Biodegradable and radiopaque caprolactone based polymer, a process of preparation and application thereof

Abstract

The present disclosure relates tobiodegradable and radiopaque polymer of formula I. More specifically, the present disclosure pivots on development of tracheal splint and self- expandable stent that is radiopaque and promotes the growth, proliferation and functioning of various types of cells – chondrocytes, epithelial cells and endothelial cells.

Description

[0001] BIODEGRADABLE AND RADIOPAQUE CAPROLACTONE BASED POLYMER, A PROCESS OF PREPARATION AND APPLICATION THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to a biodegradable and radiopaque caprolactone based polymer of Formula I

[0004] Formula I wherein a is in the range of 140-180; b is in the range of 30-100.

[0005] Particularly, the present invention relates to a process for the preparation of the biodegradable and radiopaque caprolactone based polymer of Formula I. More particularly, the present invention relates to the biodegradable and radiopaque caprolactone based copolymer of Formula I for the development of tracheal splint and / or self-expandable stent that is radiopaque and promotes the growth, proliferation and functioning of various types of cells - chondrocytes, epithelial cells and endothelial cells.

[0006] BACKGROUND OF THE INVENTION

[0007] Multiple etiology such as tracheal stenosis, tracheoesophageal fistula, tracheal obstruction, and tracheomalacia which can permanently damage the trachea is commonly seen in all age groups. There is no good treatment for severe or life-threatening diffuse tracheomalacia condition, other than surgical options such as bypassing (tracheostomy), aortopexy and stenting the area with a tracheotomy tube. However, all these treatment options have high complication and failure rates. Treatment like tracheal resection and reanastomosis is often impossible as the extent of tracheal damage risk associated with these surgical procedures are very high (refer, Auchincloss, H. G. et al., J. Thorac. Dis. 2016, 8 (Suppl 2), S160-S167). Theone of reports says the annual incidence of respiratory arrest after tracheostomy is as high as 43% and it is considered as one of the high-risk surgical procedures in the pandemic of COVID 19 (refer, Krishnan, A. et al., Era. Ann. Thorac. Surg. Short Reports 2023).

[0008] External stabilization or splinting is one of techniques which can overcome the drawbacks involved in current treatment producers involved. With the aid of techniques like 3D Bio printing, one of the latest technologies in additive manufacturing which can contribute for the design and development of optimized and personalized medical splints which adapt to patient anatomy and can aid in the restoration of the structure and functioning of malacia trachea.

[0009] Polymeric biomaterials with radiopacity are employed in medical applications such as implants, prostheses, and controlled drug-release devices due to their ability to allow postoperative monitoring of the device's fate using X-radiography. These devices are frequently rendered radiopaque by introducing metal powders like tantalum or metal salts of barium, bismuth, and uranium into the systems. Conventional polymers cannot be detected by X-ray or ultrasound because they contain elements with low electron density and specific gravity, such as C, H, O, and N. As a result, initiatives for developing radiopaque polymers concentrate on techniques to boost polymer average electron density and specific gravity. Furthermore, the probability of inorganic ions seeping into human fluids in the case of long-term use renders radiopacity a transient phenomenon, in addition to causing systemic toxicity.

[0010] The commonly used metal stents lack the ability to expand and may cause long-term issues like hyperplasia because they are permanently inserted inside the body. An appealing possibility is bioresorbable polymers, which have been extensively studied for their potential use in stent devices. A superior method for creating radiopaque polymers is to synthesise monomers with covalently attached heavy halogen atoms such as iodine or bromine and then use these monomers as building blocks for new polymers with inherent radiopacity (refer, El Habnouni, S. et al., Macromol. Rapid Commun. 2009, 30 (3), 165— 169; and Kiran, S. et al., J. Biomed. Mater. Res. - Part A 2015, 103 (7), 2214-2224).

[0011] Even though many biodegradable polymers like Polycaprolactone (PCL), Polylactic acid (PLA), Poly lactic-co-glycolic acid (PLGA), and Polyglycolic acid (PGA), provide an attractive conduit structure for tracheal stents, it does not have an optimal environment for regenerating tissues because of its hydrophobicity and absence of pendant functional groups. So, most of the current research activity in the regeneration of tracheal tissue with above mentioned biodegradable polymers attained limited success. Moreover, these polymers are non-radiopaque in nature, making the post-operative assessment of the polymer challenging by non-invasive technique.

[0012] The approach to solving the aforementioned problems of a radiolucent properties of splints and lack of functional groups on the polymer backbone has been addressed by the present invention. The fundamental scientific understanding developed from the present disclosure have the potential to be taken forward for development into a medical implant for the treatment of air way disorders.

[0013] Based on the above it is desired to address the above mentioned disadvantages or other shortcomings or at least provide a useful alternative.

[0014] Accordingly, the present disclosure pivot on development of tracheal splint and selfexpandable stent that is radiopaque and promotes the growth, proliferation and functioning of various types of cells - chondrocytes, epithelial cells and endothelial cells. This solution proves to be beneficial, safe, and more affordable and accessible polymers which ultimately provide effective splint and self-expandable stent.

[0015] OBJECTS OF THE INVENTION

[0016] Main object of the present invention is to provide a biodegradable and radiopaque caprolactone based polymer of Formula I.

[0017] Another object of the present invention is to provide an amino acid initiated copolymerized caprolactone and iodocaprolactone polymer of Formula I, which is biodegradable and radiopaque.

[0018] Yet another object of the present invention is to provide a process of preparation of biodegradable and radiopaque caprolactone based polymer of Formula I.

[0019] Yet another object of the present invention is to provide a process for preparing amino acid initiated copolymerized caprolactone and iodocaprolactone polymer of Formula I.

[0020] Yet another object of the present invention is to provide a process for introducing functionality to the PCL polymer backbone leading to improvement in surface characteristics, radiopacity and hydrophillicity. Yet another object of the present inventionis to achieve polymer having inherent radiopacity without the addition of any additives.

[0021] Yet another object of the present invention is toprovide 3D printable nature of functional radiopaque polycaprolactone making them suitable for fabricating complex scaffolds.

[0022] Yet another object of the present invention is to develop customized 3D printed splints and stents using said polymer.

[0023] Yet another object of the present invention is to place the tracheobronchial splint externally and avoiding second surgical procedure for removal as the splint is bioresorbable.

[0024] SUMMARY OF THE INVENTION

[0025] Accordingly, present invention provides a caprolactone-based polymer of Formula I

[0026] Formula I wherein a is in the range of 140-180; b is in the range of 30-100; and said polymer is biodegradable and radiopaque.

[0027] In an embodiment of the present invention, the polymer shows inherent radiopacity without the addition of additive(s).

[0028] In yet another embodiment, present invention provides a process for the preparation of caprolactone-based polymer of Formula I comprising the steps of: a) copolymerizing a-iodocaprolactone with caprolactone in the presence of an initiator, at a temperature in the range of 180 to 220 °C for time period in the range of 12 to 20 hours to obtain a said polymer of Formula I; and b) purifying the polymer as obtained in step a) to obtain a pure polymer of Formula In yet another embodiment of the present invention, the concentration of E- iodocaprolactone is in range of 0.02 to 0.08 mol; and wherein the concentration of initiator is in the range of 1 .2-1 .5 mmol.

[0029] In yet another embodiment of the present invention, initiator is selected from amino acid containing one or two primary amine groups selected from the group consisting of lysine, arginine, asparagine or glutamine.

[0030] In yet another embodiment of the present invention, purification process of the polymer at step b) comprising the steps of: a) solubilizing the polymer of Formula I as obtained in step a) in an organic solvent to obtain a solution; b) precipitating the solution as obtained in step a) in a cold mixture of solvents, followed by drying to obtain the pure polymer of Formula I.

[0031] In yet another embodiment of the present invention, the organic solvent at step a) is selected from aromatic solvents including benzene, toluene, and mixtures thereof, alcohols including methanol, ethanol, propanol, butanol, ester, ether, ketone, amine, nitrated and halogenated hydrocarbon solvent.

[0032] In yet another embodiment of the present invention, mixture of solvents at step b) is based on alcohol selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol and water with a ratio in the range of 1 :1 to 1 :5, respectively.

[0033] In yet another embodiment, present invention provides a process for the preparation of 3D printing products, wherein said process comprises the steps of: a) preheating the polymer of Formula I at a temperature in the range of 50 to 90°C for a time period in the range of 8 to 15 min in a cartridge of a 3D printer machine to form a paste; b) 3D printing the paste of step a) at a temperature in the range of 70-100 °C, pressure in the range of 20-100 PSI, and layered with a nozzle at printing speed of 1 -3 mm / s to obtain 3D printed products. In yet another embodiment of the present invention, 3D printing products comprises of 3D stent products, splint models, self-expandable stents, tracheal splints, 3D scaffolds for tissue engineering and the self-expandable stents and tracheal splints are radiopaque, bioresorbable and help to maintain internal architecture and normal cervical motions.

[0034] In yet another embodiment of the present invention, functionality to the PCL backbone is introduced, leading to improvement in surface characteristics, radiopacity and hydrophillicity.

[0035] In yet another embodiment of the present invention, Inherent radiopacity is achieved without the addition of any additives.

[0036] In yet another embodiment of the present invention, printable nature of functional radiopaque polycaprolactone make them suitable for fabricating self-expandable stents and tracheal splints.

[0037] In yet another embodiment of the present invention, 3D printed self-expandable stents and tracheal splints of functional radiopaque polycaprolactone are prepared.

[0038] In some embodiments, the self-expandable stents and tracheal splints are radiopaque.

[0039] In yet another embodiment of the present invention, the self-expandable stents and tracheal splints are bioresorbable and help to maintain internal architecture and normal cervical motions, thereby avoiding second surgical procedure for removal.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 illustrates the XPS analysis at: a) Full scan XPS spectrum of iodinated PCL, and b) I3d XPS spectra of iodinated PCL.

[0042] Figure 2 illustratesthe a) TGA analysis and b) DTGA analysis of polymers.

[0043] Figure 3 illustrates the DSC analysis of polymeric samples at a) Heating cycle b) Cooling.

[0044] Figure 4 illustrates the contact angle measurements of iodinated PCL polymer.

[0045] Figure 5 illustrates the Invitro cytotoxity studies on iodinated PCL. Figure 6 illustrates the a) 3D printed disc of iodinated PCL b, c) Micro CT imaging of 3D printed discs.

[0046] Figure 7 illustrates the CAD modelling and 3D printing of iodinated PCL.

[0047] Figure 8 illustrates the crimping and delivery of the 3D printed prototype, (a) 3D printed stent (b) 3D printed stent placed inside crimping device, (b) Stent crimping to an approximate diameter of 5 mm. (c) Stent transferred to the transapical delivery device with an internal diameter of 12 mm. (d) Self-expansion as the stent is pushed out of the delivery system.

[0048] Figure 9 illustrates the X-ray imaging of 3D printed iodinated self-expandable stents and splint.

[0049] Figure 10 shows the 1 H NMR spectra of synthesized polymers at different concentrations of ICL (5, 10, 15 and 20%).

[0050] Figure 11 shows IR spectrum of synthesized PCL, and polymers at different concentrations of ICL (5, 10, 15 and 20%).

[0051] ABBREVIATIONS USED

[0052] CL: £-caprolcatone

[0053] ICL: lodo-£-caprolactone

[0054] PCL: poly-£-caprolcatone

[0055] IPCL: lodo-poly-£-caprolactone

[0056] CAD: Computer Aided Design

[0057] Transapical delivery: A delivery system and method for deliveringself expandable stent.

[0058] Crimping: A tool to crimp a stent onto a catheter.

[0059] TGA: Thermogravimetric Analysis

[0060] DTGA: Derivative Thermogravimetric analysis

[0061] XPS: X-ray photoelectron spectroscopy

[0062] DETAILED DESCRIPTION OF THE INVENTION The present invention provides functional radiopaque polycaprolactone by using L-lysine amino acid as initiators for ring opening copolymerization of s-caprolactone and a- iodocaprolactone to obtain functional radiopaque polycaprolactone.

[0063] The present invention relates to a caprolactone based polymer of formula I which is biodegradable and radiopaque, the Formula I is represented by:

[0064] Formula I wherein a is in range of 140-180; and b is in range of 30-100.

[0065] In an embodiment of the present invention, the polymer shows inherent radiopacity without the addition of additive(s).

[0066] In another embodiment of the present invention, process for preparing Amino acid initiated copolymerization of caprolactone and iodocaprolactone is provided.

[0067] In another embodiment, present invention provides a process of preparation of caprolactone based polymer of Formula I which is biodegradable and radiopaque, wherein the process comprising the steps of: a) copolymerizing a-iodocaprolactone with caprolactone in presence of initiator at temperature in the range of 180 to 220eC for time period in the range of 12 to 20 hrs to obtain said polymer of Formula I; and b) purifying the polymer as obtained in step a) to obtain pure polymer of Formula I.

[0068] In yet another embodiment of the present invention, the amount / concentration of £- caprolactone is of around 0.876 mol.

[0069] In yet another embodiment of the present invention, the amount / concentration of £- iodocaprolactone is in range of 0.02 to 0.08 mol or is in the range of 5 to 20 % w / w. In yet another embodiment of the present invention, the amount / concentration of initiator is in the range of 1 .2-1 .5 mmol.

[0070] In yet another embodiment of the present invention, the amount / concentration of initiator is of around 1 .4 mmol, or 2 mg / mL.

[0071] In yet another embodiment of the present invention, the initiator is selected from amino acid containing one or two primary amine groups.

[0072] In yet another embodiment of the present invention, the amino acid containing two primary amine groups is selected from lysine, arginine, aspargine, glutamine, and so on.

[0073] In yet another embodiment of the present invention, the purification step b) is done by solubilizing the polymer of Formula I as obtained in step a) in organic solvent followed by precipitating the solution in cold mixture of solvents based on alcohol and water with ratio in the range of 1 :1 to 1 :5, which is then dried to obtain pure polymer of Formula I.

[0074] In yet another embodiment of the present invention, the organic solvent is selected from aromatic compounds (e.g. benzene, toluene, etc.), alcohol (e.g. methanol, ethanol, propanol, butanol, etc.), ester, ether, ketone (e.g. acetone, etc.), amine, nitrated and halogenated hydrocarbon (e.g. dichloromethane, dichloroethane, etc.) and so on.

[0075] Specifically, the solvent is dichloromethane.

[0076] In yet another embodiment of the present invention, the alcoholic solvent used in mixture of solvents is selected from methanol, ethanol, propanol, butanol, pentanol, hexanol, and so on.

[0077] In yet another embodiment of the present invention, the present invention relates to a process of preparation of 3D printing product comprising steps of: a) preheating the polymer at atemperature in the rang of 50 to 90°C for a time period in the range of 8 to 15 min of Formula I in a cartridge of 3D printer machine to form a paste; and b) 3D printing the paste of step a) at temperature in the range of 70-100°C, pressure in the range of 20-100 PSI, and layered with a nozzle at printing speed of 1 -3 mm / s to obtain 3D printed products. In yet another embodiment of the present invention, the 3D products formed by said process is selected from but not limited to 3D stent products, splint models, self expandable stents, 3D scaffolds for tissue engineering, and so on.

[0078] In yet another embodiment of the present invention, process for preparing Amino acid initiated copolymerization of caprolactone and iodocaprolactone is provided.

[0079] In yet another embodiment of the present invention, functionality to the PCL backbone is introduced, leading to improvement in surface characteristics, radiopacity and hydrophillicity.

[0080] In yet another embodiment of the present invention, Inherent radiopacity is achieved without the addition of any additives.

[0081] In yet another embodiment of the present invention, printable nature of functional radiopaque polycaprolactone make them suitable for fabricating self-expandable stents and tracheal splints.

[0082] In yet another embodiment of the present invention, 3D printed self-expandable stents and tracheal splints of functional radiopaque polycaprolactone are prepared.

[0083] In yet another embodiment of the present invention, the self expandable stents and tracheal splints are radiopaque.

[0084] In yet another embodiment of the present invention, the self-expandable stents and tracheal splints are bioresorbable and helps to maintain internal architecture and normal cervical motions, thereby avoiding second surgical procedure for removal.

[0085] In yet another embodiment of the present invention, the present invention relates to one pot solvent free amino acid modified radiopaque PCL polymer, which can be 3D printed to specific structures as per the requirement, to become noninvasively monitored and low- cost radiographic technique over other (reported) implantable tracheal splints and selfexpandable stents available in the market for airway growth and to treat airways related diseases / conditions.

[0086] In order to improve the bio interaction of the scaffold material and improve the X-ray visibility of scaffold a different strategy of polymer synthesis is carried out by amino acid initiated copolymerization of CL and lodo-CL with varying concentration of monomers. Thus developed PCL will be further subjected for additive manufacturing for developing splints. Splints thus developed were expected to exhibit the following properties: i. Amino acid functionalized radiopaque PCL splints favors better biointeraction. ii. The bioresorbable nature of amino acid modified radiopaque PCL avoid additional surgical procedure for removal. ill. Modified external splint nature helps to maintain mucociliary architecture and normal cervical range of motions. iv. Post-operative assessment on the fate of the implant can be analyzed by non- invasive technique.

[0087] The present invention relates to:

[0088] • Amino acid initiated copolymerization of caprolactone and iodocaprolactone.

[0089] • Introduction of functionality to the PCL backbone leading to improvement in surface characteristics, radiopacity and hydrophillicity.

[0090] • Inherent radiopacity achieved without the addition of any additives.

[0091] • 3D printable nature of functional radiopaque polycaprolactone making it suitable for fabricating complex scaffolds.

[0092] • Customized 3D printed splint is developed using computer aided design software.

[0093] • The tracheobronchial splint is placed externally and avoids second surgical procedure for removal as the splint is bioresorbable. It helps maintain internal architecture and normal cervical motions.

[0094] This solution proves to be beneficial and safe and it possible to have solutions that are affordable and accessible for Indian surgeons. Therefore, the study's rationale pivots on the suitability of one pot solvent free amino acid modified radiopaque PCL, which can be 3D printed to patient-specific structures and noninvasively monitored with low-cost radiographic technique will be a better and safer alternative to otherimplantable tracheal splints and self-expandable stents available in the market for airway growth. More specifically, the present invention relates to the use of L-lysine amino acid as initiator for ring opening copolymerization of s-caprolactone and a-iodocaprolactone to obtain functional radiopaque polycaprolactone.

[0095] In a nutshell, the present invention provides radiopaque bioresorbable stents and splint as 3D printed products which is obtained using modified polymer based on amino acids as initiators for copolymerization of s-CL and a-iodo-s-CL. A variety of splints and stents pattern were successfully designed, fabricated and characterized with encouraging results regarding radiopacity and biological properties that find applications in tracheal tissue engineering.

[0096] EXAMPLES

[0097] The following examples are given as a way of illustration only and should not be construed to limit the scope of the present invention.

[0098] Materials / Chemicals used

[0099] CL (s-Caprolactone, >99.0%; Aldrich Co., Poland) was dried under calcium hydride for 24hrs and distilled under reduced pressure. Anhydrous Lysine (>98.0%; SRL chemicals) was dried over vacuum prior to use. Lithiumdiisopropylamide (LDA, 2.0 M in tetrahydrofuran / heptane / ethylbenzene, Aldrich), iodine monochloride (ICI, 1.0 M in methylenechloride, Aldrich) were used as received. Tetrahydrofuran (THF, Fluka) was dried by refluxing over a benzophenone- sodium mixture and distilled, Dichloromethane (anhydrous, >99.8%; Merck), chloroform (>99.0%; Merck), (methanol (anhydrous, 99.8%; Merck) were used as received.

[0100] Example 1 : Synthesis of a- lodoCaprolactone (Monomer)

[0101] A functional caprolactone was synthesized reported by Habnouni et al [Macromol. Rapid Commun.2009, 30 (3), 165-169] according to Scheme 1. To a solution of 2.0 M lithium diisopropyl amide (13.1 mL, 26.3 * 10’3mol) in anhydrous tetrahydrofuran (50 mL) at -78°C was added a solution of £-caprolactone (2.5 g, 21.9 * 10'3mol) in anhydrous tetrahydrofuran (10mL) drop wise under an inert atmosphere. After 1 hr, 1.0M solution of iodine monochloride in dichloromethane (26.2 mL, 26.3 * 10'3mol) was added to the lactone enolate and stirred for 3h at -30 °C. Further, the reaction mixture was quenched with a saturated ammonium chloride solution, and thoroughly washed successively with a saturated ammonium chloride solution and with aqueous sodium thiosulfate. The organic layer was dried using anhydrous sodium sulphate and evaporated under reduced pressure and further purification of crude product was carried out using column chromatography at (Petroleum ether / EtOAc 5%) which yielded yellowish oil (70-80%).

[0102] Scheme 1 : Synthesis of a- lodo-caprolactone monomer

[0103] The functional lactone was successfully synthesized using a non-nucleophilic strong base (lithium diisopropyl amide) to extract a methylene proton in the a-position of the ester carbonyl. Afterwards, the generated lithium carbanion was quenched with iodine monochloride to achieve a-iodo-caprolactone. Monomer a-l-s-CL was isolated after purification using silica gel column chromatography as yellowish oil with a 70% yield.

[0104] Nuclear magnetic resonance spectrometry (NMR): For characterization of PCL samples, the polymers were dissolved in chloroform-d at room temperature. NMR spectra were recorded on Bruker 400 MHz spectrometer with 2%w / v concentration of solution. Fourier- transform infrared spectroscopy: FTIR spectroscopic analysis was done on Perklin Elmer FTIR spectrometer at ambient temperature. FTIR-ATR spectra were recorded by acquiring 20 scans in the range of 400-4000cnr1.

[0105] The peaks at 5 =4.97 for1H NMR spectrum of a-l-s-CL (dd, 1 H, J1 / 46.6, 6.3, -CH (I)-), 4.69 (m, 1 H, -CH2-O-), 4.38 (m, 1 H, -CH2-O-), 1.84-2.01 (m, 6H, -CH (l)-CH2-CH2- CH2-)was consistent with assigned structure. The structure was also confirmed by IR spectroscopy, the infrared spectrum showed an intense peak at 1711cm-1corresponding to the carbonyl group of lactone and 2932 cm-1representing C-H stretching.

[0106] Example 2: Lysine initiated copolymerization of CL and ICL (modified polymer or copolymer)

[0107] Bulk copolymerization of a-iodocaprolactone and caprolactone was carried out using lysine as initiator as shown in Scheme 2. Monomer percentage of s-CL (2g, 0.0175mol), a-iodocaprolactone (5,10,15,20 %) w / w, and L-lysine concentration of 1 mg / ml was used for the study. The reaction was carried out at 200°C for 16h. After appropriate reaction time the polymer was solubilized in DCM and precipitated in cold methanol / water (4:1). The final polymer was dried under a vacuum.

[0108] Scheme 2: Lysine initiated copolymerization of e-CL and a-l-s-CL for formation of modified polymer

[0109] The synthesis of iodinated PCL was initiated using amino acid containing different concentrations of 5% to 20% of iodine containing monomer. The results are tabulated in Table no.1. Molecular weight determination of amino acid initiated iodinated PCL was done by gel permeation chromatography using polystyrene calibration standards. Molecular weights (relative Mn and Mw) and dispersity values were determined by size exclusion chromatography on a waters apparatus by eluting PCL samples at a concentration of 4mg / ml in chloroform. Chloroform was used as an eluent at a flow rate of 1 ml / min at 25°C and refractive index detector was used to determine different molecular weight fractions. Molecular weights were calculated using polystyrene calibration.

[0110] The molecular weight of polymer was found to be Mn(number average molecular weight)=17000 to 22000g / mol and Mw(weight average molecular weight)=29000 to 34000g / mol, and polydispersivity index (PDI) of 1.5 to 1.9.

[0111] It was observed that as the iodine concentration in the polymer was increased, the molecular weight decreased. This might be attributed to the bulky iodine group that caused steric hindrance for ring opening polymerization of caprolactone. It would also be apparent to a person skilled in the art that reaction mechanism is complicated by addition of iodine to the e-CL rings, either by attack of an iodine species on cationic intermediates or through electrophilic addition.

[0112] Table 1 : Molecular characterization of polymers aMolecular weight determined from gel permeation chromatography;bTheoretical Molecular weight calculated using andcCalculated from XPS.

[0113] 1H NMR spectra (of figure 10) of Poly[(a-iodo-£-caprolactone)-co-(£-caprolactone)] shows peaks at 5=4.23 (t, -CH(I)-C(O)-), 4.08 (t, -CH2-O-), 3.65(m, -CH2-OH), 2.29 (t, - CH2-C(O)-), 1.58-1.68 (m, -CH2-CH2 -CH2-CH2-O-), 1.31-1.41 (m, -CH2-CH2- CH2-CH2-O-),3-6(t-CH2-NH), and 4.23(CH-NH). This clearly demonstrates that l-lysine is effective in copolymerization of CL and ICL, and incorporation happens during polymerization process.

[0114] In the infrared spectrum (of figure 11), the characteristic absorption spectra appeared at 1735 cm-1corresponding to the carbonyl stretching mode of PCL and the peak at 1294 cm-1was assigned to the backbone C-C and C-0 stretching modes in the crystalline phase of PCL and the peaks at 1178 cm’1and 1168 cm’1is due to symmetric C-O-C stretching and C-O, C-C stretching in amorphous phase respectively. The strong bands at 2997 and 2947 cm-1are attributed to the stretching vibration of aliphatic C-H chain of PCL, respectively. Furthermore, the bands at 1680-1640 cm-1and 1550-1520 cm-1are assigned to amide I and II vibrations, thus indicating the presence of lysine amino acid in the polymer macromolecule. The characteristic peak at 500 cm’1corresponding to C-l stretch demonstrates the incorporation of iodine moiety to the polymer backbone.

[0115] XPS analysis

[0116] To further clarify the structure of lysine initiated iodinated PCL, XPS analysis was carried out. The XPS spectrum of iodinated polymers shown in Figure 1a exhibits four main peaks (284.6, 398.9, 532.5, and 620.0 eV) which are assigned to C 1 s, N 1 s, O 1 s, and I 3d, respectively. Referring to the spectra with a high-resolution scan of Lysine initiated iodinated PCL, I 3d doublet is detected at ~619eV (l3d5 / 2) and ~632eV (l3d3 / 2). As can be seen in Figure 1b, there is shift in this binding energy. This shift towards higher binding energy of 619eV for polymers containing 15% and 20% of iodinated monomer corroborates the formation of strong C-l bond. Thermal analysis

[0117] Thermogravimetric analyzer (PerkinElmer, Pyris Diamond System) was used to determine the thermal stability of lysine based PCL. The samples of around 10 mg were heated and tested with the thermal ramp from 50 to 900° C, with a heating rate of 10° min-1, under constant nitrogen flow (50 cm3min'1).

[0118] Thermal behaviour of iodinated lysine based PCL was investigated by means of TGA under nitrogen atmosphere. Figure 2a and b shows characteristic TGA and Derivative TGA curves of iodinated PCL having different concentrations of iodinated monomer percentage ranging from 5-20%. The initial degradation temperature and maximum degradation temperature was tested and tabulated in Table no. 2. Lysine initiated iodinated PCL exhibits single degradation stage with initial degradation around 273-301 °C at T onse t( first temperature, at which the sample starts losing weight)and Tmax(the temperature, at which maximum weight loss occurs) .of around 365-408°C leading to total decomposition of polymeric chain. The DTGA curve in Fig 2b illustrates the maximum temperature for thermal degradation of lysine initiated iodinated PCL. Increase in degradation temperature was ascribed by inclusion of higher concentration of iodine into the polymer chain due to the covalent bond formed between carbon and iodine.

[0119] Table 2: Thermogravimetric analysis of lysine based PCL

[0120] Dynamic scanning calorimetry

[0121] A differential scanning calorimeter DSC Q10 from Waters was used for determination and measurement of the calorimetric behaviour of PCL. The calibration of the temperature was done with the extrapolated onset temperature of the phase transition of indium. The samples with a weight of about 10 mg were put into an aluminium pan. All the experiments were conducted in nitrogen (flow rate 50 mL / min), in order to prevent oxidative degradation.

[0122] In order to analyse melting and crystallization properties of iodinated PCL samples, DSC first melting runs was analysed. The results of thermal characterization are shown in Figure 3. The attention was paid on Tm, AHtand degree of crystallinity. As seen in Figure 3a, the DSC curves showed single melting endotherm, the melting temperatures of PCL copolymers prepared from L-lysine was found in the range of 47 to 55°C. The thermal properties seem to be affected by the increased addition of a-ls-CL. The melting temperature of polymers increased with decrease in the concentration of a-ls-CL. While the crystallinity of polymers increased in decrease in the concentration of iodinated monomers, this may be due to hindrance caused by the bulky iodine atom during ordered arrangement of polymeric chain.

[0123] Contact angle measurements

[0124] Contact angle were measured using a goniometer system equipped with a digital camera for droplet image capture using the sessile drop method. A drop of distilled water (30 pl) was carefully placed on the polymer film and the contact angle was recorded over time. The mean contact angle was calculated as average angle of 2 to 5 sec after the droplet was set on the surface.

[0125] The hydrophillicity and hydrophobicity plays an important role for cell adhesion and migration, mechanical properties and degradation of polymers. Figure 4 shows the contact angle mesaurements of PCL and Lysine based iodinated PCL. Angles> 90° indicate hydrophobic property and < 90° indicates hydrophillic property. According to contact angle values for pure PCL was found to be 90 ± 0.96° and gradual decrease in the contact angle values from 71.6±3.3° to 42±1.6° was observed for polymers with increase in iodine monomer concentartion. The drastic difference in the contact angle of pure PCL and Lysine initiated Iodinated based was 18.4±2.340to 48 ± 0.64° respectively.The addition of amino acids and iodine to the polymers resulted in decrease in contact angle values. Since amino acid introduce functionality to the polymer the carboxy group of amino acid intend to induce hydrophillicty to the polymer that causes absorption of water. The reduced contact angle can be explained for amino acid incorporated PCL has charged amino acids are known to reduce hydrophobicity of polymers.

[0126] Invitro cytotoxicity assay

[0127] The ability of the cells to adhere on scaffold is the key in tissue engineering applications. L929 fibroblast cells are commonly used cell lines in invitro study to assess the potential of scaffold in tissue engineering . In order to analyse Cytotoxicity of iodinated PCL, MTT assay was performed. L929 cells were cultured on PCL films for 24hrs and viable cell densities were recorded by MTT assay as shown in Figure 5. The cell viability was estimated to be >70%. Hence it confirmed non Cytotoxicity nature of developed polymers as per ISO standards.

[0128] Example 3: Manufacturing of products based on the prepared polymer or copolymer

[0129] Lysine based iodinated polycaprolactone stent and splint models were fabricated using Allevi 3 Bioprinter (by 3D systems). The iodinated PCL polymer (Mn 22-20KDa) were put into the printing cartridge and preheated. Subsequently the paste was heated to 80°C, and layered with a nozzle (diameter, 25gauge) at a printing speed of I mm / s. Different 3D stent and splint models of PCL were printed and analysed for 3D printability of lysine initiated iodinated PCL.

[0130] Cell viability assay

[0131] L929 is a mouse fibroblast cell line. It is recommended by ISO 10993 as the preferred cell line for preliminary Cytotoxicity testing of biomaterials. L929 (Purchased from National Centre for Cell Science (NCCS), Pune, 411007, Maharashtra, India). Cells were maintained in DMEM (Gibco) with 10% FBS (Gibco). PCL films were sterilized by UV light for 3h, soaked and washed with ethanol. The L929 cells 10,000 cells / ml were seeded in 96 well plates for 24 hrs. Further PCL film was placed in plate and incubated in a CO2 incubator at 37 °C. After 24hrs of incubation, the films were discarded and 100 pl of MTT solution was added followed by further incubation for 4h. The working MTT solution was discarded and the dark blue formazan crystals formed was solubilised in 100 pl of DMSO. The optical density (OD) of the solution was measured at 550 nm using an absorbance plate reader to assess the cell viability of each sample. The obtained values were compared with the values of plate control.

[0132] 3D printing and Micro CT imaging of iodinated PCL disc

[0133] Table 3: Printing parameters for 3D printing iodinated PCL

[0134] Table 3 indicates the printing parameters used for 3D printing of lysine based PCL and iodinated PCL discs. The printing temperature ranged between 75- 80°C and pressure between 5-75 PSI to get desired 3D discs. The 3D printed discs were further used for micro CT analysis. Figure 6a shows the 3D printed iodinated PCL. The radiopacity property of PCL was studied by micro CT analysis. Figure 6b and 6c indicates the micro CT analysis of 3D printed PCL. Interestingly it was observed that as the percentage of iodocaprolactone monomer increased the radiopacity of polymers increased. The maximum of 7% transmission of X- ray was observed in the polymer containing 20% a- iodo-s-CL monomer.

[0135] CAD modelling and 3D printing iodinated PCL

[0136] Table 4: Optimization of printing parameters

[0137] In this experiment, Lysine based iodinated PCL of containing 5% and 10% iodinated monomer concentration were chosen for study as these polymers were 3D printable. For model design, a computer aided design (CAD model) was designed using FreeCAD software. The model was exported in STL file and used for 3D printing. For additive manufacturing the different iodinated PCL was fed into the nozzle of high temperature dispensing head and dispensed through cylindrical metal needle with inner diameter of 25 gauge using pneumatic pressure, crosshead speed and temperature presented in Table 4. It was clearly observed that the PCL with lower molecular weight was difficult to print as the viscosity of the polymer was lower compared to higher molecular weight iodinated PCL. It should be noted that the flow ability of polymer plays an important role in additive manufacturing. The splint and self expandable stent was successfully printed using Allevi 3 Bioprinter as shown in Figure 7.

[0138] Example 4: Demonstration of self-expandable stent To illustrate the performance of the optimal design, the 3D printed stent prototype was inserted in a transapical delivery device. The crimping method before implantation consists on placing the stent inside a crimping device (Figure 8b) and reducing its diameter to ~5mm to facilitate insertion in the delivery tool. Once the stent has been transferred from the crimping device to the delivery tool, its OD is 10 mm (Figure 8c). To illustrate the transapical stent delivery, the crimped stent was gradually pushed from the delivery system and allowed for self-expansion (Figure 8d).

[0139] Example 5: X-ray Imaging of 3D printed Stents and Splints

[0140] The X-ray intensity was measured using XRD machine (Rigaku, T okyo, Japan) in 20 range from 10 to 30° using the radiation of CuKa 1.54 A at a scan rate of 0.05 s-1.

[0141] X-Ray Photon spectroscopy (XPS) analysis were carried out using Thermo Fisher Instrument, UK (K ALPHA+) using 1486.6eV. X-ray energy with power 72 W. The beam was set at 400 microns to scan sample surface held at 90°. The photoelectron intensity (counts / s) acquisition was done against binding energy to detect hydrocarbons of polymer and iodine content.

[0142] The 3D printed iodinated PCL discs was analysed by X-ray tomographic imaging using Xradia Versa 510 X-ray microscope (Zeiss X-ray Microscopy, Pleasanton, California).X- ray projection image was obtained to understand the % transmittance of X-rays.

[0143] X-radiographs were obtained using a standard clinical X-ray machine (General Electric, USA) equipped with 2.5 aluminium filtration set at 45 kV with 10 mA current for 0.2 s.

[0144] Different types of stents and splints were imaged using clinical X-ray in order to evaluate their radiopacity. Figure 9 presents the resulting reconstruction for all stents. Qualitatively, iodine-containing stents appear to be more radiopaque compared to commercial PCL stent. On the other hand, there are clear differences on x-ray visibility of stent and splint as the thickness of splint was higher compared to that of self-expandable stent leading to high intensity of x-rays. Hence the highly visible radiopaque functional PCL splint and selfexpandable stents for tracheal tissue regeneration was successfully demonstrated.

[0145] ADVANTAGES OF THE INVENTION

[0146] • The present disclosure provides a process for Amino acid initiated copolymerization of caprolactone and iodo-caprolactone. • The present disclosure provides an amino acid initiated copolymerized caprolactone and iodocaprolactone.

[0147] • The present disclosure provides a process for introducing functionality to the PCL backbone leading to improvement in surface characteristics, radiopacity and hydrophillicity.

[0148] • The present disclosure achieves Inherent radiopacity without the addition of any additives.

[0149] • The present disclosure provides a 3D printable nature of functional radiopaque polycaprolactone making them suitable for fabricating complex scaffolds. • The present disclosure develops customized 3D printed splints using computer aided design software.

[0150] The present disclosure places the tracheobronchial splint externally and avoiding second surgical procedure for removal as the splint is bioresorbable.

Claims

WE CLAIM1 . A caprolactone-based polymer of Formula Iwherein a is in the range of 140-180; b is in the range of 30-100; and said polymer is biodegradable and radiopaque.

2. The polymer as claimed in claim 1 , wherein the polymer shows inherent radiopacity without the addition of additive(s).

3. A process for the preparation of caprolactone-based polymer of formula I as claimed in claim 1 , wherein the process comprising the steps of: a) copolymerizing a-iodocaprolactone with caprolactone in the presence of an initiator, at a temperature in the range of 180 to 220 °C for time period in the range of 12 to 20 hours to obtain a said polymer of Formula I; and b) purifying the polymer as obtained in step a) to obtain the pure polymer of Formula I.

4. The process as claimed in claim 3, wherein the concentration of c-iodocaprolactone and initiator is in range of 0.02 to 0.08 mol and 1.2-1.5 mmol respectively.

5. The process as claimed in claim 3, wherein the initiator is selected from amino acid containing one or two primary amine groups selected from the group consisting of lysine, arginine, asparagine or glutamine.

6. The process as claimed in claim 3, wherein purification process of the polymer at step b) comprises the steps of:a) solubilizing the polymer of formula I as obtained in step a) of claim 3 in an organic solvent to obtain a solution; b) precipitating the solution as obtained in step a) in a cold mixture of solvents, followed by drying to obtain the pure polymer of Formula I.

7. The process as claimed in claim 6, wherein the organic solvent at step a) is selected from aromatic solvent selected from the group consisting of benzene, toluene, and mixtures thereof, alcohols selected from the group consisting of methanol, ethanol, propanol, butanol, ester, ether, ketone, amine, nitrated and halogenated hydrocarbon.

8. The process as claimed in claim 6, wherein mixture of solvents at step b) is based on alcohol selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol and water with a ratio in the range of 1 :1 to 1 :5.

9. A process for the preparation of 3D printing products, wherein said process comprises the steps of: c) preheating the polymer of formula I as claimed in claim 1 at a temperature in the range of 50 to 90 °C for time period in the range of 8 to 15 min in a cartridge of a 3D printer machine to form a paste; d) 3D printing the paste of step a) at a temperature in the range of 70-100 °C, pressure in the range of 20-100 PSI, and layered with a nozzle at printing speed of 1 -3 mm / s to obtain 3D printed products.

10. The process as claimed in claim 9, wherein 3D printing products comprises of 3D stent products, splint models, self-expandable stents, tracheal splints, 3D scaffolds for tissue engineering and the self expandable stents and tracheal splints are radiopaque, bioresorbable and help to maintain internal architecture and normal cervical motions.

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