Material suitable for use as a vitreous substitute and related methods

Abstract

There is provided a material comprising a multi-block thermogelling polymer, said multi-block thermogelling polymer comprising a hydrophilic polymer block; a thermosensitive polymer block; and a hydrophobic polymer block, wherein the hydrophilic polymer block, the thermosensitive polymer block and the hydrophobic polymer block are chemically coupled together by at least one of urethane/carbamate, carbonate, ester linkages or combinations thereof, and wherein the material is suitable for use as a vitreous substitute. Also provided are a method of preparing said material and a synthetic vitreous humour or part thereof comprising said material.

Claims

1. A material comprising a multi-block thermogelling polymer, said multi-block thermogelling polymer consisting of one or more of a first poly(alkylene glycol) block; one or more of a second poly(alkylene glycol) block; and one or more of a polyester block, wherein the first poly(alkylene glycol) block, the second poly(alkylene glycol) block and the polyester block are chemically coupled together by at least a urethane/carbamate linkage, wherein the first poly(alkylene glycol) block is different from the second poly(alkylene glycol) block, and wherein the material is suitable for use as a vitreous substitute.

2. The material of claim 1, wherein the first poly(alkylene glycol) block is poly(ethylene glycol) (PEG) and the second poly(alkylene glycol) block is poly(propylene glycol) (PPG).

3. The material of claim 1, wherein the polyester block is poly(caprolactone) (PCL).

4. The material of claim 1, wherein the molar ratio of the first poly(alkylene glycol) block to the second poly(alkylene glycol) block is in the range of 1:1 to 10:1.

5. The material of claim 1, wherein the polyester block is in an amount of from 1 wt % to 10 wt % of the multi-block thermogelling polymer.

6. The material of claim 1, wherein the material comprises 1% to 30% w/v of the multi-block thermogelling polymer in an aqueous medium.

7. The material of claim 1, wherein the material has a high water content of more than 60% by weight.

8. The material of claim 1, wherein the material has a pH value in a range of from 7.1 to 7.7.

9. The material of claim 1, wherein the material is in a flowable state at a temperature falling in the range of 20° C. to 30° C. and is in a non-flowable gel-like state at a temperature falling in the range of 36° C. to 40° C.

10. The material of claim 1, wherein the material is substantially transparent and/or has a refractive index falling in the range of from 1.20 to 1.48.

11. The material of claim 1, wherein the material is substantially devoid of a metal.

12. A method of preparing a material of claim 1, the method comprising: coupling one or more hydrophilic polymer, one or more thermosensitive polymer and one or more hydrophobic polymer together such that the hydrophilic polymer block, the thermosensitive polymer block and the hydrophobic polymer block are chemically coupled together by at least one of urethane/carbamate, carbonate, ester linkages or combinations thereof to form a multi-block polymer.

13. The method according to claim 12, wherein the one or more hydrophilic blocks, one or more thermosensitive blocks and one or more hydrophobic blocks are mixed in a molar ratio of 1-10: 1: 0.01-1.5, optionally wherein the mixing step is performed at an elevated temperature in the range of from 70° C. to 150° C., and optionally wherein the mixing step is carried out for at least 12 hours.

14. The method according to claim 12, wherein the coupling step is carried out in the presence of a coupling agent and the coupling agent comprises an isocyanate monomer that contains at least two isocyanate functional groups, optionally wherein the coupling agent is a diisocyanate selected from the group consisting of hexamethylene diisocyanate, tetramethylene diisocyanate, cyclohexane diisocyanate, tetramethylxylene diisocyanate, dodecylene diisocyanate, tolylene 2,4-diisocyanate and tolylene 2,6-diisocyanate.

15. The method according to claim 12, wherein the coupling step is carried out in the presence of an anhydrous solvent selected from the group consisting of toluene, benzene and xylene and/or wherein the coupling step is carried out in the presence of a tin catalyst selected from the group consisting of alkyltin compounds, aryltin compounds and dialkyltin diesters.

16. The method according to claim 12, the method further comprises removing the multi-block polymer of contaminants; and solubilizing the multi-block polymer in aqueous medium to form a multi-block thermogelling polymer.

17. A synthetic vitreous humour or part thereof comprising the material of claim 1.

18. The material of claim 1, wherein the first poly(alkylene glycol) block, the second poly(alkylene glycol) block and the polyester block are each linked to a respective adjacent block by a urethane/carbamate linkage.

19. The material of claim 2, wherein the molar ratio of PEG to PPG is in a range of 2:1 to 6:1.

20. The material of claim 19, wherein the molar ratio of PEG to PPG is 4:1.

21. The material of claim 3, wherein the PCL is derived from PCL-diol.

22. The material of claim 1, wherein the first poly(alkylene glycol) block is poly(ethylene glycol) (PEG), wherein the second poly(alkylene glycol) block is poly(propylene glycol) (PPG), wherein the polyester block is poly(caprolactone) (PCL), wherein the molar ratio of PEG to PPG is in a range of 2:1 to 6:1, and wherein the PCL is derived from PCL-diol.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 is a schematic diagram 100 for illustrating the application of a polymer as a vitreous substitute in the clinical repair of retinal detachment in accordance with an example embodiment disclosed herein.

(2) FIG. 2 is a graph showing the results obtained from oscillatory stress sweep measurement experiments performed on an exemplary hydrogel designed in accordance with various embodiments disclosed herein.

(3) FIG. 3 is a graph showing the changes in the viscosity of an exemplary hydrogel designed in accordance with various embodiments disclosed herein with varying temperature and polymer concentration. The symbols (.square-solid.) to (.diamond-solid.) represent the different concentrations of EPC polymer in balanced salt solution (BSS) ranging from 7 wt % to 2 wt %. EPC stands for PEG-PPG-PCL triblock polymer. For the EPC samples, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer. The symbol (∘) represents EPE, which stands for the PEG-PPG-PEG triblock polymer.

(4) FIG. 4 is a graph showing the changes in the loss in mass (in %) of an exemplary hydrogel designed in accordance with various embodiments disclosed herein, as measured at pH 7.4 and 37° C. in a balanced salt solution (BSS). Cross (x) represents EPC3%, square (.square-solid.) represents EPC7%, triangle (.box-tangle-solidup.) represents EPC12%.

(5) FIG. 5 is a graph showing the surface tension of thermogelling solutions having 3% (w/v), 5% (w/v) and 7% (w/v) of gel in water measured at specific temperatures of 24° C. and 37° C. respectively. In the thermogelling solutions, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer.

(6) FIG. 6 is a graph showing the changes in the viscosity of thermogelling solutions having 3% (w/v), 5% (w/v) and 7% (w/v) of gel in water with varying temperature, measured at a shear rate=1.25 s.sup.−1 and temperature ramp rate=3° C./min. In the thermogelling aqueous solutions, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer.

(7) FIG. 7 is a graph showing the conductivity of thermogelling solutions having 3% (w/v), 5% (w/v) and 7% (w/v) of gel in water measured respectively after pH adjustment (7.2-7.3), at specific temperatures of 24° C. and 37° C. respectively. The pH of the endosol buffer is 7.6. In the thermogelling solutions, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer.

(8) FIG. 8 is a graph showing the cell viability (%) of diluted gel solutions. Gel solutions used for the in vitro study are 5% (w/v) of PEG/PPG/PCL copolymer gel in water, 5% (w/v) of PEG/PPG/PHB copolymer gel in water and 20% (w/v) of PEG/PPG copolymer gel in water. The gel solutions were diluted with BSS solutions in the dilution ratio of 1:1, 1:2, 1:4, 1:10 and 1:20 respectively.

(9) FIG. 9 is a graph showing the intraocular pressure (IOP) measurements obtained for thermogels namely, PEG/PPG/PCL copolymer (1), PEG/PPG/PHB copolymer (2) and PEG/PPG copolymer (3) over 28 days after surgery. BSS was used as the control (4).

(10) FIG. 10 shows images taken during slit lamp assessment on rabbit eyes injected with hydrogel without pH adjustment. Images were captured on day 1, day 7 and day 14 after surgery. The white arrows pointed retinal detachment on fundus image.

(11) FIG. 11 shows spectral-domain optical coherence tomography (SD-OCT) images of rabbit eyes injected with normal BSS, PVA without pH adjustment, thermogels with pH adjustment and thermogels without pH adjustment (white arrow indicates area of retinal necrosis). The images were captured 1 month after surgery.

(12) FIG. 12 shows spectral-domain optical coherence tomography (SD-OCT) images and histology of rabbit eyes injected with normal BSS, PEG/PPG/PCL copolymer, PEG/PPG/PHB copolymer.

(13) FIG. 13 shows scotopic and photopic electroretinograms of rabbit eyes injected with PEG/PPG/PCL copolymer, alongside together with a non-operated control. The electroretinograms were recorded on day 28, day 56 and day 72 after surgery.

(14) FIG. 14 is a schematic flowchart 200 for illustrating the experiment set-up for demonstrating biocompatibility/non-toxicity of the polymer designed in accordance with various embodiments disclosed herein.

(15) FIGS. 15A, 15B and 15C are graphs showing the temperature ramp of EPC thermogelling solutions with concentration range from 3 to 12 wt %, measured at strain=1%, frequency=1 Hz and temperature ramp rate=3° C./min. In the thermogelling aqueous solutions, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer. The crossover point of G′ and G″ is the point when sol-gel transition occurred.

(16) FIG. 15D is a graph showing the complex viscosity of solutions determined in an oscillation temperature sweep experiment. The y-axis is put in log scale for clarity. Commercial available silicone (Oxane® 1300, Bausch & Lomb, USA) oil was used as a control.

(17) FIG. 15E is a graph showing the surface tension of solutions conducted with tensiometer using a DuNouy ring at specific temperature.

(18) FIG. 15F is a graph showing the swelling counter force (N) of solutions as determined from sweep experiments displayed over temperature from 10 to 50° C.

(19) FIG. 16 shows strain amplitude sweep of thermogelling solutions with concentrations of 3 wt. %, 7 wt. % and 12 wt. %, from 0.1 to 100% strain, at both and 37° C. respectively.

(20) FIG. 17 shows frequency sweep of thermogelling solutions with concentrations of 3 wt. %, 7 wt. % and 12 wt. %, from 0.1 to 100 Hz, at both 25 and 37° C. respectively.

(21) FIG. 18 shows spectral-domain optical coherence tomography (SD-OCT) images captured during live in vivo imaging of rabbits implanted with different hydrogels at Day 7 post-operation. Scale bars represent 200 μm in B4, C4, D4 and E4.

(22) FIG. 19 shows in vivo imaging and ex vivo retinal analysis of rabbits at 3-months post-implantation of the EPC gels. Scale bars represent 200 μm in A3, B3, D3. Scale bars represent 50 μm in A4, B4, D4, A5, B5 and D5.

(23) FIG. 20 shows the functional assessment of rabbit retina by electroretinography (ERG). Rabbit eyes were implanted with EPC 7 wt % and monitored over 1 month (herein termed “1M”), 2 months (herein termed “2M”) and 3 months (herein termed “3M”).

(24) FIG. 21 shows the intraocular pressure (IOP) after implantation of EPC thermogels. (A) IOP follow up in rabbits. Data are plotted as the mean±s. e. m in all groups. (B) IOP follow up in two NHPs.

(25) FIG. 22A shows ophthalmic images taken post-retinal detachment surgery. Images A1 to A6 were taken at 3-months post-retinal detachment surgery. Images B1 to B6 were taken at 12-months post-retinal detachment surgery.

(26) FIG. 22B shows pattern ERG of EPC-7% gel filled eye.

(27) FIG. 22C shows H&E analysis of the macular of EPC-7% filled eye.

(28) FIGS. 22D and 22E show overview of macular structure, with scale bar=50 μm.

(29) FIG. 23 shows auto-fluorescence images and SD-OCT images of retina post-retina detachment surgery (white arrow indicates sealed retinotomy site).

(30) FIG. 24 shows proteome profile of EPC reformed vitreous-like body (A) Gross dissection of rabbit eye implanted with 7% EPC gel at 3-month post implantation: a vitreous like body of consistency similar to native vitreous (B) was observed.

(31) FIGS. 25A to 25D show mass-spectrometry (MS) analysis of EPC 7% thermogel at 3-months post-operation. FIG. 25A shows mass-spectrometry (MS) analysis of native vitreous. FIG. 25B shows mass-spectrometry (MS) analysis of native vitreous spiked in with EPC-7% polymer as a positive control. FIG. 25C shows mass-spectrometry (MS) analysis of fluid sampled from vitreous cavity of operated control at 3-months post operation. FIG. 25D shows mass-spectrometry (MS) analysis of vitreous-like body samples from vitreous cavity of EPC-7% filled at 3-months post-operation, indicating the absence of detectable EPC-7% polymer.

(32) FIGS. 26A to 26C show .sup.1H NMR spectra (in CDCl.sub.3, 25° C.) of EPC-7% reformed vitreous-like body samples from rabbit eyes at 2 and 3-months post-operation. FIG. 26A shows .sup.1H NMR spectra (in CDCl.sub.3, 25° C.) of pure EPC-7% thermogel to serve as a control, Peaks “a to d” indicates presence of all PCL, PEG, PPG segments and isocyanate moieties. FIG. 26B shows .sup.1H NMR spectra (in CDCl.sub.3, 25° C.) of EPC-7% vitreous-like body at 2-month post-operation, indicating that EPC gel is still present as shown by the presence of all segments of the thermogel (i.e. PCL, PEG, PPG and isocyanate moieties). FIG. 26C shows .sup.1H NMR spectra (in CDCl.sub.3, 25° C.) of EPC-7% vitreous-like body at 3-months post-operation, indicating that EPC 7% gel is not detected at all (as shown by the absence of any peaks corresponding to “a, b, c or d”). *solvent impurity (acetone).

(33) FIG. 27 shows a comparison of ranked protein abundances from highest to lowest based on label-free quantification (LFQ) intensities across the native, EPC reformed and BSS control proteome. The inset represents the top 10 proteins in each group according to protein intensities.

(34) FIG. 28 is a heat map showing the expression of known vitreous structural components in EPC reformed vitreous-like body, compared to native vitreous and operated controls.

EXAMPLES

(35) Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following examples, tables and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, and chemical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new example embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

Example 1: Design and Application of Polymer

(36) A chemical structure of an exemplary polymer designed in accordance with various embodiments disclosed herein is shown in Scheme 1. The polymer is a tri-component multi-block thermogelling polymer which consists of hydrophilic poly(ethylene glycol) (PEG), thermosensitive poly(propylene glycol) (PPG), and hydrophobic biodegradable polyesters such as, but not limited to, biodegradable poly(ε-caprolactone) (PCL) segments linked together via urethane bonds.

(37) ##STR00001##
Synthesis of Polymer

(38) The general steps for preparing a polymer in accordance with various embodiments disclosed herein include: mixing one or more hydrophilic polymers, one or more hydrophobic polymers and one or more thermosensitive polymers with a coupling agent (in the example below, 1,6-diisocyanatohexane was used) in the presence of a metal catalyst (in the example below, dibutyltin dilaurate was used) and a suitable solvent (in the example below, toluene was used), as shown in Scheme 1.

(39) An example of preparing a polymer designed in accordance with various embodiments disclosed herein is described in detail as follows.

(40) Poly(PEG/PPG/PCL urethane) was synthesized from PEG, PPG, and PCL-diol using 1,6-Diisocyanatohexane as a coupling reagent. The amount of 1,6-Diisocyanatohexane added was equivalent to the reactive hydroxyl groups in the solution. Typically, 0.25 g of PCL-diol (Mn=2040, 1.23×10.sup.−4 mol), 5 g of PEG (Mn=1890, 2.65×10.sup.−3 mol), and 5 g of PPG (Mn=1880, 2.66×10.sup.−3 mol) were dried in a 250-mL two-neck flask at 50° C. under high vacuum overnight. Then, 200 mL of anhydrous 1,2-toluene was added to the flask, and any trace of water in the system was removed through azeotropic distillation with only 40 mL of toluene being left in the flask. When the flask was cooled down to 75° C., 0.912 g of 1,6-Diisocyanatohexane (5.43×10.sup.−3 mol) and two drops of dibutyltin dilaurate (˜8×10.sup.−3 g) were added sequentially. The reaction mixture was stirred at 60-110° C. under a nitrogen atmosphere for 24-48 h. The resultant copolymer was precipitated from diethyl ether and further purified by re-dissolving into chloroform, followed by precipitation in a mixture of methanol and diethyl ether. The yield was 85% after isolation and purification.

(41) Advantageously, the polymer designed in accordance with various embodiments disclosed herein rapidly forms a gel upon contact with the intra-ocular cavity at 37° C. Even more advantageously, as embodiments of the polymer disclosed herein gel inside the eye (or within the vitreous cavity), the polymer may be introduced into an eye vitreous cavity to be used as a surgical adjunct for treating ophthalmological disorders and to replace the vitreous. Embodiments of the polymer disclosed herein may therefore be used as vitreous substitute for maintaining the shape of the eye and provides an internal tamponading effect. They may be used as vitreous substitute for vitrectomy to treat ophthalmological disorders of the eye such as, but not limited to, retinal detachments, vitreous haemorrhage, epi-retinal membranes, macular holes and stem cell transplantations. In addition, embodiments of the polymer are capable of remaining in the eye for long periods of time (i.e 3 to 6 months) before biodegradation. Further, embodiments of the polymer are transparent and have the same refractive index as vitreous. Embodiments of the polymer disclosed herein are capable of providing physical support for the retina, and acts as an internal tamponade.

(42) Importantly, as will be shown in the following examples, embodiments of the polymers/gels disclosed herein overcome the inherent issues of conventional preformed hydrogels in the art.

(43) Application of Polymer as a Vitreous Substitute

(44) FIG. 1 is a schematic diagram 100 for illustrating the application of a polymer as a vitreous substitute in the clinical repair of retinal detachment in accordance with an example embodiment disclosed herein.

(45) (A1) of FIG. 1 shows retinal detachment with retinal tear. The vitreous is attached to anterior lip of retina tear causing traction. Native vitreous are represented by reference numeral 102. (A2) of FIG. 1 shows core vitrectomy (removal of vitreous). (A3) of FIG. 1 shows endolaser around retinal tear after air-fluid exchange. The air-filled vitreous cavity is indicated by reference numeral 104. (A4) of FIG. 1 shows injection of thermogel 106 in the eye to provide internal tamponade. (A5) of FIG. 1 shows that the thermogel 106 supports retina and allows chorio-retinal adhesion to occur at the site of retinal laser. (A6) of FIG. 1 shows that the thermogel is replaced by vitreous-like body 108 after biodegradation.

(46) The gel is introduced into the eye as a liquid (by way of an injection) and is then allowed to gelate within the vitreous cavity, after a pars plana vitrectomy to remove residual vitreous, as well as retinal tractional bands. The retina is reattached by conventional means. The vitreous fluid will be replaced initially with air (fluid air exchange) followed by injecting the gel. These gels are biodegradable, and thus there is no need for a second surgery to remove them in the postoperative period. Because of the consistency of the gel and their softness, they provide gentle support for the retinal structure and prevent accumulation of fluid, cells and subsequent membrane formation, which is often seen with the use of silicone oil or other liquid materials (due to the dead spaces left between these materials and the retina).

Example 2: Polymer Development and Characterisation

(47) The hydrogel designed in accordance with various embodiments disclosed herein was developed and characterized. The results obtained from the characterisation studies are provided as follows.

(48) i) Oscillatory Stress Sweep Measurement

(49) Oscillatory stress sweep measurement experiments were performed on an exemplary hydrogel designed in accordance with various embodiments disclosed herein and the results are provided in FIG. 2. As shown, the mechanical viability of the hydrogel can be maintained over 3 magnitudes order of stress.

(50) ii) Dependence of Viscosity on Temperature and Polymer Concentration

(51) FIG. 3 shows the changes in the viscosity of an exemplary hydrogel designed in accordance with various embodiments disclosed herein with varying temperature and polymer concentration. As shown, viscosity can be tuned by (1) varying individual component ratios; or (2) varying polymer concentration in the solution.

(52) iii) Mass Loss (%) of Hydrogels Measured at pH 7.4 and 37° C. (in BSS)

(53) FIG. 4 shows the changes in mass loss (in %) of an exemplary hydrogel designed in accordance with various embodiments disclosed herein, as measured at pH 7.4 and 37° C. in a balanced salt solution (BSS). Degradation time can be varied to fit any specific clinical applications. For example, the hydrogel may be designed and developed as a short-term, medium-term or long-term tamponade for retinal detachment (RD) repair.

(54) iv) Surface Tension of Thermogelling Solutions

(55) Surface tension of thermogelling solutions having 3% (w/v), 5% (w/v) and 7% (w/v) of gel in water were measured respectively with a tensiometer which was equipped with DuNouy ring, at specific temperatures of 24° C. and 37° C. respectively. In the thermogelling solutions, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer. The results are shown in FIG. 5.

(56) v) Viscosity Temperature Ramp

(57) FIG. 6 shows the changes in the viscosity of thermogelling solutions having 3% (w/v), 5% (w/v) and 7% (w/v) of gel in water with varying temperature, measured at a shear rate=1.25 s.sup.−1 and temperature ramp rate=3° C./min. In the thermogelling aqueous solutions, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer.

(58) As shown, the viscosity of the thermogelling solutions at 37° C. for 3% (w/v) polymer is 8.3 Pa.Math.s, the viscosity of the thermogelling solutions at 37° C. for 5% (w/v) polymer is 38.8 Pa.Math.s, and the viscosity of the thermogelling solutions at 37° C. for 7% (w/v) polymer is 107 Pa.Math.s.

(59) vi) Conductivity of Thermogelling Solutions

(60) Conductivity of thermogelling solutions having 3% (w/v), 5% (w/v) and 7% (w/v) of gel in water were measured respectively after pH adjustment (7.2-7.3), at specific temperatures of 24° C. and 37° C. respectively. The pH of the endosol buffer is 7.6. In the thermogelling solutions, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer. The results are shown in FIG. 7.

(61) vii) Temperature Ramp

(62) The temperature dependence of thermogelling solutions having 3% (w/v), 5% (w/v) and 7% (w/v) of gel in water was measured at strain=1%, frequency=1 Hz and temperature ramp rate=3° C./min. In the thermogelling aqueous solutions, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer.

(63) For 3% (w/v) polymer, the modulus crossover mod is about 3.018 Pa, temperature is about 27.7° C. and G′ at 37° C. is 8.2 Pa. For 5% (w/v) polymer, the modulus crossover mod is about 11.64 Pa, temperature is about 18.8° C. and G′ at 37° C. is 184 Pa. For 7% (w/v) polymer, the modulus crossover mod is about 14.79 Pa, temperature is about 16.3° C. and G′ at 37° C. is 448 Pa. As can be seen, increasing the concentration of polymer in the solution resulted in higher gel storage modulus at 37° C.

(64) viii) Strain Amplitude Sweep

(65) Strain amplitude sweep experiments were performed on thermogelling solutions having 3% (w/v), 5% (w/v) and 7% (w/v) of gel in water, at specific temperatures of 25° C. and 37° C. respectively. In the thermogelling aqueous solutions, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer.

(66) The thermogelling solutions at different concentrations were not affected by change in strain even in large amplitude oscillation of 10%-100%, therefore indicating that the hydrogel designed in accordance with various embodiments disclosed herein is a stable gel without macroscopic collapse at high strain.

(67) ix) Frequency Sweep

(68) Frequency sweep experiments were performed on thermogelling solutions having 3% (w/v), 5% (w/v) and 7% (w/v) of gel in water, at specific temperatures of 25° C. and 37° C. respectively. In the thermogelling aqueous solutions, the ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the polymer.

(69) x) Elemental Analysis Using Inductively Coupled Plasma Mass Spectrometry

(70) Samples of 10 wt % polymer were sent to TUV SOD PSB Pte. Ltd. for Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis. The sample was dissolved in water, followed by elemental analysis using ICP-MS. Table 2 shows the ICP-MS results provided by TUV SOD.

(71) TABLE-US-00002 TABLE 2 Elemental Analytical Results for 10 wt % polymer Permitted Parenteral Test Result Concentration (μg/g).sup.b Inferred Result Antimony, Sb Not Detected .sup.a 9 Pass Arsenic, As 0.005 1.5 Pass Cadmium, Cd Not Detected .sup.a 0.2 Pass Cobalt, Co Not Detected .sup.a 0.5 Pass Copper, Cu 0.02  30 Pass Lead, Pb Not Detected .sup.a 0.5 Pass Lithium, Li Not Detected .sup.a 25 Pass Mercury, Hg Not Detected .sup.a 0.3 Pass Nickel, Ni Not Detected .sup.a 2 Pass Vanadium, V Not Detected .sup.a 1 Pass .sup.a The method detection limit was 0.0001 ppm (1 μg/g = 1 ppm). .sup.bThe permitted concentration limits of elements across drug product companies for drug products with daily intakes of not more than 10 grams. Note: The elements for the leachable test were selected for the parenteral assessment with reference to FDA Q3D Elemental Impurities Guidance for Industry.

(72) Results indicated a lack of heavy metals in the polymer developed in accordance with various embodiments disclosed herein.

(73) xi) Organic Residual Solvent Test

(74) Samples of 10 wt % polymer were sent to TUV SOD PSB Pte Ltd for organic residual solvent test. Approximately 1 gram of sample was sealed in a 20 ml glass vial and heated at 90° C. for 20 minutes before being analysed by Headspace-Gas Chromatograph with Flame Ionisation Detector (GC-FID). Table 3 shows the GC-FID results provided by TUV SOD.

(75) TABLE-US-00003 TABLE 3 Analytical Results for 10 wt % polymer Residual Solvent* Components Results (ppm) Class 1 benzene Not Detected .sup.a Class 1 carbon tetrachloride Not Detected .sup.a Class 1 1,2-dichloroethane Not Detected .sup.b Class 1 1,1-dichloroethene Not Detected .sup.b Class 1 1,1,1-trichloroethane Not Detected .sup.b Class 2 acetonitrile Not Detected .sup.c Class 2 chlorobenzene Not Detected .sup.c Class 2 chloroform Not Detected .sup.c Class 2 cyclohexane Not Detected .sup.c Class 2 1,2-dichloroethene Not Detected .sup.c Class 2 dichloromethane Not Detected .sup.c Class 2 1,2-dimethoxyethane Not Detected .sup.c Class 2 N,N-dimethylacetamide Not Detected .sup.c Class 2 N,N-dimethylformamide Not Detected .sup.c Class 2 1,4-dioxane Not Detected .sup.c Class 2 2-ethoxyethanol Not Detected .sup.c Class 2 ethyleneglycol Not Detected .sup.c Class 2 formamide Not Detected .sup.c Class 2 hexane Not Detected .sup.c Class 2 methanol Not Detected .sup.c Class 2 2-methoxyethanol Not Detected .sup.c Class 2 methylbutylketone Not Detected .sup.c Class 2 methylcyclohexane Not Detected .sup.c Class 2 N-methylpyrrolidone Not Detected .sup.c Class 2 nitromethane Not Detected .sup.c Class 2 pyridine Not Detected .sup.c Class 2 sulfolane Not Detected .sup.c Class 2 tetrahydrofuran Not Detected .sup.c Class 2 tetralin Not Detected .sup.c Class 2 toluene Not Detected .sup.c Class 2 1,1,2-trichloroethene Not Detected .sup.c Class 2 xylene (m-, p-, o-isomers) Not Detected .sup.c Class 3 acetic acid Not Detected .sup.c Class 3 acetone Not Detected .sup.c Class 3 anisole Not Detected .sup.c Class 3 1-butanol Not Detected .sup.c Class 3 2-butanol Not Detected .sup.c Class 3 butyl acetate Not Detected .sup.c Class 3 tert-butylmethyl ether Not Detected .sup.c Class 3 cumene Not Detected .sup.c Class 3 dimethyl sulfoxide Not Detected .sup.c Class 3 ethanol Not Detected .sup.c Class 3 ethyl acetate Not Detected .sup.c Class 3 ethyl ether Not Detected .sup.c Class 3 ethyl formate Not Detected .sup.c Class 3 formic acid Not Detected .sup.c Class 3 heptane Not Detected .sup.c Class 3 isobutyl acetate Not Detected .sup.c Class 3 isopropyl acetate Not Detected .sup.c Class 3 methyl acetate Not Detected .sup.c Class 3 3-methyl-1-butanol Not Detected .sup.c Class 3 methylethylketone Not Detected .sup.c Class 3 methylisobutylketone Not Detected .sup.c Class 3 2-methyl-1-propanol Not Detected .sup.c Class 3 pentane Not Detected .sup.c Class 3 1-pentanol Not Detected .sup.c Class 3 1-propanol Not Detected .sup.c Class 3 2-propanol Not Detected .sup.c Class 3 propyl acetate Not Detected .sup.c *The residual solvents (Class 1, Class 2 and Class 3) are based on USP 467 Organic solvent residual. .sup.a The method detection limit was 2 ppm (1 μg/g = 1 ppm). .sup.b The method detection limit was 5 ppm (1 μg/g = 1 ppm). .sup.c The method detection limit was 10 ppm (1 μg/g = 1 ppm).

(76) Results indicated a lack of solvent contaminants in the polymer developed in accordance with various embodiments disclosed herein.

Example 3: Biocompatibility of Polymer

(77) In vitro and in vivo studies were conducted to assess the biocompatibility of the polymer designed in accordance with various embodiments disclosed herein. As will be shown in the following examples, the hydrogel is biocompatible (in the eye), and therefore suitable for ophthalmic usage.

(78) i) In Vitro Study: Cell Viability Test on ARPE-19 Cells

(79) Diluted gel solutions were applied to confluent retinal pigment epithelium ARPE-19 cells (a human cell line) on 96-well plate and incubated for 24 h. Cell viability was performed by MTT assay. In brief, cultures are replaced with fresh medium before MTT assay. 10 μl MTT solution (5 mg/ml) was added to each well and incubated at 37° C. for 4 hours. After removal of MTT solution, 100 μl DMSO was added in each well to dissolve the formazan crystals. The plate was kept in dark for 10 minutes at room temperature before absorbance reading at 570 nm using a spectrophotometer. Gel solutions used for the in vitro study are (1) 5% (w/v) of PEG/PPG/PCL copolymer gel in water (2) 5% (w/v) of PEG/PPG/PHB copolymer gel in water and (3) 20% (w/v) of PEG/PPG copolymer gel in water. The gel solutions were diluted with BSS solutions in the dilution ratio of 1:1, 1:2, 1:4, 1:10 and 1:20 respectively. The results are shown in FIG. 8.

(80) As shown, there is no difference of cell viability between gel groups. The mean ratios were 80.87±12.61% among all groups.

(81) ii) In Vivo Study

(82) For the in vivo study, 22 albino rabbits were used. Vitrectomy and (0.5-1.0 ml) thermogel was injected into the experimental eye. Vitrectomy and balanced salt solution (BSS) filled eyes served as controls. A 3 months follow-up in vivo study was conducted which include performing imaging such as slit-lamp, spectral-domain optical coherence tomography (SD-OCT), color fundus; and function test such as electroretinography (ERG) and intraocular pressure (IOP).

(83) (a) Intraocular Pressure (IOP) Measurements

(84) Thermogels used for IOP measurements are (1) PEG/PPG/PCL copolymer (2) PEG/PPG/PHB copolymer and (3) PEG/PPG. BSS was used as the control. Measurements were taken over 28 days and the results are shown in FIG. 9.

(85) As shown, IOP measurements remained in the normal range (between 8.4 and 13.9 mmHg). The mean value was 11.2±2.6 mmHg.

(86) (b) Biocompatibility of Hydrogel without pH Adjustment:

(87) Images taken during slit lamp assessment on rabbits are provided in FIG. 10. It is shown that retinal detachment occurred after one-week post surgery.

(88) Post-Operation Day 1:

(89) No evidence of inflammation. Thermogel optically clear. Fundus attached retina at POD1.
Post-Operation Day 7: Retina was spontaneously detached (indicated by white arrow in FIG. 10).
Post-Operation Day 14: Retinal detachment was enlarged (indicated by white arrow in FIG. 10).

(90) (c) Biocompatibility of Hydrogel with pH Adjustment (pH 7.2-7.4)

(91) SD-OCT at 1 Month after Surgery

(92) Spectral-domain optical coherence tomography (SD-OCT) images of rabbit eyes taken 1 month after surgery are provided in FIG. 11. SD-OCT images and histology are provided in FIG. 12.

(93) SD-OCT images showed normal reflection bands referring to retinal layers among BSS control and thermogel filled eyes. Normal retinal structure was presented on histology after haemotoxylin and eosin staining (H&E staining).

(94) Scotopic Electroretinography (ERG)

(95) Scotopic electroretinograms of rabbit eyes are provided in FIG. 13. Decreased b waves were observed on electro-retinography (ERG) post surgery, which was slowly recovering till 3 months. Scotopic and photopic ERG waveforms in rabbits at 3 months post-vitrectomy, and replacement with thermogel, showed both normal retinal structure and retinal function.

Example 4: Demonstration of Biocompatibility of EPC Thermogels

(96) FIG. 14 is a schematic flowchart 200 for illustrating the experiment set-up for demonstrating biocompatibility/non-toxicity of the polymer designed in accordance with various embodiments disclosed herein. At step 202, a sol-gel transition occurs upon a change in temperature from 25° C. to 37° C. The polymer in the sol state 210 rapidly forms a thermogel 212 at 37° C., which may transit back to its original state 210 upon cooling to 25° C. At step 204, thermogel 212 is applied to retinal pigment epithelium (RPE) cells and cell viability test is performed on said cells 214. At step 206, thermogel 212 is injected into rabbit eye 216 during vitrectomy surgery. At step 208, the rabbit eye is followed up post-operatively for at least up to 6 months.

(97) EPC Thermogels

(98) In the following examples 4, 5 and 6, the developed copolymer thermogel consists of hydrophilic PEG, thermosensitive poly(propylene glycol) (PPG) and hydrophobic, biodegradable poly(ε-caprolactone) (PCL) segments linked together via urethane bonds (herein termed “EPC”). The ratio of PEG:PPG is 4:1 and the concentration of PCL is 1 wt % of the EPC polymer.

(99) Rheological Characterisation of EPC Thermogels

(100) The sol-gel transition properties of different concentrations of thermogels (3 wt %, 7 wt %, 12 wt %) were characterized by rheological analysis.

(101) The storage and loss moduli (G′ and G″) were determined from 10° C. to 70° C., as shown in FIG. 15A to 15C. The gelation temperature decreases from 27.7° C. to 12.3° C., over a range of 3 to 12 wt % EPC concentrations.

(102) Complex viscosity of solutions were determined in an oscillation temperature sweep experiment (FIG. 15D). At gel state, the EPC thermogels exhibit a complex viscosity higher than that of silicone oil.

(103) Surface tension of solutions were conducted with tensiometer using a DuNouy ring at specific temperature (FIG. 15E). EPC-12% was too viscous for the test. Data represents mean±SD of 10-30 data points. Statistical analysis was performed using Student's t-test: # p<0.05 compared to Endosol buffer at 24° C. *p<0.05 compared to Endosol buffer at 37° C.

(104) Swelling counter force (N) of solutions as determined from sweep experiments were displayed over temperature from 10 to 50° C. (FIG. 15F). Swelling counter force generally increases with temperature, with EPC 12% (2.031 N)>EPC 7% (0.843 N)>EPC 3% (−0.066 N), silicon oil (−0.023 N) at 37° C.

(105) Oscillation frequency sweep and oscillation amplitude sweep experiments were also performed (FIG. 16 and FIG. 17). FIG. 16 shows strain amplitude sweep of thermogelling solutions with concentrations of 3 wt. %, 7 wt. % and 12 wt. %, from 0.1 to 100% strain, at both 25 and 37° C. respectively. FIG. 17 shows frequency sweep of thermogelling solutions with concentrations of 3 wt. %, 7 wt. % and 12 wt. %, from 0.1 to 100 Hz, at both 25 and 37° C. respectively.

(106) In Vivo Biocompatibility of EPC Thermogels

(107) In vivo biocompatibility of EPC thermogels was demonstrated using ophthalmic surgical models in New Zealand White (NZW) rabbits. 23 gauge core-vitrectomy were performed in NZW rabbits and 25 eyes were injected with sol-state EPC thermogel. All EPC thermogels were observed to form a gel in-situ within the vitreous during vitreo-retinal surgery. These rabbits were followed up post-operatively for up to 6 months for potential complications.

(108) In the eyes filled with gel, on examination, there was clear cornea and lens, with normal retinal appearance and normal intra-ocular pressure (IOP). In addition, normal retinal architecture was preserved on haematoxylin and eosin (H&E) stain.

(109) FIG. 18 shows live in vivo imaging of rabbits implanted with different hydrogels at Day 7 post-operation. Slit lamp pictures show negligible inflammation in the anterior chamber (see column B1 to E1), absence of cataract formation (see column B2 to E2), and optically clear hydrogel in the vitreous cavity. The optic disc and retinal vascular architecture appear normal (see column B3 to E3). The white lines indicate the position at which SD-OCT images were taken (1-2 optic disc distance superior to the disc). Rabbits implanted with all three concentrations (3 wt %, 7 wt % and 12 wt %) of EPC hydrogels have normal retinal architecture as observed in SD-OCT images (see column D4, E4).

(110) FIG. 19 shows in vivo imaging and ex vivo retinal analysis of rabbits at 3-months post-implantation of the EPC gels. In the 1.sup.st column (A1, B1 and D1), the slit lamp images of non-operated rabbits, operated BSS controls and EPC 7% thermogels showed no significant inflammation and cataract formation. In the 2.sup.nd column (A2, B2 and D2), fundus images revealed normal optic disc appearance and vessel morphology in all groups. In the 3.sup.rd column (A3, B3 and D3), normal optical coherence tomography (OCT) images were obtained in all three groups. Haematoxylin and eosin (H&E) histology analysis was performed in all 3 groups which was normal, as shown in 4.sup.th column (A4, B4 and D4).

(111) In vivo functional assessment of the retina was performed by electro-retinography (ERG), which shows mild loss of scotopic b-wave and photopic 30 Hz flicker response at 1 month with complete recovery by 3 months, that was maintained at 6 months (FIG. 20). In rabbit eyes implanted with EPC-7% gel, at 1 month, there was only a mild loss of scotopic b wave-amplitude and 30 Hz flicker ERG amplitudes, but complete recovery by 3 months compared to normal.

Example 5: Confirmation of Suitability and Efficacy as Tamponade Agent

(112) The suitability and efficacy as an internal tamponade agent was confirmed in a surgical non-human primate (NHP) retinal detachment model.

(113) Briefly, retinal detachment was induced in 2 NHPs, followed by retinal detachment repair and full-fill injection gel into the vitreous cavity (approximate 1.5 to 2.0 ml).

(114) The intraocular pressure (IOP) after implantation of EPC thermogels is provided in FIG. 21. FIG. 21A shows the IOP follow up in rabbits. The mean IOP among non-operated rabbit eyes is 14.3±1.8 mmHg (range from 10.6 to 16.4 mmHg). The mean IOP in rabbits implanted with 3% and 7% EPC thermogel is within normal limits (13.5±2.6 mmHg, range from 7.6 to 18 mmHg). FIG. 21B shows the IOP follow up in two NHPs. IOP in both EPC-7% implanted eyes were maintained within normal limits (range from 12 to 17 mmHg).

(115) A non-human primate (NHP) surgical retinal detachment model demonstrated EPC-7% thermogel to be an effective endotamponade. Ophthalmic images were taken at 3-months (see images A1 to A6) and 12-months (see images B1 to B6) post-retinal detachment surgery, as shown in FIG. 22A. Color fundus images of EPC-7% filled eye showed clear vitreous, attached retina and formation of chorio-retinal adhesion around the iatrogenic retinal tear (indicated by white arrows, in A1 and B1). Fluorescein angiography images show normal retinal vasculature without any leakage (A2 and B2 were taken within 30 seconds after fluorescein injection, while A3 and B3 were taken 10 minutes later). Auto-fluorescence images (imaging the auto-fluorescence of lipofuscin to blue laser light at the level of the retinal pigment epithelium (RPE) using a SLO) showed normal retina surrounding the lasered area of retinal tear (indicated by white arrows, in A4 and B4). The SD-OCT scan obtained at the site of the retinal tear (indicated by white line in A4 and B4) demonstrated sufficient chorioretinal adhesion, and with surrounding flat (re-attached) retina at both 3 months (A5) and 12 months (B5). SD-OCT scan of the macula (indicated by white line in A4 and B4) showed both normal foveal contour and retinal architecture at 3 months (A6) and 12 months (B6). Full field ERG showed mild cone more than rod dysfunction at 1-month post-surgery, with substantial recovery at 3 months and full recovery by 12 months. In FIG. 22B, pattern ERG showed normal waveform in EPC-7% gel filled eye compared to baseline at 12 months, consistent with normal macular function. In FIG. 22C, H&E analysis of the macular of EPC-7% filled eye (FIG. 22D) is normal compared to control shown in FIG. 22E. Inserts in FIG. 22D and FIG. 22E showed overview of macular structure, with scale bar=50 μm.

(116) FIG. 23A shows that retina is flat at 1-month post-surgery with hydrogel intact. The SD-OCT images in FIG. 23B confirmed a re-attached retina.

(117) At 3-months post-operation, the vitreous cavity remained optically clear. There was no evidence of anterior segment inflammation or cataract formation. Importantly, the IOP remained within a normal range of 12-17 mmHg (FIG. 21B). Furthermore, the retina remained attached at 12 months, and this was achieved without strict prone positioning, which cannot be enforced in NHPs. This demonstrates the ability of the presently disclosed gel to function as an effective internal tamponade through surface tension and swelling counter force, without a need to rely on buoyancy as with gas or oil.

(118) Live in vivo OCT imaging of the macula at both 3 and 12 months revealed normal retinal architecture, with healed retinotomy sites. At 1-month post-surgery, on ERG, although there was mild cone and rod dysfunction on full-field ERG, there was a dramatic recovery by 3 months, and a full recovery by 12 months. Pattern ERG showed normal waveform at 12 months consistent with normal macular function. Histological analysis of Haemotoxylin and Eosin (H&E) stained tissue revealed a normal macular architecture in gel filled eyes compared to control.

Example 6: Demonstration of Biodegradability of EPC Thermogels

(119) Upon dissection of the enucleated rabbit globes at 3-months post-surgery, a vitreous-like body formed, with a consistency reminiscent of native vitreous (FIG. 24). To show that the gel is no longer present by 3-months, consistent with its biodegradability property, the gel polymer is no longer detected by either mass-spectrometry (MS) analysis (FIGS. 25A to 25D) or nuclear magnetic resonance (NMR) (FIGS. 26A to 26C) at 3 months.

(120) Mass spectrometry-based proteomics analysis was performed to characterize this vitreous-like material. Of 1177 proteins identified in native vitreous, 924 proteins were also identified in the gel induced vitreous-like body at 6-month post-implantation, indicating higher similarity between them.

(121) Comparison of ranked protein abundances from highest to lowest based on label-free quantification (LFQ) intensities across the native, EPC reformed and BSS control proteome is provided in FIG. 27. Only proteins reliably quantified across three replicates were visualized. The inset represents the top 10 proteins in each group according to protein intensities.

(122) A heat map showing the expression of known vitreous structural components in EPC reformed vitreous-like body, compared to native vitreous and operated controls is provided in FIG. 28.

(123) It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.