical gel concentrations (CGCs) for stereocomplexation of PEG-PLA12-MA and PEG-PLA12 are equal, indicating that the methacrylate end groups do not influence the stereocomplexation. PEG-PLLA, PEG-PLLA-MA and PEG-MA/PLLA single enantiomers were also able to form gels at relatively high polymer concentrations. The CGC of PEG-PLLA12-MA single enantiomer is somewhat lower compared to PEG-PLLA12 single enantiomer, which is attributed to the increased hydrophobicity of PEG-PLLA12-MA. Aqueous solutions of PEG-MA/PLLA12 single enantiomer could be prepared up to much higher polymer concentrations compared to PEG-PLLA12-MA single enantiomer. Stereo hydrogels could also be formed from PEG-MA/PLLA12 and PEG-MA/PDLA12 copolymers, but at much higher polymer concentrations compared to PEG-PLLA12-MA and PEG-PDLA12-MA copolymers. The higher CGC for stereocomplexation of PEG-MA/PLA12 compared to PEG-PLA12-MA is due to the lower crosslinking functionality (i.e. number of PLA blocks per molecule) and lower hydrophobicity of PEG-MA/PLA12 compared to PEG-PLA12-MA. Previously we have shown that the CGC for stereocomplexation of PLA-PEG-PLA triblock copolymers are higher compared to the CGC of eight-arm PEG-PLA star block copolymers.13 PEG-MA/PLA16 copolymers showed lower CGC values for stereocomplexation compared to PEG-MA/PLA12 copolymers, due to the increased PLA block length.
The mechanical properties of stereo hydrogels were studied by rheology experiments at 37° C. Stereo hydrogels were prepared by mixing aqueous solutions of equimolar amounts of PEG-PLLA12 and PEG-PDLA12, PEG-PLLA12-MA and PEG-PDLA12-MA, or PEG-MA/PLLA12 and PEG-MA/PDLA12 star block copolymers in HEPES buffered saline (pH 7) in a polymer concentration range of 12.5 to 17.5 w/v %. After mixing, the solutions were quickly applied to the rheometer and the evolutions of the storage modulus (G′) and loss modulus (G″) were recorded (
The mechanical properties of photopolymerized hydrogels were determined by combined rheology and UV-irradiation (350-400 nm, 16 mW/cm2) of PEG-PLA12-MA or PEG-MA/PLA16 stereo hydrogels (yielding stereo-photo hydrogels) or solutions of PEG-PLLA12-MA or PEG-MA/PLLA16 single enantiomers (yielding photo hydrogels) in HEPES buffered saline (pH 7) at 37° C. (
As shown earlier, a stereo hydrogel was formed within 1-2 min after mixing aqueous solutions of equimolar amounts of PEG-PLLA12-MA and PEG-PDLA12-MA copolymers. UV-irradiation of the stereo hydrogel at 1 mol % initiator and 15 w/v % polymer concentration 10 min after mixing increased the storage modulus from 5.6 to 9.6 kPa within 15 min due to photocrosslinking (
The storage modulus of the stereo-photo hydrogel is highly dependent on the stereocomplex equilibration time before UV-irradiation.
To study the influence of stereocomplexation on the morphology of photopolymerized hydrogels scanning electron microscopy (SEM) measurements were performed on freeze-dried PEG-PLA12-MA and PEG-MA/PLA16 stereo-photo and photo hydrogels. The stereo-photo and photo hydrogels were prepared by UVA-irradiation (250 mW/cm2) of PEG-PLA12-MA or PEG-MA/PLA16 stereo hydrogels (equilibrated for ca. 15 min after mixing the enantiomeric solutions) and solutions of PEG-PLLA12-MA or PEG-MA/PLLA16 single enantiomers, respectively, in HEPES buffered saline (pH 7) at 8 mol % initiator and 15 w/v % polymer concentration.
Hydrogels based on PEG-PLA-MA or PEG-MA/PLA copolymers were degradable under physiological conditions. To study the rate of degradation, stereo-photo and photo hydrogels were prepared by UVA-irradiation (250 mW/cm2) of PEG-PLA12-MA or PEG-MA/PLA16 stereo hydrogels (equilibrated for ca. 15 min after mixing the enantiomeric solutions) and solutions containing PEG-PLLA12-MA or PEG-MA/PLLA16 single enantiomer, respectively, in HEPES buffered saline (pH 7) at 8 mol % initiator concentration. After the hydrogels were formed, HEPES buffered saline was applied on top and the gels were allowed to swell at 37° C. At regular time intervals, the swelling ratio was calculated by rationing the swollen hydrogel weight after exposure to buffer with the initial hydrogel weight after preparation (Wt/W0).
The much slower degradation of the PEG-MA/PLA16 stereo-photo hydrogels compared to the PEG-PLA12-MA stereo-photo hydrogels is attributed to the slower hydrolysis of ester bonds of the polymerized methacrylate groups compared to the ester bonds of the PLA blocks, which correlates well with the results obtained by Bryant et al. for photopolymerized PEG dimethacrylate and PEG-PLA dimethacrylate hydrogels.56 PEG-PLA-MA stereo-photo hydrogels degrade mainly through hydrolysis of the ester bonds in the PLA block, upon which both physical and chemical crosslinks are lost (
PEG-PLA-MA copolymers were prepared by methacrylation of ca. 40% of the PLA hydroxyl end groups of eight-arm PEG-PLA star block copolymers. PEG-MA/PLA copolymers were prepared by ring opening polymerization of lactide initiated by eight-arm star PEG with 40% of its hydroxyl end groups methacrylated. PEG-PLA-MA and PEG-MA/PLA stereocomplexed hydrogels could be rapidly formed in situ upon mixing aqueous solutions containing equimolar amounts of PEG-PLLA-MA and PEG-PDLA-MA, or PEG-MA/PLLA and PEG-MA/PDLA copolymers. Interestingly, stereocomplexation aided in the photopolymerization of the methacrylate groups. Photocrosslinking of stereo hydrogels, yielding stereo-photo hydrogels, resulted in increased hydrogel storage moduli, compared to the hydrogels crosslinked by only stereocomplexation (stereo hydrogels) or only photocrosslinking (photo hydrogels). Moreover, photocrosslinking of stereo hydrogels already took place at very low initiator concentrations. The degradation time of PEG-PLA-MA stereo-photo hydrogels was doubled compared to PEG-PLLA-MA photo hydrogels (ca. 3 vs. 1.5 weeks). PEG-MA/PLA stereo-photo hydrogels degraded within ca. 7 to over 16 weeks, depending on the polymer concentration. In principle, PEG-PLA-MA and PEG-MA/PLA may be combined to vary the hydrogel degradation rate. The fast gelation in vitro and in vivo due to stereocomplexation circumvents the need for fast photopolymerization, thus preventing substantial heat effects due to the photopolymerization and potentiating the use of low initiator concentrations and low light intensities. Moreover, the fast gelation allows for easy handling.
It will be understood that the stereo-photo hydrogels and in particular the process for their formation in situ within the human or animal body will have a high potential for in vivo applications including tissue engineering and drug delivery.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/002538 | 3/20/2007 | WO | 00 | 2/1/2010 |
