well-defined degradable polypropylene fumarate polymers and scalable methods for their synthesis

专利摘要:
Well-defined degradable polypropylene fumarate polymers and scalable methods for the synthesis thereof The present invention provides a low molecular weight ppf polymer (and related methods) which is suitable for 3d printing and other polymer device manufacturing modalities and can be produced at low cost in commercially reasonable quantities. These new low molecular weight ppf polymers have a low molecular weight distribution (µm) and a wide variety of potential uses, particularly as a component in medical device 3d printing resins. The ability to produce low ppm ppf creates a new opportunity for reliable ppf production in accordance with good manufacturing practice (gmp). it provides low cost synthesis and synthesis scalability, well-defined mass mix and viscosity ppf and reduces solvent or heating dependence to (a) achieve mix of 3d printable resins or (b) and flowability during 3D printing. These ppf polymers are non-toxic, degradable and resorbable and can be used in tissue molds and medical devices that are implanted within a living organism.
公开号:BR112017010057A2
申请号:R112017010057-6
申请日:2015-11-18
公开日:2019-11-05
发明作者:Dean Howard；Becker Matthew；Luo Yuanyuan
申请人:Univ Ohio State；Univ Akron；
IPC主号:

专利说明:

"DEGRADABLE AND WELL DEFINED POLYPROPYLENE FUMARATE POLYMERS AND SCALABLE METHODS FOR THE SYNTHESIS OF THE SAME" [0001] This application claims the benefit of the provisional US patent application serial number 62/081219 entitled Products and Methods for Synthesis and Functionalization Materials , and use of them as a Medical Device, filed on November 18, 2014, and of the provisional US patent application serial number 62 / 139,196 entitled Well Defined Degradable Polypropylene Fumarate Polymers and Scalable Methods for their Synthesis, filed on March 27, 2015, both of which are incorporated by reference, in their entirety, in the present.
Names of Parties to a Joint Research Agreement [0002] The subject of this application was developed in accordance with a Joint Research Agreement between Akron University and Ohio State University.
Field of the Invention [0003] One or more embodiments relates to a new polypropylene fumarate polymer and methods for making polypropylene fumarate polymers. In certain embodiments, the present invention relates to a well-defined biodegradable polypropylene fumarate polymer and scalable methods for making and functionalizing it. In certain embodiments, the present invention relates to a well-defined biodegradable polypropylene fumarate polymer for use in various applications of regenerative medicine.
Background to the Invention [0004] Additive manufacturing, also known as three-dimensional (3D) printing, has the potential to revolutionize the way surgeons treat complicated reconstructive efforts in pathogenesis, congenital deformity, senescence, oral, maxillofacial and / or orthopedic trauma and repair of cancer defects, just to mention some of the many possible applications for 3D printing. While numerous 3D printing methods have been reported, photoreticulation-based printing methods, in particular, have demonstrated potential for
Petition 870170048142, of 10/07/2017, p. 9/102
2/68 reliable and highly accurate rendering of solid cure polymer molds that are designed to fit defects visualized by medical imaging. Advances in image projection through digital light processing technology (DLP) allowed 3D printing of tissue engineering molds with complex geometric designs together with very fine features (<; 50 pm).
[0005] To realize this potential, efforts have been made to develop a cost-effective, non-toxic and biodegradable polymer that works well with well-known 3D printing technologies, including photochemical cross-linking techniques. In addition, since the idea is that these 3D printed structures are implanted in the human body, the polymers used must withstand close regulatory scrutiny. While there are many inert photoreticulable resins, few of them are non-toxic, implantable and resorbable. Of this final category, the most explored are polylactides, poly (£ -caprolactone), and polypropylene fumarate (PPF). Regarding resorption profiles, it was found that polylactides undergo rapid volume degradation, leading to localized acidosis and inflammation. Poly (£ -caprolactone) is known to degrade very slowly, sometimes taking years, thus limiting the necessary remodeling or vascularization of new tissue. Polypropylene fumarate was developed, in part, due to a desire to have a material that had safe and controllable degradation properties that were expected to be useful for things like controlled release of drugs, stents, blood vessels, nerve grafts and engineering of cartilaginous tissue, especially bone tissue engineering. Since its invention using the step growth polymerization method, more than two decades ago, PPF has been very successfully investigated as a skeletal repair mold material. Subsequent reports improved the synthetic methods and the resulting materials.
[0006] A relevant factor that limits the availability of resorbable photoreticulable polymers, such as PPF, is the lack of GMP quality materials, ie, materials that meet the requirements of Good Manufacturing Practices implemented by the FDA (Food and Drug Administration), required to conduct pilot trials on humans and large animal models. PPF is traditionally synthesized using one of a variety of stepwise growth condensation reactions. So far, it has not been possible to synthesize well-defined low-molecular oligomers reliably and reproducibly in the scale required for applications and
Petition 870170048142, of 10/07/2017, p. 10/102
3/68 widespread commercialization of 3D printing. In particular, known stepwise growth methods for PPF synthesis require high energy input, high vacuum, long reaction times and result in low conversion (-35%) with uncontrolled molecular mass distribution, conjugation-addition secondary reactions and unwanted cross-linking, all of which greatly influence the mechanical properties and degradation rates of the final product. Furthermore, these methods are slow, labor-intensive and very expensive, and as a result, have not proven to be commercially viable.
[0007] Particularly problematic is the difficulty in controlling the molecular mass distribution inherent in these step growth methods. No two batches are exactly alike. These polymers tend to have a relatively high molecular weight distribution (D _m ) (also known as the Polydispersity Index (PDI)), and the colors and viscosity / mechanical properties of the polymers are inconsistent from batch to batch. This variation between batches has been shown to lead to a significant difficulty in predicting the mechanical properties that influence biological performance, such as reabsorption time, reabsorption uniformity (due to long chains acting as a link in some locations and not in others - - ie, non-uniform crosslinking mesh), as well as the incorporation of non-uniform crosslinking of other resins used as solvent (s), photoinitiator (s), dye (s), pigment (s) or component (s) ( eg, diethyl fumarate (DEF), bioactive molecules) during 3D printing. The researchers' inability to reliably predict 3D printing and the subsequent biological performance of these polymers made it difficult to obtain the regulatory approvals necessary for the use of these polymers in implants and other medical devices. In fact, it is believed that, to date, PPF has not been part of any FDA-approved device or therapy, despite more than two decades of continuous study of its use in regenerative medicine and successful experimental results.
[0008] More recently, PPF with a high molecular weight, a narrow D _m (below 1.6) and low ether bond (<; 1%) has been successfully synthesized using a chain growth mechanism under conditions smooth reaction times. In this method, maleate anhydride and epoxide are polymerized through ring-opening copolymerization with chromium salen as a catalyst at 45 ° C, and
Petition 870170048142, of 10/07/2017, p. 10/112
4/68 the produced polypropylene maleate (PPM) is then isomerized using diethylamine at room temperature for 16 hours to produce PPF. The PPF synthesized in this way was solid and had an MW of more than 4 kDa, a molecular mass distribution of 1.6 and less than 1% ether binding with 99% conversion. Compared to traditional synthesis methods, the chain growth mechanism provides PPF with better molecular properties and the reaction is more reproducible, enabling the production of PPF with controlled properties for additional mechanical, toxicity and degradation tests and large-scale production in manufacturing. Unfortunately, however, the high molecular weights, lack of fluidity and residual chrome metal of PPF polymers produced using these methods make them unsuitable for 3D printing or other applications in regenerative medicine.
[0009] What is needed in the art is a resorbable, non-toxic, fluid, low molecular weight PPF polymer with restricted and predictable material properties, and related methods for its production and use, which are suitable for 3D printing and use in medical devices and can be produced at low cost and in commercially reasonable quantities using GMP.
Summary of the Invention [0010] One or more embodiments of the present invention provide a resorbable, non-toxic, low molecular weight PPF polymer (and related methods for its production and use) that has restricted and predictable material properties suitable for 3D printing and that can be produced at low cost in commercially reasonable amounts.
[0011] In a first aspect, the present invention provides a polypropylene fumarate polymer for use in 3D printing that has an average numerical molecular mass (M _n ) of approximately 450 Daltons to approximately 3500 Daltons and a molecular mass distribution (D _m ) from 1.0 to 2.0. In some embodiments, the present invention is directed to the polypropylene fumarate polymer of the first aspect of the present invention, wherein said average numerical molecular mass (M _n ) is approximately 700 to approximately 3200. In one or more embodiments, the polypropylene fumarate polymer of the present invention includes
Petition 870170048142, of 10/07/2017, p. 10/122
5/68 any one or more of the embodiments of the first aspect of the present invention referenced above, having a glass transition temperature (T _g ) of approximately -25 ° C to approximately 12 ° C. In one or more embodiments, the polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the first aspect of the present invention referenced above, having a maximum average molecular weight of approximately 980 Daltons to approximately 5900 Daltons .
[0012] In one or more embodiments, the polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the first aspect of the present invention referenced above, having an intrinsic viscosity of approximately 0.025 dL / g approximately 0.078 dL / g. In one or more embodiments, the polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the first aspect of the present invention referenced above, wherein said polypropylene fumarate polymer contains less than 1% polymer chains of poly (propylene oxide-maleic co-anhydride) by weight. In one or more embodiments, the polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the first aspect of the present invention, wherein said polypropylene fumarate polymer does not contain poly (propylene oxide polymer chains) - maleic anhydride).
[0013] In one or more embodiments, the polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the first aspect of the present invention referenced above, having the formula:
- where n is an integer from 3 to 30.
[0014] In a second aspect, the present invention provides a method for producing a polypropylene fumarate polymer for use in 3D printing, consisting of: dissolving maleic anhydride and propylene oxide in a solvent
Petition 870170048142, of 10/07/2017, p. 10/13
6/68 suitable under an inert atmosphere; adding a suitable initiator; heating the mixture to a temperature of approximately 60 ° C to approximately 120 ° C for a period of approximately 0.5 hours to approximately 100 hours to produce a poly (propylene oxide-maleic anhydride); collection and purification of the poly (propylene oxide-maleic co-anhydride) polymer; dissolving the poly (propylene oxide-maleic co-anhydride) in a suitable solvent and adding a catalyst; heating the mixture to a temperature of approximately 5 ° C to approximately 80 ° C for a period of approximately 5 hours to approximately 100 hours to produce a polypropylene fumarate polymer.
[0015] In some embodiments, the present invention is directed to the method of producing a polypropylene fumarate polymer of the second aspect of the method of the present invention of claim 9, wherein the solvent used to dissolve maleic anhydride and oxide of propylene is selected from the group consisting of toluene, tetrahydrofuran (THF), dioxane and combinations thereof. In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the solvent used to dissolve maleic anhydride and the propylene oxide is toluene.
[0016] In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the initiator is magnesium ethoxide ( Mg (OEt) ₂ ). In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the molar ratio between maleic anhydride or oxide propylene and the initiator is approximately 3: 1 to approximately 400: 1.
[0017] In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, further comprising cooling the reaction mixture under a inert gas atmosphere; evaporation of volatile compounds from the mixture by distillation or under reduced pressure; addition of chloroform or dichloromethane; washing solution
Petition 870170048142, of 10/07/2017, p. 10/142
7/68 with an aqueous solution, thereby forming an organic layer containing the poly (propylene oxide-maleic co-anhydride) polymer intermediate and an aqueous layer; collecting and pouring this organic layer into a non-polar organic solvent, such as hexane, to cause the (propylene oxide-maleic coanhydride) polymer to precipitate; collection of poly (propylene oxide-maleic co-anhydride); dissolving the poly (propylene oxide-maleic co-anhydride) polymer in a small amount of a suitable solvent; concentration of the solution by evaporation; and drying the concentrated solution in vacuo, to produce a purified poly (propylene oxide-maleic anhydride) polymer intermediate. In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the inert atmosphere comprises nitrogen.
[0018] In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the volatile compounds are evaporated from the mixture distillation or reduced pressure. In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the poly (propylene-oxide-co polymer) - maleic anhydride) is collected by a separating funnel.
[0019] In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the suitable solvent comprises chloroform or dichloromethane .
[0020] In one or more embodiments, the present invention is directed to the method of producing a polypropylene fumarate polymer of the second aspect of the method of the present invention, wherein the solvent used to dissolve the poly (oxide) polymer intermediate propylene-maleic anhydride) is selected from the group consisting of chloroform, tetrahydrofuran (THF), dioxane and combinations thereof. In one or more modes of execution, the method for producing
Petition 870170048142, of 10/07/2017, p. 10/152
8/68 a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the solvent used to dissolve the poly (propylene oxide-co-anhydride) polymer intermediate maleic) is chloroform. In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the catalyst is diethylamine.
[0021] In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, further comprising the collection and purification of the polymer polypropylene fumarate. In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the step of collecting and purifying the polymer from polypropylene fumarate comprises: concentration of the polypropylene polymer intermediate by evaporation; washing the resulting solution with a buffered aqueous solution to remove the catalyst, thereby forming an organic layer and an aqueous layer; collection of the organic layer; concentration of the organic layer by evaporation; addition of sodium sulfate, or any inorganic drying agent, acid proton or molecular sieve to remove remaining water; filtration of sodium sulfate or other inorganic drying agent or molecular sieve; pouring the resulting mixture into a non-polar organic solvent to cause the polypropylene fumarate polymer to precipitate; collecting the polypropylene fumarate polymer and drying it under vacuum to produce a purified polypropylene fumarate polymer.
[0022] In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the poly ( dissolved propylene oxide-maleic anhydride) is concentrated by rotary evaporation or under reduced pressure. In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the aqueous solution comprises a
Petition 870170048142, of 10/07/2017, p. 10/162
9/68 phosphate buffered saline.
[0023] In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the organic layer containing the washed polymer with water it is collected by separating funnel. In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the organic layer containing the water-washed polymer is concentrated by rotary evaporation or reduced pressure. In one or more embodiments, the method for producing a polypropylene fumarate polymer of the present invention includes any one or more of the embodiments of the second aspect of the present invention referenced above, wherein the non-polar organic solvent used to precipitate the Polypropylene fumarate polymer comprises hexane.
Brief Description of the Drawings [0024] For a more complete understanding of the characteristics and advantages of the present invention, reference is now made to the detailed description of the invention together with the attached figures, in which:
[0025] Figure 1 is a scheme comparing the ¹ H NMR spectra (CDCI ₃ , 300 MHz) for a PPM intermediate (base) to a PPF polymer (top) according to one or more embodiments of the present invention, indicating the quantitative conversion from c / s stereochemistry (PPM intermediate) to the trans configuration (PPF polymer).
[0026] Figure 2 is a schematic comparing the ¹³ C NMR spectra (CDCI ₃ , 300 MHz) for a PPM intermediate and a PPF polymer according to one or more embodiments of the present invention, indicating the complete conversion of the PPM intermediate for the PPF polymer.
[0027] Figure 3 is a schematic comparing the characterizations (- 50 ° C - 50 ° C, 10 ° C / min) of Differential Scanning Calorimetry (DSC) for a PPM intermediate and a PPF polymer for one or more embodiments of the present invention.
[0028] Figure 4A is a MALDI-TOF mass spectrograph from the PPF sample
Petition 870170048142, of 10/07/2017, p. 10/172
10/68 number 3 in Table 1 showing the mass distribution in this sample.
[0029] Figure 4B is an enlarged portion of a MALDITOF mass spectrograph from the PPF sample number 3 in Table 1 showing the repeated unit in PPF and the possible chemicals of the final group, which correspond to the individual peaks in the distribution shown in the data of the mass spectrograph.
[0030] Figure 5A is a MALDI-TOF mass spectrograph of the PPF sample number 2 in Table 1 showing the mass distribution in that sample.
[0031] Figure 5B is an enlarged portion of a MALDITOF mass spectrograph from the PPF sample number 2 in Table 1 showing the repeated unit in PPF and the possible chemicals of the final group, which correspond to the individual peaks in the distribution shown in the data of the mass spectrograph.
[0032] Figures 6A-D are images showing the results of cytotoxicity experiments to confirm the in vitro biocompatibility of a PPF according to an embodiment of the present invention with mesenchymal stem cells derived from human bone marrow (RoosertBio, Frederick , MD) (hMSC). Figures 6A and 6B are a bright field image (Figure 6A) and a fluorescent image (Figure 6B) of a direct contact assay showing the PPF polymer in a hMSC monolayer. Figures 6C and 6D are a bright field image (Figure 6C) and a fluorescent image (Figure 6D) from a direct contact assay showing hMSCs grown on the PPF polymer material. It should be understood that the lightest areas in Figures 6B and 6D are the areas of green fluorescence in the color image. Scale bar = 500 pm.
[0033] Figure 7 is a kinetic plane showing almost linear growth in molecular mass over time. The average numerical molecular mass (M _n ), and the molecular mass distribution (D _m ) are displayed as a function of reaction time for PPM intermediates made according to one or more embodiments of the present invention using molar ratios between monomer and initiator of 100: 1,200: 1 and 300: 1.
[0034] Figure 8 is a schematic comparing Fourier Transform Infrared Spectroscopy (FTIR) spectra (film, KBr, CHCI, ₃ , 400 cm ^-1 - 4000 cm ^-1 ) for a PPM intermediate and a polymer of PPF according to one or more
Petition 870170048142, of 10/07/2017, p. 10/182
11/68 embodiments of the present invention, indicating complete conversion of the PPM intermediate to the PPF polymer. The conversion from c / s to trans is observed in sections C-H.
[0035] Figure 9 is a scheme comparing Spectroscopy spectra in Visible Ultraviolet (UV-Vis) (acetonitrile, 190 nm- 700 nm) for a PPM intermediate and a PPF polymer according to one or more modes of execution of the present invention, indicating complete conversion of the PPM intermediate to the PPF polymer.
[0036] Figure 10 is a graph showing q _sp / c and ln (q _r ) / c versus c for the sample of
PPF number 1 in Table 1.
[0037] Figure 11 is a graph showing q _sp / c and ln (q _r ) / c versus c for the sample of
PPF number 2 in Table 1.
[0038] Figure 12 is a graph showing q _sp / c and ln (q _r ) / c versus c for the sample of
PPF number 3 in Table 1.
[0039] Figure 13 is a graph showing q _sp / c and ln (q _r ) / c versus c for the sample of
PPF number 4 in Table 1.
[0040] Figure 14 is a graph showing q _sp / c and ln (q _r ) / c versus c for the sample of
PPF number 5 in Table 1.
[0041] Figure 15 is an image of a fabric mold made by a 3D printing process using a PPF polymer made according to one or more embodiments of the present invention. Scale bar is 2mm.
[0042] Figure 16A-B are images showing a PPF mold created with SolidWorks ™ CAD software using a Schoen Triple Periodic Gyro Minimal Surface with support thickness of 125 pm, pore diameter of 600 pm, and 93, 5% porosity Figure 16B is an enlargement of the PPF mold shown in Figure 16A.
[0043] Figure 16C is an image of a PPF template created in SolidWorks ™ computer aided design software and printed in 3D using a Perfactory ™ P3 printer.
Petition 870170048142, of 10/07/2017, p. 10/192
12/68 [0044] Figures 17A-C are computer aided design (CAD) images showing a front perspective view (Figure 17A), side view (Figure 17B), and top view (Figure 17C) of a 3D object made by a 3D printing process using a PPF polymer made according to one or more embodiments of the present invention.
[0045] Figure 17D is a photograph taken of a 3D object made by a 3D printing process with the use of a PPF polymer made according to one or more embodiments of the present invention.
[0046] Figure 17E is a schematic representation of the support structure and pore of a 3D object made by a 3D printing process using a PPF polymer made according to one or more embodiments of the present invention.
[0047] Figure 18 is a graph showing the results of a 14-day degradation experiment done on a 3D object made by a 3D printing process using a PPF polymer made according to one or more execution modes of the present invention.
[0048] Figure 19 is a graph showing the results of dynamic mechanical testing done on a 3D object made by a 3D printing process and using a PPF polymer made according to one or more modes of execution of the present invention. displaying the loss module as a function of the frequency.
[0049] Figure 20 is a graph showing the results of dynamic mechanical testing done on a 3D object made by a 3D printing process and using a PPF polymer made according to one or more embodiments of the present invention. displaying the storage module as a function of the frequency.
[0050] Figure 21 is a graph showing the results of dynamic mechanical testing done on a 3D object made by a 3D printing process and using a PPF polymer made according to one or more modes of execution of the present invention displaying the complex module as a function of frequency.
[0051] Figure 22 is a graph showing the results of dynamic mechanical testing done on a 3D object made by a 3D printing process and using a PPF polymer made according to one or more modes of execution of this
Petition 870170048142, of 10/07/2017, p. 10/20
13/68 invention showing Tan Δ as a function of frequency.
[0052] Figure 23 is a graph showing the results of the compression to rupture test done on a 3D object made by a 3D printing process and using a PPF polymer made according to one or more modes of execution of the present invention exhibiting pressure as a deformation function for non-degraded samples (C1-C5), samples degraded for 7 days (AE) and samples degraded for 14 days (KO).
[0053] Figure 24 is a bar graph showing the results of the burst compression test made on a 3D object made by a 3D printing process and using a PPF polymer made according to one or more modes of execution of the present invention showing Yield pressure for non-degraded samples, samples degraded for 7 days and samples degraded for 14 days.
Detailed Description of Illustrative Modes of Execution [0054] One or more embodiments of the present invention provide a resorbable, non-toxic, low molecular weight PPF polymer (and related methods for its production and use) that has a molecular mass and a well-defined molecular mass distribution, as well as predictable viscosity properties that is suitable for 3D printing and can be produced at low cost in commercially reasonable quantities. These PPF polymers provide predictable and reliable mechanical performance, and resorption profiles can also reduce the amount of solvent needed to ensure sufficient material flow during 3D printing. MALDI mass spectrometry accurately displays final group fidelity and size exclusion chromatography (SEC) demonstrates numerical average molecular mass distributions (<; 1.6) of a series of low molecular mass oligomers (M _n = 700 -3000 Da). In one or more modes of execution, the corresponding instrinsic viscosities range from 0.0288 ± 0.0009 dL / g to 0.0780 ± 0.0022 dL / g. In addition, standardized ISO 10993-5 tests have shown that 3D materials printed from the PPF polymers of embodiments of the present invention are non-toxic to rat fibroblast L929 and mesenchymal stem cells.
[0055] In a first aspect, the present invention is directed to a new low molecular weight resorbable PPF polymer that has a distribution of
Petition 870170048142, of 10/07/2017, p. 10/212
14/68 low molecular weight (D _m ) and a wide variety of potential uses, particularly as a component in resins for 3D printing. The PPF polymers of the present invention are non-toxic and can be used in tissue molds and other medical devices that are implanted in a human body or other living organism. In addition, the PPF polymer is degradable and resorbable. The polymer is degradable or biodegradable so as to break in vivo into its component parts within a period of time suitable for therapeutic purposes. The rate of degradation for a particular PPF polymer according to embodiments of the present invention will depend on its molecular mass, crosslink density and geometric considerations (eg, relative amount of surface area) of the material traditionally formed or printed on 3D. The PPF polymer of embodiments of the present invention is also resorbable, so that its degradation products are well tolerated by the body and can be metabolized by the body or excreted in a period of time suitable for therapeutic purposes. In the case of PPF polymers according to the embodiments of the present invention, the degradation products are fumaric acid (a normal metabolic product) and propane-1,2-diol, which is a common diluent in drug formulations and is excreted by the body.
[0056] The structure of the PPF polymers of the present invention was confirmed by Nuclear Magnetic Resonance spectroscopy ( ¹ H NMR) and Carbon 13 Nuclear Magnetic Resonance spectroscopy ( ¹³ C NMR). (See Figures 1 and 2) In some embodiments, the PPF polymers of the present invention have the formula:
- where "n" is an integer from 3 to 30. In some modes of execution, n can be an integer from 5 to 30. In some modes of execution, n can be an integer from 15 to 30. In some execution modes, n can be an integer from 3 to
25. In some modes of execution, n can be an integer from 3 to 20. In some modes of execution, n can be an integer from 3 to 15. In some modes
Petition 870170048142, of 10/07/2017, p. 10/22
15/68 execution, n can be an integer from 5 to 15. In some execution modes, n can be an integer from 3 to 10. In some execution modes, n can be an integer from 3 to 6 .
[0057] The molecular mass and mass distribution properties of the PPF polymers according to various embodiments of the present invention were characterized by Size Exclusion Chromatography (SEC). In one or more embodiments, the PPF polymer (I) will have an average numerical molecular mass (M _n ) of approximately 450 Da to approximately 3500 Da. In some embodiments, the PPF polymer may have an M _n of approximately 500 Da to approximately 3000 Da. In some embodiments, the PPF polymer may have an M _n of approximately 750 Da to approximately 2500 Da. In some embodiments, the PPF polymer may have an M _n of approximately 1000 Da to approximately 2000 Da. In some embodiments, the PPF polymer may have an M _n of approximately 1000 Da to approximately 1500 Da. In some embodiments, the PPF polymer may have an M _n of approximately 450 Da to approximately 1000 Da. In some embodiments, the PPF polymer may have an M _n of approximately 1000 Da to approximately 3500 Da. In some embodiments, the PPF polymer may have an M _n of approximately 1500 Da to approximately 3500 Da Da. In some embodiments, the PPF polymer may have an M _n of approximately 2000 Da to approximately 3000 Da.
[0058] In some embodiments, the PPF polymer may have an M _n of approximately 700 Da (M _p : 980 Da). In some embodiments, the PPF polymer may have an M _n of approximately 1269 Da (M _p : 1711 Da). In some embodiments, the PPF polymer may have an M _n of approximately 1362 Da. In some embodiments, the PPF polymer may have an M _n of approximately 1856 Da (M _p : 2573 Da). In some embodiments, the PPF polymer may have an M _n of approximately 2367 Da (M _p : 3190 Da). In some embodiments, the PPF polymer may have an M _n of approximately 3200 Da (M _p : 5974 Da). In some embodiments, the PPF polymer may have an M _n of approximately 1496 Da.
[0059] In one or more embodiments of the present invention, the PPF polymer will have an average numerical molecular mass (M _w ) of approximately 450 Daltons
Petition 870170048142, of 10/07/2017, p. 10/23
16/68 to 3500 Daltons. In one or more embodiments of the present invention, the PPF polymer will have an average numerical molecular mass (M _w ) of approximately 900 Daltons to 7000 Daltons. In one or more embodiments of the present invention, the PPF polymer will have an average numerical molecular mass (M _w ) of approximately 1000 Daltons to approximately 1500 Daltons. In one or more embodiments of the present invention, the PPF polymer will have an average numerical molecular mass (M _w ) of approximately 1000 Daltons to 2000 Daltons. In one or more embodiments of the present invention, the PPF polymer will have an average numerical molecular mass (M _w ) of approximately 1000 Daltons to approximately 3000 Daltons. In one or more embodiments of the present invention, the PPF polymer will have an average numerical molecular mass (M _w ) of approximately 2000 Daltons to approximately 3000 Daltons. In one or more embodiments of the present invention, the PPF polymer will have an average numerical molecular mass (M _w ) of approximately 2000 Daltons to 4000 Daltons. In one or more embodiments of the present invention, the PPF polymer will have an average numerical molecular mass (M _w ) of approximately 2000 Daltons to approximately 6000 Daltons.
[0060] As stated above, PPF polymers according to embodiments of the present invention also have a well-defined and relatively low molecular mass distribution (D _m ), which can be defined as the ratio between M _w and M _n . As used herein, the term well-defined as applied to molecular mass distribution means 2.0 or less. In some embodiments, the PPF polymer will have a D _m of approximately 1.0 to approximately 2.0. In some embodiments, the PPF polymer will have a D _m of approximately 1.0 to approximately 1.8. In some embodiments, the PPF polymer will have a D _m of approximately 1.0 to approximately 1.6. In some embodiments, the PPF polymer will have a D _m of approximately 1.0 to approximately 1.4. In some embodiments, the PPF polymer will have a D _m of approximately 1.0 to approximately 1.2.
[0061] In some embodiments, the PPF polymer has a D _m of approximately 1.35. In some embodiments, the PPF polymer has a D _m of approximately 1.57. In some embodiments, the PPF polymer has a D _m of approximately 1.78. In some embodiments, the PPF polymer
Petition 870170048142, of 10/07/2017, p. 10/242
17/68 has a D _m of approximately 1.46. In some embodiments, the polymer of
PPF has a D _m of approximately 1.64. In some embodiments, the PPF polymer has a D _m of approximately 1.50. In some embodiments, the PPF polymer has a D _m of approximately 1.60. In some embodiments, the PPF polymer has a D _m of approximately 1.70.
[0062] As will be appreciated by those skilled in the art, the PPF polymers of the present invention will have a glass transition temperature (T _g ). (See also, Figure 3). AT _g of polymers according to embodiments of the present invention is not particularly limited. In some embodiments, the T _g of the PPF polymer can be approximately -30 ° C to 20 ° C. In some embodiments, the T _g of the PPF polymer can be approximately -25 ° C to 12 ° C. In some embodiments, the T _g of the PPF polymer can be approximately -10 ° C to 5 ° C. In some embodiments, the T _g of the PPF polymer can be -25 ° C. In some excretion modes, the T _g of the PPF polymer can be -19 ° C. In some embodiments, the T _g of the PPF polymer can be -3 ° C. In some embodiments, the T _g of the PPF polymer may be 3 ° C.
[0063] In some embodiments, the PPF may have an M _n of 700 Da, D _m of 1.6, a T _g of -25 ° C. In some embodiments, PPF may have an M _n of 1270 Da, D _m of 1.5, and a T _g of -3 ° C. In some embodiments, the PPF may have an M _n of 1860 Da, D _m of 1.6, and a T _g of 0 ° C. In some embodiments, the PPF may have an M _n of 2450 Da, D _m of 1.6, and a T _g of 6 ° C. In some embodiments, the PPF may have an M _n of 3200 Da, D _m of 1.7, and a T _g of 12 ° C.
[0064] In some embodiments, the PPF polymers of the present invention have the characteristics set out in Table 1, below.
Table 1
Polymer Data with Temperature, Time, Rates M _w , M _p , M _n , T _g , and Viscosity
Intrinsic
PPF MAn or PO (mol) C (mol / L) Molar ratio between Monomer and Mg (OEt) ₂ Time(H) Temp.(° C) Molar ratio between PPM and Temp.(° C) Time(H) Yield (Gives) Dm T _g (° C) [η] (dL / g)
Petition 870170048142, of 10/07/2017, p. 10/252
18/68
DEA (%) 1 6,962 7.14 5.7 6 r.t. 6.67 50 16 51 700 1.6 -25 0.0288 ± 0.0009 2 2,856 7.14 24 40 80 6.67 60 16 65 1270 1.5 -3 0.0490 ± 0.0001 3 2,856 7.14 48 40 80 10 60 24 48 1860 1.6 0 0.0529 ± 0.0013 4 2,856 7.14 200 42 80 10 60 22 AT 2450 1.6 6 0.0622 ± 0.0006 5 0.714 7.14 200 138 80 6.67 55 20 AT 3160 1.7 12 0.0780 ± 0.0022
[0065] At room temperature, PPF polymers of embodiments of the present invention are a viscous fluid and can be further described in terms of viscosity. (See Table 1, above) The intrinsic viscosity is measured here in THF using an Ubbelodhe viscometer at 35 ° C.
[0066] In some embodiments, the PPF polymer has an intrinsic viscosity of approximately 0.025 dL / g to approximately 0.090 dL / g. In some embodiments, the PPF polymer has an intrinsic viscosity of approximately 0.049 dL / g to approximately 0.078 dL / g. In some embodiments, the PPF polymer has an intrinsic viscosity of approximately 0.0520 dL / g to approximately 0.0630 dL / g. In some embodiments, the PPF polymer has an intrinsic viscosity of approximately 0.0288 dL / g. In some embodiments, the PPF polymer has an intrinsic viscosity of approximately 0.0490 dL / g In some embodiments, the PPF polymer has an intrinsic viscosity of approximately 0.0529 dL / g. In some embodiments, the PPF polymer has an intrinsic viscosity of approximately 0.0622 dL / g. In some embodiments, the PPF polymer has an intrinsic viscosity of approximately 0.0780 dL / g.
[0067] Mass Spectroscopy by Matrix-Assisted Laser Desorption / Lonization - Flight-Time (MALDI-TOF) is able to precisely determine the mass of the individual materials and the populations of the final group. At low molecular weights, MALDI is able to determine molecular mass more precisely than
Petition 870170048142, of 10/07/2017, p. 10/26
19/68 size exclusion chromatography. Figure 4A is a MALDI-TOF mass spectrograph from PPF sample number 3 in Table 1 showing the mass distribution in that sample. Figure 4B is an enlarged portion of the MALDI-TOF mass spectrograph of the PPF sample number 3 in Table 1 showing the repeated unit in PPF and the possible chemicals of the final group, which correspond to the individual peaks in the distribution in the mass spectrograph data. . As seen in Figure 4B, there are three groups (labeled with A, B, C) of possible final groups in this sample (PPF sample number 3 in Table 1). The m / z = 156 between two adjacent peaks shows the mass of repeated unit, which is equivalent to the mass of maleic anhydride and propylene oxide. The predominant final group population is an ethoxy group (A). These characteristics support the successful synthesis of PPF. Figure 5A is a MALDI-TOF mass spectrograph from PPF sample number 2 in Table 1 showing the mass distribution in that sample and Figure 5B is an enlarged portion of a MALDI-TOF mass spectrograph from PPF sample number 2 in Table 1 showing the repeated unit in PPF and the possible chemicals of the final group, which correspond to the individual peaks in the distribution shown in the mass spectrograph data.
[0068] As stated above, at room temperatures, PPF polymers of embodiments of the present invention are a viscous fluid. Such polymers, however, can be cross-linked using any suitable method known in the art for this purpose to form a solid that has known mechanical properties. Suitable means for crosslinking the PPF polymers of embodiments of the present invention include, but are not limited to, radically initiated photoreticulation. In some embodiments, the polymer can be cross-linked to form 3D shapes using conventional manufacturing techniques, such as molds, electrospinning, or CNC, in addition to 3D printing methods, such as photoreticulation, cross-linking by in situ heating , FDM (fused deposition modeling), laser sintering or bioprinting. (See Examples 10, 12 and 13, below) These crosslinked PPF polymers are degradable and resorbable and may be suitable for use in surgical implants and other implantable medical devices. Cell toxicity tests conducted on cross-linked PPF polymers in accordance with embodiments of the present invention indicate that these polymers are non-toxic. (See Examples 16-20; Figures 6A-D).
Petition 870170048142, of 10/07/2017, p. 10/272
20/68 [0069] In another aspect, the present invention is directed to a new method of synthesizing PPF polymers, as described above. The present new method allows the production of large quantities of low molecular weight PPF polymer suitable for traditional formation, use as an injectable or for 3D printing and implant, among other things, without the problems identified above with respect to known PPF polymers . And while the new method of synthesizing PPF polymers described here can, in some embodiments, be used to synthesize the PPF polymers described above, in some other embodiments the method can be used to synthesize PPF polymers very bigger. It is believed that the method of various embodiments of the claimed invention can be used to synthesize PPF polymers with an M _n as large as 10,000 Da.
[0070] In some modes of execution, this new method is aimed at synthesizing PPF polymers using the two-step process shown in Scheme 1, below.
- where A and E are one or more initiators (A) or catalysts (E), B and F are each one or more solvents, C and G are each a reaction temperature, D and H are , each, a reaction time, and n is the number of units of propylenoco oxide-maleic anhydride (propylene maleate) repeated (Step I) or units of propylene fumarate (Step II).
[0071] In some modes of execution, n is an integer from 3 to 90. In some modes of execution, n is an integer from 3 to 30. In some modes of execution, n is an integer from 3 to 20 In some modes of execution, n is a number
Petition 870170048142, of 10/07/2017, p. 10/282
21/68 integer from 3 to 10. In some embodiments, n can be an integer from 5 to 30. In some embodiments, n can be an integer from 15 to 30. In some embodiments, n it can be an integer from 3 to 25. In some modes of execution, n can be an integer from 3 to 20. In some modes of execution, n can be an integer from 3 to 15. In some modes of execution, n can be an integer from 5 to 15. In some embodiments, n can be an integer from 3 to 6.
[0072] In Step I, maleic anhydride (MAn) (ii) is reacted with propylene oxide (PO) (ill) in the presence of an initiator A and one or more solvents B, at a reaction temperature C for a while reaction D, to form the intermediate (iv) of poly (propylene oxide-maleic co-anhydride) (also known as polypropylene maleate) (PPM). As will be apparent to those of ordinary skill in the art, PPM is the cis isomer of PPF (i). In Step II, the PPF polymer (iv) is isomerized to form the trans isomer (PPF) (i) in the presence of a catalyst E and one or more solvents F, at a reaction temperature G for a reaction time H.
[0073] The term isomerization is used here to refer to a reaction that converts the cis isomer (PPM) (iv) to the form of the trans isomer (PPF) (i) in the presence of a catalyst. While the isomerization step (Step II) does not result in some other changes in the polymer, it should be apparent that most of the general aspects of the PPF polymers (i) of embodiments of the present invention, such as the variations of M _n , D _m , and approximate T _g , are determined in the first reaction (Step I).
[0074] Turning now to the execution mode shown in Step I of Scheme 1 above, the starting materials for the reaction are MAn (ii) and PO (ill). While other modes of execution are possible, it was found that the MAn (ii) and PO (ill) of Step I react in a 1: 1 molar ratio.
[0075] The reaction shown in Step I additionally requires one or more initiators A. While other modes of execution are possible, A is preferably magnesium ethoxide (Mg (OEt) _2. Magnesium ethoxide has the advantage of degrading into magnesium oxide MgO and ethanol, which are generally considered to be non-toxic in this context, as will be clarified for those of ordinary skill in the art, the molar ratio between the monomer and the initiator also plays an important role in the nature and kinetics of the reaction. 2 below shows M _n results,
Petition 870170048142, of 10/07/2017, p. 10/292
22/68
M _p and D _m for PPM polymers made according to the reaction of Step I above in reaction times of 3, 6, 12, 24 and 48 hours using molar ratios between monomer and initiator of 100: 1, 200: 1 and 300: 1. (7.14 mol MAn / 1L Toluene, 80 ° C) (See also
Figure 7).
Table 2
Ratio (Molar) between Monomer and Initiator time(H) M „(Da) Average STDEV of M _n (Da) M _p (Da) Average STDEV of M _p (Da) D _m Average STDEV'sDm 100: 1 3 550 40 1020 260 1.65 0.13 6 720 90 1300 390 1.58 0.02 12 990 40 1850 560 1.64 0.03 24 1570 80 2780 840 1.66 0.19 48 2100 250 2760 280 1.66 0.14 200: 1 3 520 70 870 120 1.53 0.12 6 600 30 840 150 1.71 0.34 12 860 50 1930 240 1.72 0.2 24 1360 140 3040 400 1.7 0.23 48 2740 180 3890 870 1.67 0.14 300: 1 3 480 30 810 80 1.48 0.11 6 640 40 1050 60 1.42 0.02 12 860 50 1560 60 1.5 0.05 24 1200 180 2420 290 1.62 0.03 48 2060 490 3930 700 1.62 0.02
[0076] In Figure 7, M _n of PPMs increased almost linearly as the polymerization time increased from 3 h to 48 h, supporting a chain growth mechanism. The small deviation of Mn and Dm in multiple reactions demonstrates the reproducibility of this reaction. The molecular mass distribution of all polymerizations was around 1.6 without fractionation, further demonstrating that the chain growth method provides more precise control over the molecular mass distribution, compared to a stepwise growth mechanism, in that D _m is usually 2 or more. In addition, the yields for some of these reactions approach 65 percent, which is significantly higher than the yields for low molecular weight oligomers in stepwise growth processes.
[0077] In some embodiments, the molar ratio between the monomer and the
Petition 870170048142, of 10/07/2017, p. 10/30
23/68 primer is approximately 3: 1 to approximately 400: 1. In some embodiments, the molar ratio between the monomer and the initiator is approximately 3: 1 to 300: 1. In some embodiments, the molar ratio between the monomer and the initiator is approximately 3: 1 to approximately 200: 1. In some embodiments, the molar ratio between the monomer and the initiator is approximately 3: 1 to 100: 1. In some embodiments, the molar ratio between the monomer and the initiator is approximately 10: 1 to approximately 124: 1.
[0078] The reaction shown in Step I of Scheme 1 above is carried out in one or more solvents B. In one or more embodiments, B can be any suitable solvent including, without limitation, toluene, tetrahydrofuran (THF), dioxane and combinations thereof. It is anticipated that any solvent that is selected will be able to be removed without undue difficulty or cost. In some embodiments, B is toluene. In some embodiments, the molar ratio between the monomer and the solvent is approximately 5: 1 to 10: 1. In some embodiments, the molar ratio between the monomer and the solvent is approximately 6: 1 to 9: 1. In some embodiments, the molar ratio between the monomer and the solvent is approximately 7: 1 to 8: 1. In some embodiments, the molar ratio between the monomer and the solvent is approximately 5: 1 to approximately 8: 1. In some embodiments, the molar ratio between the monomer and the solvent is approximately 7.14: 1.
[0079] In some embodiments of the present invention, the monomers and the selected solvent B are placed in a suitable container, such as a round bottom flask, and the monomers are dissolved at room temperature with the use of a magnetic stirrer. It should be understood, however, that any method known in the art can be used to dissolve the monomers in the solvent, as long as it does not deactivate the initiator. Additionally, it should be appreciated by those skilled in the art that the monomers must be dissolved and reacted in an atmosphere of inert gas. An individual of ordinary skill in the art will be able to select an inert gas for the inert atmosphere without undue experimentation. Suitable inert gases include, without limitation, nitrogen, argon or helium. In some modes of execution, the system is cooled to room temperature under an atmosphere of nitrogen or argon.
Petition 870170048142, of 10/07/2017, p. 10/312
24/68 [0080] In these embodiments, the container can be connected to a condenser and the mixture is then heated to a reaction temperature C. In some embodiments, the condenser can be a water reflux condenser or other conventional cooling system. The method used to bring the temperature of the mixture to the reaction temperature is not particularly limited and may include, without limitation, a bath in silicone oil, a bath in water or an electrical coating. It should be apparent that reaction temperature C plays an important role in the nature and kinetics of the Step I reaction and is generally in the range of approximately 60 ° C to approximately 120 ° C, but can also be performed at room temperature in some modes. execution. (See Tables 3 and 6, below). It must be appreciated, however, that at lower temperatures (below 50 ° C) there may be random polymerization. In some embodiments, C can be from approximately 60 ° C to approximately 120 ° C. In some embodiments, C can be from approximately 70 ° C to approximately 100 ° C. In some embodiments, C can be from approximately 70 ° C to approximately 90 ° C. In some embodiments, C can be from approximately 75 ° C to approximately 80 ° C. In some embodiments, C is approximately 80 ° C.
Table 3
T (° C) 80 90 100 M _n (Da) 550 650 770 M _p (Da) 650 900 910 D _m 1.2 1.5 1.5
[0081] Additionally, as seen in Table 2 above and Table 4 below, reaction time D also plays an important role in the nature and kinetics of the reaction in Step I. In general, the longer the reaction time, the greater the M _n for the PPM produced. As will be apparent to those skilled in the art, in very short reaction times (less than 0.5 h) the reaction is highly inefficient, as there is little polymer produced and large amounts of unreacted monomer that must be removed. In reaction times greater than 100 hours, the polymer can become so viscous that it cannot be stirred with the use of a stirrer
Petition 870170048142, of 10/07/2017, p. 10/32
25/68 magnetic and polymerization becomes more difficult to control. In some modes of execution, D can be from 0.5 hours to 100 hours. In some modes of execution, D can be from 3 hours to 75 hours. In some modes of execution, D can be from 3 hours to 50 hours. In some modes of execution, D can be 12 hours to 50 hours. In some modes of execution, D can be 40 hours to 60 hours. In some modes of execution, D is 40 hours.
Table 4
Name Temp o (h) M „(Da) Dm Synthesis Conditions MAn / Toluene (mol / L) Molar ratio between MAn / PO in the feed Molar ratio between MAn and Mg (OEt) ₂ Temp.(° C) Mol of MAn (mmol) PPM20140919 20 1060 1.76 7.14 1 200 80 714 50 2900 1.53 70 3600 1.48 90 3240 1.58 114 3310 1.64 138 3740 1.57
[0082] In some embodiments, A is magnesium ethoxide (Mg (OEt) ₂ ), B is toluene, C is 80 ° C, D is 2 hours and the PPM produced had an M _n of 1700 Daltons, D _m 1.64 and yield of 58.97%. In some embodiments, A is magnesium ethoxide (Mg (OEt) ₂ ), B is toluene, C is 80 ° C, D is 40 hours and the PPM produced had an M _n of 1192 Daltons and D _m of 1, 42. In some modes of execution, Aer magnesium ethoxide (Mg (OEt) ₂ ), B is toluene, C is 80 ° C, D is 2 hours and the PPM produced has an M _n of 1206 Daltons and a D _m of 1. In some embodiments, A is magnesium ethoxide (Mg (OEt) ₂ ), B is toluene, C is 80 ° C, and D, M _n , M _p , and D _m of the produced PPM polymers are all as established in Table 2. In some embodiments, A is magnesium ethoxide (Mg (OEt) ₂ ), B is toluene, C is 80 ° C, and D, M _n , and D _m of the produced PPM polymers are all as established in Table 4.
[0083] When the reaction is complete, the PPM intermediate can be isolated and purified by any suitable methods known in the art for this
Petition 870170048142, of 10/07/2017, p. 10/33
26/68 purpose. Suitable methods may include, without limitation, extraction and concentration. In some embodiments, once the designated polymerization time has elapsed, the system is cooled to a temperature of approximately 80 ° C to approximately 20 ° C under an inert atmosphere. The method for cooling the system is not particularly limited and may include, without limitation, an ice bath, recirculation bath or ambient air temperature. Similarly, an individual of ordinary skill in the art will be able to select an inert gas for the inert atmosphere without undue experimentation. Suitable inert gases include, without limitation, nitrogen, argon or helium. In some modes of execution, the system is cooled to room temperature under an atmosphere of nitrogen or argon.
[0084] Then, in these embodiments, volatile compounds are removed by evaporation using any method known in the art for this purpose. In some embodiments, volatile compounds can be removed by distillation, rotary evaporation or evaporation under reduced pressure. In some of these embodiments, the resulting polymer is then diluted with an organic solvent such as chloroform (CHCl ₃ ) or dichloromethane CH ₂ CL ₂ . In some embodiments, the polymer can be diluted with chloroform.
[0085] The polymer solution in these embodiments is then washed with water or an aqueous solution. In some embodiments, the polymer solution is washed with water that contains an oxidizer or an acidic solution to remove inorganic compounds. In some embodiments, the polymer solution is washed with water that contains a trace amount of HCI. As should be appreciated, in embodiments where the polymer solution is washed with water or an aqueous solution, the polymer solution will separate to form an organic layer that contains the polymer and an aqueous layer that contains dissolvable impurities in Water. In these embodiments, the organic layer containing the polymer can then be collected by any conventional means, including, but not limited to, a separating funnel. It should be noted that in some embodiments, the steps of diluting the polymer with an organic solvent such as chloroform or dichloromethane and washing it with water or an aqueous solution can be repeated. In some embodiments, the PPM polymer can be washed with water 1 to 10 times.
Petition 870170048142, of 10/07/2017, p. 10/34
27/68 [0086] In some of these modes of execution, after the desired number of washing steps has been carried out, the resulting organic layer containing the PPM polymer is then poured in an excessive amount of a non-organic solvent polar, such as hexane, heptane, pentane, toluene, diethyl ether or octane to precipitate the PPM polymer out of solution. It should be appreciated that in modes of execution in which there is an M _n of less than approximately 4000 Daltons, the PPM polymer will be a viscous fluid and will separate from the non-polar organic solvent again, forming two layers. The fluid polymer layer can then be collected by any conventional means, including, but not limited to, a separating funnel. In embodiments where the polymer is a solid, it can be removed from the organic solvent by any conventional means for isolation and for collecting solids, including, but not limited to, filtration or centrifugation.
[0087] In these embodiments, the resulting polymer can again be dissolved in a minimal amount of an organic solvent, such as chloroform or dichloromethane, and then concentrated by distillation or rotary evaporation. Finally, in these embodiments, the purified PPM intermediate can then be obtained by drying the product under vacuum overnight at room temperature to remove all volatiles.
[0088] In some embodiments, the Step I reaction in Scheme 1 may comprise the dissolution of molar equivalents of maleic anhydride and propylene oxide in a suitable solvent, such as toluene, at room temperature under nitrogen. After all monomers are dissolved in toluene with magnetic stirring, Mg (OEt) ₂ is added to the mixture in a ratio of 1 mol of Mg (OEt) ₂ for every 24 moles of monomers and the vial is moved into a bath of silicone oil equipped with a water reflux condenser to initiate polymerization at 80 ° C for 40 h. After the designated polymerization time of 40 hours has elapsed, the system is then cooled to room temperature under nitrogen and all volatiles are removed by evaporation. In these embodiments, the resulting polymer is then diluted with CHCI ₃ , washed with water that contains a trace amount of HCI to remove the inorganic compounds. The organic layer is then poured into hexane after rotary evaporation, and the precipitated polymer mixture is again dissolved in a minimum amount of CHCl ₃ and then concentrated by rotary evaporation. The PPM intermediate is then obtained after
Petition 870170048142, of 10/07/2017, p. 10/35
28/68 vacuum drying the product overnight at room temperature to remove all volatiles.
[0089] As stated above, the second reaction (Step II) in Scheme 1 involves isomerization of the PPM synthesized in Step I into the trans isomer to form PPF. It has been found that even a relatively small amount of PPM polymer chains remain in the PPF polymer, this will adversely affect the polymer's ability to be cross-linked, making it unsuitable for 3D printing and other similar applications. Thus, it is important that essentially the entire PPM is converted to PPF. Figure 1 is a schematic comparing ¹ H NMR spectra (CDCI3, 300 MHz) for the PPM intermediate and the PPF polymer according to one or more embodiments of the present invention, indicating confirmation that no measurable PPM remains in the polymer. The residual solvent used in the purification step can be removed additionally with longer times under vacuum. The spectra in Figure 1 show that the PPM was successfully isomerized into PPF, with the location of the proton resonances c / s-alkenes (δ = 6.2) in C = C bonds changing to the expected position for the protons in the trans configuration (δ = 6.8).
[0090] FTIR and UV-vis spectrophotometries were used to additionally support the chemical structures of PPM and PPF. Figure 8 is a scheme comparing Fourier Transform Infrared Spectroscopy (FTIR) spectra (film, KBr, CHCI ₃ , 400 cm ^-1 - 4000 cm ^-1 ) for the PPM intermediate and the PPF polymer according to one or more embodiments of the present invention, confirming that no measurable PPM remains in the PPF polymer. In the PPM spectra in Figure 8, the peak at 1715-1740 cm ^-1 represented the C = O (ester) unsaturated stretch, which demonstrated the formation of the ester bond in the PPM synthesis process. Excerpts at 2988 cm ^-1 , 1642 cm ^-1 , 1162 cm ^-1 , 814 cm ^-1 exhibited CH section, C = C (alkene) section, OC (alkoxy) section, and (broad) CH (cis alkene curve) patterns ) separately. In the PPF spectra, the peak at 1715-1740 cm ^-1 represented the peak of unsaturated stretch C = O (ester). Stretches at 2986 cm ^-1 , 1646 cm ^-1 , 1156 cm ^-1 , 984 cm ^-1 were CH section, C = C (alkene) section, OC (alkoxy) section and CH (trans alkene) curve patterns, respectively. The appearance of curved sections CH (trans alkene) at 960-990 cm ^-1 in the solid line curve demonstrated the isomerization process. These characteristic signs supported the synthesis well
Petition 870170048142, of 10/07/2017, p. 36/102
29/68 successful PPM and PPM isomerization into PPF. See Figure 8. Spectroscopy in the Visible Ultraviolet clearly displays (acetonitrile, 190 nm - 700nm) for an intermediate of PPM and PPF. In Figure 9, the dashed line curve shows the spectra in the Visible Ultraviolet of the PPM intermediate and the solid line curve displays the spectra in the Visible Ultraviolet of the PPF polymer. As can be seen, the dashed line curve has a strong absorption at λ = 192 nm, which corresponded with the π-π * transition of the C = C connections with PPM configuration. In the PPF solid line spectrum, there is a strong absorption at λ = 210 nm, which has the transition π - π * of the C = C connection of trans configuration in PPF. The change results from converting C = C bonds from a higher energy cis configuration to a lower energy trans configuration.
[0091] In some embodiments, the conversion rate from PPM to PPF is approximately 96 percent by mass to approximately 100 percent by mass. In some embodiments, the conversion rate from PPM to PPF is approximately 98 percent by mass to approximately 100 percent by mass. In some embodiments, the conversion rate from PPM to PPF is approximately 99 percent by mass to approximately 100 percent by mass. In some embodiments, the PPF polymer of the present invention does not contain residual PPM polymer chains.
[0092] In the embodiments of the present invention shown in Step II of Scheme 1 above, the PPM intermediate is placed in a suitable container, such as a round bottom flask, and dissolved in a suitable solvent F. In one or more embodiments, F can be any suitable solvent including, but not limited to, chloroform, tetrahydrofuran (THF), dioxane, diethyl ether, and combinations thereof. It is anticipated that any solvent that is selected will be able to be removed without undue difficulty or cost. In some embodiments, F can be chloroform.
[0093] In some modes of execution, Step II is performed under an inert atmosphere. It is reiterated, an individual of ordinary skill in the technique will be able to select an inert gas for the inert atmosphere without undue experimentation. Suitable inert gases include, without limitation, nitrogen, argon or helium. In some modes of execution, the system is cooled to room temperature under a
Petition 870170048142, of 10/07/2017, p. 37/102
30/68 atmosphere of nitrogen or argon.
[0094] Once the PPM intermediate is dissolved, an E catalyst is added. While other embodiments are possible, catalyst E is preferably diethylamine. In one or more embodiments, the container is then connected to a condenser and heated to a reaction temperature G. In some embodiments, the condenser may be a water reflux condenser or other conventional cooling system used in the technique for this purpose. The method used to bring the temperature of the mixture to the reaction temperature G is not particularly limited and may include, without limitation, a bath in silicone oil, a bath in water or an electrical coating.
[0095] In some embodiments, G can be a reaction temperature of approximately 5 ° C to approximately 80 ° C. In some embodiments, G can be a reaction temperature of approximately 5 ° C to approximately 80 ° C. In some embodiments, G can be a reaction temperature of approximately 20 ° C to approximately 70 ° C. In some embodiments, G can be a reaction temperature of approximately 20 ° C to approximately 60 ° C. In some embodiments, G can be a reaction temperature of approximately 30 ° C to approximately 60 ° C. In some embodiments, G can be a reaction temperature of approximately 40 ° C to approximately 60 ° C. In some embodiments, G can be a reaction temperature of approximately 50 ° C to approximately 60 ° C. In some embodiments, G can be a reaction temperature of approximately 20 ° C. In some embodiments, G can be a reaction temperature of approximately 55 ° C. In some embodiments, G can be a reaction temperature of approximately 58 ° C. In some embodiments, G can be a reaction temperature of approximately 60 ° C. In some embodiments, G can be a reaction temperature equivalent to room temperature.
[0096] In some modes of execution, H can be a reaction time of approximately 5 to approximately 100 hours. In some embodiments, H can be a reaction time of approximately 15 to approximately 50 hours. In some embodiments, H can be a reaction time of approximately 20 to approximately 50 hours. In some modes of execution, H can be a
Petition 870170048142, of 10/07/2017, p. 38/102
31/68 reaction time approximately 20 hours. In some modes of execution, Η can be a reaction time of approximately 24 hours. In some modes of execution, H can be a reaction time of approximately 40 hours. In some modes of execution, H can be a reaction time of approximately 48 hours.
[0097] When the isomerization reaction is complete, the PPF polymer can be isolated and purified by any suitable methods known in the art for this purpose. In some modes of execution, once the reaction time has elapsed, the reaction mixture containing the PPF polymer can first be concentrated by evaporation. In some of these modes of execution, the reaction mixture can be concentrated by rotary evaporation or by evaporation under reduced pressure. In these embodiments, the concentrated reaction mixture can then be washed with a buffered aqueous solution to remove the catalyst. While other modes of execution are possible, it is anticipated that in these modes of execution, the buffered aqueous solution will buffer at a neutral pH in the range of approximately 6 to approximately 8. In some modes of execution, the concentrated reaction mixture can be washed with a phosphate buffered saline solution. In some embodiments, the concentrated reaction mixture can be washed with a saline solution buffered with 0.5 molars of phosphate, configured to buffer at a pH of approximately 6 to approximately 8.
[0098] In these execution modes, it should be understood that the reaction mixture will separate into an organic layer containing the PPF polymer and an aqueous layer containing impurities dissolved in water. The organic layer can then be collected by any conventional means, including, but not limited to, a separating funnel. It should be noted that in some embodiments, the washing steps of the mixture reaction with a buffered aqueous solution can be repeated. In some embodiments, the reaction mixture can be washed with a buffered aqueous solution 1 to 10 times. In some embodiments, the reaction mixture can be washed three times with BPS and then three times with saturated saline). Once these washing steps are completed, the solution is concentrated by evaporation. In some embodiments, the organic layer containing the polymer can be concentrated by rotary evaporation or under reduced pressure. In these modes of execution, an inorganic drying agent, acid proton or molecular sieve is then added to the concentrated polymer solution.
Petition 870170048142, of 10/07/2017, p. 10/39
32/68 to remove any remaining water. In some embodiments, the inorganic drying agent, acidic proton or molecular sieve may comprise sodium sulfate. The solution is then filtered to remove the inorganic drying agent, acidic proton or molecular sieve.
[0099] Once the remaining water has been removed, the solution is added to an excess of a non-polar organic solvent, such as hexane, causing the PPF polymer to precipitate. It should be appreciated that in modes of execution in which there is an M _n of less than approximately 4000 Daltons, the PPF polymer will be a viscous fluid and will separate from the non-polar organic solvent again, forming two layers. The fluid polymer layer can then be collected by any conventional means, including, but not limited to, a separating funnel. In embodiments where the polymer is a solid, it can be removed from the organic solvent by any conventional means for isolation and for collecting solids, including, but not limited to, filtration or centrifugation.
[0100] The collected precipitation is then kept in a vacuum for 12 to 24 hours to remove all remaining volatile compounds. In some modes of execution, it is kept at room temperature overnight to remove all remaining volatile compounds.
[0101] In some embodiments, the Step II reaction in Scheme 1 may comprise the addition of a catalyst, such as diethylamine (0.1 eq.) To the PPM intermediate, after dissolving the PPM polymer in CHCI ₃ (1 mol / L) in a round-bottomed flask equipped with a water reflux condenser. Isomerization is carried out at approximately 55 ° C for approximately 20 hours under a nitrogen atmosphere. The resulting mixture is then concentrated by rotary evaporation and washed with phosphate-buffered saline (0.5M, pH = 6) to remove diethylamine. The organic layer is then collected after separation and sodium sulfate is added to the organic layer to remove water. The concentrated organic layer is then precipitated in hexane several times to remove impurities. The precipitation is collected and kept in vacuo overnight at room temperature to remove all volatiles, to leave a PPF polymer according to one or more embodiments of the present invention.
Petition 870170048142, of 10/07/2017, p. 40/102
33/68 [0102] Resorbable, non-toxic, low molecular weight PPF polymers and the new methods for producing the PPF polymers described above, represent a significant improvement compared to polymers and methods known in the art. The PPF polymers described above overcame the difficulties of controlling molecular mass distribution inherent in the various growth polymerization methods in stages known in the art. The resorbable, non-toxic, low molecular weight PPF polymers produced using the methods described above have low polydispersity and properties that are consistent from batch to batch. It is believed that this consistency may be sufficient to meet the requirements of Good Manufacturing Practices (GMP) implemented by the FDA, required for cytotoxicity testing, material property testing, tests on small animal models, on large animal models and on humans and / or applicable ASTM and ISO standards, as well as FDA guidelines.
[0103] As will become apparent to those skilled in the art, the viscosity and therefore the fluidity of the fluid polymer resin used is an important variable in certain 3D printing methods. In general, the more viscous (ie less fluid) the polymer resin used, the more time is required to print the 3DD object using a photoreticulation method (eg 3D Systems (Rock Hill, SC), stereolithography, or using a Digital Chipe Light Processing ™ Texas Instruments (Dallas, TX) In some embodiments, the fluidity of 3D printing resins prepared using polymers according to the embodiments of the present invention can be increased by heating the resin or by adding of non-toxic solvents, such as DEF. In practice, these viscosity reduction methods are limited, since overheating can result in self-catalysis of the polymer and if too much DEF is used, it can dramatically reduce the material properties of the part Additionally, because M _n and D _m are predictable and well known, batches with different M _n can be mixed to achieve viscosity , degradation and / or other desired characteristics. It is believed that it is possible to use a low molecular weight PPF as a solvent to reduce the viscosity of the mixed polymer and, with it, the fluidity of 3D printing resins made with PPF polymers of the present invention.
[0104] Additionally, the new methods described above are scalable and
Petition 870170048142, of 10/07/2017, p. 41/102
34/68 are an unexpected and innovative improvement in the time and costs required to synthesize PPF polymers, compared to the previous PPF step growth polymerization technique. In particular, stepwise synthesis methods for known PPF polymers are slow, labor intensive and very expensive. Using these methods, it takes approximately two weeks to produce a varying amount of PPF polymer. This process requires almost constant monitoring. Energy input (heat), high vacuum, long reaction times are required and result in low conversion with uncontrolled molecular mass distribution, side reactions of conjugation-addition and unwanted cross-linking, all of which greatly influence the mechanical properties and rates degradation of the final product. What used to take weeks before using growth polymerization methods in stages of the prior art, can be achieved in 3 to 7 days depending on the quantity with standard (inexpensive) equipment, using the methods according to the modes of implementation of the present invention. , as set forth here. With the use of standard laboratory equipment the cost per gram is dramatically reduced. Furthermore, the scalability of these methods largely makes up for time and cost savings. The new methods described here are believed to greatly reduce the cost per kilogram, using GMP-level procedures and equipment. In fact, it is believed that these methods can make the activity commercially feasible.
[0105] Additionally, the present invention overcomes problems inherent in the use of known PPF polymers prepared with the use of ring opening methods for 3D printing. PPF polymers according to embodiments of the present invention that have relatively low molecular masses, even viscous, are flowable.
Examples [0106] The following examples are offered to illustrate the invention more fully, but should not be construed as limiting the scope of the invention. Furthermore, while some examples may include conclusions about how the invention can work, the inventors do not intend to link to those conclusions, but present them only as possible explanations. Furthermore, except
Petition 870170048142, of 10/07/2017, p. 42/102
35/68 if commented using the past tense, the presentation of an example does not imply that an experiment or procedure was, or was not, conducted, or that results were, or were not, actually obtained. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. quantities, temperatures), but some errors and experimental deviations may be present. Unless otherwise stated, parts are parts by mass, molecular weight is average molecular weight, temperature is in degrees centigrade and the pressure is equal to or close to atmospheric pressure.
Methods and Materials [0107] Unless otherwise stated, the materials used are those set out in Table 5.1, below.
Table 5.1
Materials / Reagents Used
Name Formula Purity Source Maleic Anhydride (MAn) C4H2O3 99% Fluka Propylene Oxide(POWDER) C ₃ H ₆ O 99.5% Aldrich Magnesium ethoxide Mg (OEt) ₂ 98% Aldrich Diethylamine C4H10N 99%, extra pure Sigma-Aldrich Hydrochloric acid HCI ACS, 37% Sigma-Aldrich Toluene (Tol) c ₇ h ₈ anhydrous, 99.8% Sigma-Aldrich Tetrahydrofuran (THF) c ₄ h ₈ o ACS grade Sigma-Aldrich Chloroform CHCI3 ACS grade Sigma-Aldrich Hexane CeHi ₂ 98.5% Sigma-Aldrich Sodium PhosphateDibasic At ₂ HPO ₄ BioXtra,> 99.0% Sigma-Aldrich Sodium PhosphateMonobasic NaH ₂ PO ₄ BioXtra,> 99.0% Sigma-Aldrich
Petition 870170048142, of 10/07/2017, p. 43/102
36/68 [0108] Unless otherwise stated, the analytical methods described here were performed using the equipment and the conditions set out in Table 5.2, below.
Table 5.2
Analytical Methods / Equipment used
Analytical Methods Type / Equipment Ή NMR Varian Mercury 300 spectrometer ¹³ C NMR Varian Mercury 300 spectrometer Ubbelohde viscometer Cannon State College, PA, 16804,0016, USA, 50 L79 UV spectra HP Hewlett Packard 8453 InstrumentUV-Vis FTIR (Fourier Transform Infrared Spectroscopy) Excalibur Manual Spectrometer (FTS3000 and FTS 4000 Series) DSC (Differential Scanning Calorimetry) Instrument TA DSC Q2000 SEC (Exclusion Chromatography)Size) GPCmax VE 2011 (with Waters 2414Reflective Index Detector) MALDI-TOF (Matrix-Assisted Laser Desorption / Lonization Flight Time) Bruker tandem mass spectrometer UltraFlex III MALDI tandem Time-of-Flight (TOF / TOF) (Bruker Daltonics, Billerica, MA, USA) equipped with a 355 nm Nd: YAG laser emitter.
[0109] The ¹ H and ¹³ C nuclear magnetic resonance (NMR) spectra were recorded with a Varian NMRS 300 MHz instrument. Deuterated chloroform (CDCI ₃ ) was used as a solvent. Chemical changes, δ (ppm), were referenced
Petition 870170048142, of 10/07/2017, p. 44/102
37/68 with the residual proton signal.
[0110] The chemical structures of PPF samples were further analyzed by a Bruker Ultraflex III mass spectrometer for laser desorption assisted by matrix / time-of-flight flight (MALDI-ToF / ToF). The samples were dissolved in CHCI ₃ at a final concentration of 10 mg / mL. The sandwich method was used with trans-2- [3- (4-tert-Butylphenyl) -2-methyl-2-propenylidene] malononitrile (DCTB) as a matrix and NaTFA as a 10: 1 salt. Final groups were identified for characterization of absolute molecular mass.
[0111] FTIR spectra were recorded for film samples released on potassium bromide (KBr) disks from the CHCI ₃ solution by an Excalibur Spectrometer (FTS 3000 and FTS 4000 Series) with variations in wave numbers from 400 cm ^-1 to 4000 cm ' ¹ . The molecular mass and molecular mass distribution of each polymer were determined by size exclusion chromatography (SEC). SEC analysis in THF at 35 ° C was performed on a Viscotek GPCmax VE 2011 GPC Solvent Sample Module with a Waters 2414 Reflective Index Detector, with polystyrene patterns of narrow molecular weight distributions (with M _w (g / mol) : 580; 1280; 3180; 4910; 10440; 21,810; 51,150; 96,000; 230,900). The thermal properties of PPF were characterized by DSC using a TA Q2000 differential scanning calorimeter from -100 ° C to 100 ° C at a scanning rate of 10 ° C / min in order to obtain the glass transition temperature ( T _g ).
Example 1.1
Representative Synthesis of Poly (propylene oxide-maleic co-anhydride) [0112] 70.06 g (714 mmol) of Maleic anhydride (MAn) and 50.0mL (714 mmol) of propylene oxide (PO) were dissolved in 100 mL of toluene in a 500 mL round-bottom flask at room temperature under a nitrogen atmosphere. After all monomers were dissolved in toluene with constant magnetic stirring, 272.34 mg (2.38 mmol, molar ratio of MAn / Mg (OEt) ₂ = 300: 1, Mg (OEt) ₂ was added to the mixture and the The flask was moved into a silicone oil bath equipped with a reflux condenser to initiate polymerization at 80 ° C. The polymerization was allowed to proceed and aliquots were taken at defined points of time (3 h, 6 h, 18 h, 24 h and 48 h) Similar studies incorporating the molar ratio of MAn / Mg (OEt) ₂ = 200: 1, 100: 1
Petition 870170048142, of 10/07/2017, p. 45/102
38/68 driven. After the designated polymerization time, the system was cooled to room temperature under nitrogen and subjected to conditions of reduced pressure to remove volatile materials. The residue was diluted with chloroform (CHCI ₃ ) washed with water containing trace amounts of hydrochloric acid (HCI) to remove the inorganic compound of Mg (OEt) ₂ . The organic layer was poured into hexane after rotary evaporation, and the precipitated polymer mixture was again dissolved in a minimum amount of CHCl ₃ . The residue was then concentrated by rotary evaporation. Poly (propylene oxide-maleic co-anhydride) was obtained after drying the product under vacuum overnight at room temperature to remove all volatiles, and then the molecular mass and molecular mass distribution properties were characterized by Size Exclusion Chromatography (SEC) at each time point after Ή NMR characterization. Ή NMR (300 MHz, Chloroform-c / δ ppm 1.13 -1.41 (d, 3H, OCH ₂ CH (CH ₃ ) O), 4.04 -4.36 (m, 2H, OCH ₂ CH ( CH ₃ ) O), 5.23 -5.30 (m, 1H, OCH ₂ CH (CH ₃ ) O), 6.24 -6.42 (m, 2H, CH = CH (configuration / s)) See Figure 1.
Example 1.2
General Procedure for Isomerization of Poly (propylene oxide-maleic anhydride) [0113] Diethylamine (0.15 eq.) Was added to the poly (propylene oxide-maleic anhydride) after dissolving the polymer in CHCI ₃ in a round-bottomed flask equipped with a water reflux condenser to initiate isomerization at 55 ° C for 24 h under a nitrogen atmosphere. The mixture was then concentrated by rotary evaporation and washed with phosphate-buffered saline (0.5M, pH = 6) to remove diethylamine. The organic layer was then precipitated in hexane several times to remove impurities. Precipitation was collected and kept in vacuo overnight at room temperature to remove all volatiles. Then, ¹ H NMR was used for characterization. ¹ H NMR (300 MHz, Chloroform io-d) δ ppm 1.11-1.43 (d, 3H, OCH ₂ CH (CH ₃ ) O), 4.09-4.39 (m, 2H, OCH ₂ CH (CH ₃ ) O), 5.21-5.35 (m, 1H, OCH ₂ CH (CH ₃ ) O), 6.83-6.91 (m, 2H, CH = CH (trans configuration)) .
Example 2
Large Batch Synthesis (M _n = 1.5 kDa)
Petition 870170048142, of 10/07/2017, p. 46/102
39/68
Synthesis of Poly (propylene oxide-maleic co-anhydride) [0114] Maleic anhydride (2856 mmol) and propylene oxide (2856 mmol) were dissolved in toluene (400 mL) in a 2 L round bottom flask at room temperature under nitrogen. After all monomers were dissolved in toluene with magnetic stirring, Mg (OEt) ₂ (119 mmol; molar ratio of MAn: Mg (OEt) ₂ = 24: 1) was added to the mixture and the flask was moved into a silicone oil bath equipped with a water reflux condenser to initiate polymerization at 80 ° C for 40 h. After the designated polymerization time, the system was cooled to room temperature under nitrogen, evaporated to remove all volatiles and then diluted with CHCl ₃ , washed with water containing a trace amount of HCI to remove the inorganic compound. The organic layer was poured into hexane after rotary evaporation, and the precipitated polymer mixture was again dissolved in a minimum amount of CHCl ₃ and was then concentrated by rotary evaporation. Poly (propylene oxide-maleic anhydride) (PPM) was obtained after drying the product under vacuum overnight at room temperature to remove all volatiles and then the molecular mass and distribution properties of molecular mass were characterized by SEC after the characterization of ¹ H-NMR characterization and the characterization of ¹³ C NMR (SEC: M _n 1200 Da; Ή NMR please see Figure 1); ¹³ C NMR shown in Figure 2). ¹³ C NMR (300 MHz, Chloroform-d) δ (ppm): 164.64, 164.63, 164.35; 130.42, 129.92, 129.78, 129.25; 69.15; 66.37; 16.19.
Poly (propylene oxide-maleic anhydride) isomerization [0115] Diethylamine (0.15 equivalent) was added to the poly (propylene oxide-maleic coanhydride) after dissolving the polymer in CHCI ₃ (1 mol / L) in a round-bottomed flask equipped with a water reflux condenser to initiate isomerization at 55 ° C for 20 h under nitrogen. The mixture was then concentrated by rotary evaporation and washed with phosphate-buffered saline (0.5M, pH = 6) to remove diethylamine. The organic layer was collected after separation and sodium sulfate was added to the organic layer to remove water. The concentrated organic layer was then precipitated in hexane several times to remove impurities. Precipitation was collected and kept in a vacuum overnight at room temperature to remove all volatiles, and then
Petition 870170048142, of 10/07/2017, p. 47/102
40/68 molecular mass and mass distribution properties were characterized by
SEC after the characterization of ¹ H-NMR. See Table 1 (PPF sample number 2,
M _n 1270Da, D _m 1.5) and Figure 1.
Example 3
Large Batch Synthesis of PPF Polymers at 5 M _n Levels (M _n = 0.7 kDa, 1.27 kDa, 1.86 kDa, 2.45 kDa, and 3.16 kDa) [0116] PPF Polymers that have M _n of 0.7 kDa, 1.27 kDa, 1.86 kDa, 2.45 kDa, and 3.16 kDa were synthesized using the large PPF batch procedures described above in Example 2 using polymerization parameters set out in Table 6, below.
Table 6
P PF# MAn orPOWDER(mmol) Monomer o / Toluen o (mol / L) Molar ratio between Monomer and Mg (OEt) ₂ t (h) T (° C) Molar ratio between PPM andDEA PPM / CHCh(mol / L) T (° C) t (h) 1 6962 7.14 5.7 ~ 6 without heating,29-86 6.67 1 50 16 2 2856 7.14 24 40 80 6.67 1 60 16 3 2856 7.14 48 40 80 10 1 60 24 4 2856 7.14 200 42 80 10 1 60 22 5 714 7.14 200 138 80 6.67 1 55 20
- It should be noted that, for PPF N ° 1 the reaction started at room temperature and reached a temperature of approximately 86 ° C. No heating was applied.
[0117] The positions and relative intensities of each characteristic peak or range in ¹ H NMR, ¹³ C NMR, Time-of-Flight, Lonization and Matrix-Assisted Laser Desorption (MALDI), FTIR and UV-Vis spectra were used to prove the chemical structures of the products. Nuclear magnetic resonance (NMR) proton spectra
Petition 870170048142, of 10/07/2017, p. 48/102
41/68 and nuclear magnetic resonance (NMR) carbon spectra were recorded with a Varian NMRS 300 instrument. Deuterated chloroform (CDCI ₃ ) was used as a solvent. Chemical changes, δ (ppm), were referenced with the residual proton signal. The chemical structures of PPF samples were further analyzed by a Bruker Ultraflex III MALDI-ToF / ToF mass spectrometer. The samples were dissolved in CHCI ₃ at a final concentration of 10 mg / mL. The sandwich method was used with trans-2- [3- (4-tert-Butylphenyl) -2-methyl-2propenylidene] malononitrile (DCTB) as a matrix and NaTFA as a 10: 1 salt. FTIR spectra were recorded for film samples released on potassium bromide (KBr) discs from the CHCI ₃ solution by an Excalibur Spectrometer (FTS 3000 and FTS 4000 Series) with variations in wave numbers from 400 cm ^-1 to 4000 cm ' ¹ . UV-visible spectra were obtained by diluted solutions of polymers in acetonitrile using an HP Hewlett Packard 8453 UV-Vis instrument with a wavelength variation from 190 nm to 700nm.
[0118] The molecular mass and molecular mass distribution of each polymer were determined by SEC. SEC analysis in THF at 35 ° C was performed on a Viscotek GPCmax VE 2011 GPC Solvent Sample Module with a Waters 2414 Reflective Index Detector, with polystyrene standards of narrow molecular weight distributions (with M _w (g / mol) : 580, 1280, 3180, 4910, 10440, 21810, 51150, 96000, 230900).
[0119] The thermal properties of PPF were characterized by DSC using a TA Q2000 differential scanning calorimeter from -100 ° C to 100 ° C at a scan rate of 10 ° C / min in order to obtain the temperature of glass transition (T _g ). The intrinsic viscosity of the PPF samples at five levels of molecular mass was tested in THF by the Ubbelohde viscometer at 35 ° C, using the procedures set out in Example 4, below. See Table 1, above. (See also, Figure 3).
Example 4
General Procedures for Measuring Intrinsic Viscosity of PPF Polymers [0120] Unless otherwise stated, the intrinsic viscosity of PPF samples synthesized in Example 3 was measured in THF using a Ubbelohde viscometer at 35 ° C. Each PPF sample (M _n : 0.7 kDa, 1.27 kDa, 1.86 kDa, 2.45 kDa,
Petition 870170048142, of 10/07/2017, p. 49/102
42/68 and 3.16 kDa) was weighed and diluted in THF in a volumetric flask (10mL). Freshly distilled THF was added to the volumetric flask up to the 10 mL mark with a 0.45 µm filter and sealed. The capillary viscometer was cleaned with pure THF. A thermostatic water bath was heated to maintain a temperature of 35 ° C. The capillary viscometer was pre-equilibrated in the thermostatic bath for at least 15 minutes to establish thermal equilibrium. An injector was used to make the liquid fill up to more than 1/3 of the upper ball of the capillary viscometer and then the liquid was allowed to flow downwards. A stopwatch was used to record the time when the liquid passed over the first line on the capillary viscometer and stopped recording when the liquid passed over the second line on the capillary viscometer. The time for this period has been recorded. The flow time was recorded at least 5 times. The capillary viscometer was refilled by a filter with 5.0 mL of the prepared solution of PPF and THF. The capillary viscometer was placed back in the thermostatic bath. The flow was measured and recorded at least 3 times, as described above. Then, 5.0 mL, 3.0 mL and additionally 1.8 mL or 2.0 mL (dependent results) of pure THF solvent were added to the capillary viscometer using a filter respectively, and the corresponding flow time was measured and recorded at least 3 times each. The calculations and experimental details for each PPF polymer are noted in Examples 5-9.
Example 5
Intrinsic Viscosity of PPF Polymer (M _n = 700 Da) [0121] Materials and Equipment. Thermostatic bath, Ubbelohde capillary viscometer (Cannon State College, PA, 16804, 0016, USA, 50 L79), stopwatch (accuracy: 0.01 s), polypropylene fumarate (PPF) samples, pure THF solvent, analytical balance, flasks volumetric (10 mL), filter (0.45 pm).
[0122] Preparation. Each PPF sample was weighed and diluted in THF in a volumetric flask (10 mL). Pure THF was added to the volumetric flask to the 10 mL line with a filter and then a stopper was plugged.
[0123] Measure. The capillary viscometer was taken to be rinsed with pure THF in the first place, which was then filled with pure THF to an appropriate level by a filter. The thermostatic bath was heated to maintain a temperature of 35 ° C. The capillary viscometer was kept in the thermostated bath for at least 15 minutes
Petition 870170048142, of 10/07/2017, p. 50/102
43/68 for the establishment of thermal balance. An injector was used to make the liquid fill up to more than 1/3 of the upper ball of the capillary viscometer and then the liquid was allowed to flow downwards. A stopwatch was used to record the time when the liquid flowed upwards from the first line on the capillary viscometer and stopped recording when the liquid passed from the second line on the capillary viscometer. The time for this period has been recorded. The flow time was measured at least 5 times to take 3 times At among which none exceeded 0.2 s. Then the THF in the capillary viscometer was spilled out. The capillary viscometer was refilled by a filter with 5 mL of the prepared solution of PPF and THF. The capillary viscometer was placed back in the thermostatic bath. The flow was measured and recorded at least 3 times, according to the procedures above. Then, 5 mL, 3 mL and an additional 1.8 mL (dependent results) of pure THF solvent were added to the capillary viscometer using a filter respectively, and the corresponding flow time was measured and recorded at least 3 times each , as per the procedures above.
Results and Discussion [0124] Flow times. The flow times of the solutions with different concentrations (c ₀ , Ci, c ₂ , c ₃ , and c ₄ ) were obtained in the experiment. The mean values of the flow times and errors were calculated. Representative 700 Da PPF data is shown in Table 7.
Table 7
Flow times of 700 Da PPF solutions with different concentrations
PPF of700 Da THF Ci(5mL) c ₂ (10mL) c ₃ (13mL) c ₄ (14.8mL)C (g / L) 0.00 410.00 205.00 157.69 138.51 t (s) you 123.56 693.03 264.97 219.28 201.88 ^ 2 123.66 692.90 264.82 219.28 201.84 t ₃ 123.46 695.22 264.92 219.25 201.79maybe 123.56 693.72 264.90 219.27 201.84at) 0.10 1.30 0.08 0.02 0.05
Petition 870170048142, of 10/07/2017, p. 51/102
44/68 [0125] Based on the data obtained from the experiment, a series of quantities were calculated using the following equations:
Hi ti q _r = - = - no t ₀ ns _P = nr ~ ¹ lnq _r Ί _η ι = - (1) (2) (3) n _sp (4)
- where: q _r is the relative viscosity, q _sp is the specific viscosity, q _in h is the inherent viscosity, q _r d is the reduced specific viscosity, q is the solution viscosity and q ₀ is the solvent viscosity; ti is the solution flow time and t ₀ is the solvent flow time; and c is the concentration of the solution. The results are shown in Table 8.
Table 8
Calculation results
solutions key (S) Hr = t / t ₀ Ιηη _Γ (lnq _r ) / c ns _P = (Hr-1) Hsp / C solvent 123.56c ₂ 264.90 2.143925 0.762638 0.003720 1.143925 0.005580 c ₃ 219.27 1.774603 0.573577 0.003637 0.774603 0.004912 c ₄ 201.84 1.633511 0.490732 0.003543 0.633511 0.004574
[0126] The intrinsic viscosity ([q]) can then be obtained by the Huggins equation and the Kraemer equation where [q] is the intrinsic viscosity and k ', k are constant.
Huggins equation:
q _sp / c = [q] + k '[q] ² c (5)
The Kraemer Equation:
Petition 870170048142, of 10/07/2017, p. 52/102
45/68
In (n _r ) / c = [n] + k [n] ² c (6)
- q _sp / c and In (q _r ) / c were both plotted versus c, as shown in Figure 10 by the origin 8.0. (See also Table 9, below). For the adjusted line of q _sp / c versus c in the
Figure 10, the linear adjustment was obtained by the origin 8.0.
Table 9
Linear adjustment values of 700 Da PPF solutions
(In r] _r ) / c ~ cValue Standard Error Interception 0.00322 0.000120255 Slope 2.49391E-06 7.09936E-07 R-SquareAdjusted 0.85008 Value Standard Error Hsp / C-C Interception 0.00253 1.34E-04 Slope 1.49E-05 7.89E-07 R-SquareAdjusted 0.99444
[0127] According to Figure 10, the relationship between reduced viscosity and concentration is q _sp / c = 0.00253 + 0.0000149 c. In comparison with equation 5, it is possible to achieve:
0.8001341¾ fj ^ (a _! 0Q253 ± a _f O0O134) L / g
- Similarly, the relationship between intrinsic viscosity and concentration is lnq _r / c = 0.00322+ 0.0000025c, and compared to equation 6, obtained:
[rç] ₂ = (o, 00322 ± 0.00012) L / g
- The average of [q] is treated as the final result:
Petition 870170048142, of 10/07/2017, p. 53/102
46/68 | _η | _{ΤΗρ =} Μ1 ± ί ₌ θ · θθ322.0.00253 _{= 0 002875} σ _[π1 = - 7θ, 00012 ² +0,000134 ² = 0.0001 wIthf 2 [η] = 0.0029 ± 0.0001 L / g [0128] Error analysis. Mistakes can arise from several aspects. (1) The concentration of the solutions may not be accurate; (2) Flow time may not be accurate due to eye error; (3) The temperature on the viscometer may not match that of the thermostatic bath.
Example 6
Intrinsic Viscosity of PPF Polymer (M _n = 700 Da)
Experimental Materials and Equipment [0129] The Materials and Equipment and Preparation used in this Example were the same as those set out in Examples 4 and 5, above.
[0130] Measure. The capillary viscometer was taken to be rinsed with pure THF in the first place, which was then filled with pure THF to an appropriate level by a filter. The thermostatic bath was heated to maintain a temperature of 35 ° C. The capillary viscometer was kept in the thermostatic bath for at least 15 minutes to establish thermal balance. An injector was used to make the liquid fill up to more than 1/3 of the upper ball of the capillary viscometer and then the liquid was allowed to flow downwards. A stopwatch was used to record the time when the liquid flowed upwards from the first line on the capillary viscometer and stopped recording when the liquid passed from the second line on the capillary viscometer. The time for this period has been recorded. The flow time was measured at least 5 times to take 3 times At which none exceeded 0.2 s. Then the THF in the capillary viscometer was spilled out. The capillary viscometer was refilled by a filter with 5 mL of the prepared solution of PPF and THF. The capillary viscometer was placed back in the thermostatic bath. The flow was measured and recorded at least 3 times, according to the procedures above. Then, 5 ml, 3 ml and additionally 2 ml (dependent results) of pure THF solvent were added to the capillary viscometer using a filter respectively, and the corresponding flow time was measured and recorded at least 3 times each, as the
Petition 870170048142, of 10/07/2017, p. 54/102
47/68 above procedures.
Results and Discussion [0131] Flow times. The flow times of the solutions with different concentrations (c ₀ , Ci, c ₂ , c ₃ , and c ₄ ) were obtained in the experiment. The mean values of the flow times and errors were calculated. Representative 1270 Da PPF data is shown in Table 10.
Table 10
Flow times of 1270 Da PPF solutions with different concentrations
PPF of 1270Gives THF Ci c ₂ c ₃ c ₄ c (g / L) 0.00 117.4 58.7 45.15 39.13 t (s) you 123.56 224.75 165.41 155.07 150.37 t2 123.66 224.56 165.48 154.94 150.28 t ₃ 123.46 224.63 165.5 155.06 150.43maybe 123.56 224.65 165.46 155.02 150.36at) 0.1 0.10 0.05 0.07 0.07
[0132] Based on the data obtained from the experiment, a series of quantities were calculated using the following equations:
Π. t _i n _r = - = 7θ to n _sp = n _r - ¹ lnq _r (1) (2) (3) (4)
Petition 870170048142, of 10/07/2017, p. 55/102
48/68
- where: q _r is the relative viscosity, q _sp is the specific viscosity, q _in h is the inherent viscosity, q _re d is the reduced specific viscosity, q is the solution viscosity and q ₀ is the solvent viscosity; t, is the flow time of the solution and t ₀ is the flow time of the solvent; and c is the concentration of the solution. The results are shown in Table 11.
Table 11
Calculation results
solutions key (S) f] r = t / to Ιηη _Γ (ln _n r) / c Hsp = (Hr-1) Hsp / c solvent 123.56 AT AT AT AT AT Ci 224.65 1.818118 0.597802 0.005092 0.818118 0.006969 c ₃ 155.02 1.254640 0.222649 0.005024 0.254640 0.005639 C4 150.36 1.216899 0.1996306 0.005016 0.216899 0.005543
[0133] The intrinsic viscosity ([q]) can then be obtained by the Huggins equation and the Kraemer equation where [q] is the intrinsic viscosity and k ', k are constant.
The Huggins Equation:
q _sp / c = [q] + k '[q] ² c (5)
The Kraemer Equation:
ln (q _r ) / c = [q] + k ”[q] ² c (6)
- q _sp / c and In (q _r ) / c were both plotted versus c, as shown in Figure 11 by origin 8.0. (See also Table 12, below). For the adjusted line of q _sp / c versus c in Figure 11, the linear adjustment was obtained by the origin 8.0.
Table 12
Linear adjustment values of 1270 Da PPF solutions
(In η _r ) / c ~ cValue Standard Error InterceptionO 0.00322 0.000120255
Petition 870170048142, of 10/07/2017, p. 56/102
49/68
Slope 2.49391E-06 7.09936E-07 R-SquareAdjusted 0.85008 Value Standard Error Osp / C-C InterceptionO 0.00253 1.34E-04 Slope 1.49E-05 7.89E-07 R-SquareAdjusted 0.99444
[0134] According to Figure 11, the relationship between reduced viscosity and concentration is q _sp / c = 0.00498 + 0.000000957c. In comparison with equation 5, it is possible to achieve:
[rç] = 0.00498 L / g
J _N = 0.00000159L / g [/ ^ = (0.00498 + 0.00000159)] ^ [0135] Similarly, the relationship between intrinsic viscosity and concentration is lnq _r / c = 0.00482 + 0.0000183c , and compared to equation 6, obtained:
[rç] ₂ = (0.00482 + 0.0000117) L / g [0136] The mean of [q] is treated as the final result:
[^] ι + [^] ₂ _ 0.00498 + 0.00482 = 0.00490 σ _[η1 = - Vo, 00000159 ² +0.0000117 ² = 5.9 x 10 ' ⁶
Nthf 2 [q] = 0.004910.00001 L / g
Example 7
Intrinsic Viscosity of PPF Polymer (M _n = 1860 Da)
Petition 870170048142, of 10/07/2017, p. 57/102
50/68
Experimental Materials and Equipment [0137] The Materials and Equipment and Preparation used in this Example were the same as those set out in Examples 4 and 5, above.
[0138] Measure. The capillary viscometer was taken to be rinsed with pure THF in the first place, which was then filled with pure THF to an appropriate level by a filter. The thermostatic bath was heated to maintain a temperature of 35 ° C. The capillary viscometer was kept in the thermostatic bath for at least 15 minutes to establish thermal balance. An injector was used to make the liquid fill up to more than 1/3 of the upper ball of the capillary viscometer and then the liquid was allowed to flow downwards. A stopwatch was used to record the time when the liquid flowed upwards from the first line on the capillary viscometer and stopped recording when the liquid passed from the second line on the capillary viscometer. The time for this period has been recorded. The flow time was measured at least 5 times to take 3 times At among which none exceeded 0.2 s. Then the THF in the capillary viscometer was spilled out. The capillary viscometer was refilled by a filter with 5 mL of the prepared solution of PPF and THF. The capillary viscometer was placed back in the thermostatic bath. The flow was measured and recorded at least 3 times, according to the procedures above. Then, 5 mL, 3 mL and additionally 2 mL (dependent results) of pure THF solvent were added to the capillary viscometer using a filter respectively, and the corresponding flow time was measured and recorded at least 3 times each, as the above procedures.
Results and Discussion [0139] Flow times. The flow times of the solutions with different concentrations (c ₀ , Ci, c ₂ , c ₃ , and c ₄ ) were obtained in the experiment. The mean values of the flow times and errors were calculated. Representative 1860 Da PPF data is shown in Table 13.
Table 13
Flow times of 1860 Da PPF solutions with different concentrations.
Petition 870170048142, of 10/07/2017, p. 58/102
51/68
PPF of1860 Da THF Ci c ₂ c ₃ C4C (g / L) 0.00 123.60 61.80 47.54 41.20 t (s) you 123.56 288.66 182.10 164.82 159.47 t2 123.66 288.46 182.09 164.81 159.40 t ₃ 123.46 288.50 181.94 164.78 159.44maybe 123.56 288.54 182.04 164.80 159.44at) 0.10 0.11 0.09 0.02 0.04
[0140] Based on the data obtained from the experiment, a series of quantities were calculated using the following equations:
Π. t _i n _r = - = Πθ n _sp = n _r - ¹ lnq _r (1) (2) (3) (4)
- where: q _r is the relative viscosity, q _sp is the specific viscosity, q _in h is the inherent viscosity, q _re d reduced specific viscosity, q is the solution viscosity and q ₀ is the solvent viscosity; t, is the flow time of the solution and t ₀ is the flow time of the solvent; and c is the concentration of the solution. The results are shown in Table 14.
Table 14
Calculation results
solutions key (S) Hr = t / to Ιηη _Γ (lnrj _r ) / c ns _P = (nr-i) Hsp / C solvent 123.56 AT AT AT AT AT Cl 288.54 2.335222 0.848107 0.006862 1.335222 0.010803 c ₂ 182.04 1.473319 0.387518 0.006271 0.473319 0.007659
Petition 870170048142, of 10/07/2017, p. 59/102
52/68
c ₃ 164.80 1.333792 0.288026 0.006059 0.333792 0.007022 c ₄ 159.44 1.290358 0.254920 0.006187 0.290358 0.007048
[0141] The intrinsic viscosity ([η]) can then be obtained by the Huggins equation and the Kraemer equation where [η] is the intrinsic viscosity and k ', k are constant.
The Huggins Equation:
q _S p / c = [F |] + k '[r |] ² c (5)
The Kraemer Equation:
In (n _r ) / c = [n] + k ”[n] ² c (6)
- r | sp / c and In (q _r ) / c were both planned versus c, as shown in Figure 12 by source 8.0. (See also Table 15, below). For the adjusted line of q _sp / c versus c in Figure 12, the linear adjustment was obtained by the origin 8.0.
Table 15
Linear adjustment values of 1860 Da PPF solutions
(lnr] r) / c ~ cValue Standard Error Interception 0.00571 1.09E-04 Slope 9.21 E-06 1.44E-06 R-SquareAdjusted 0.92991 Value Standard Error Osp / C-C Interception 0.00487 2.40E-04 Slope 4.76E-05 3.16E-06 R-SquareAdjusted 0.98694
Petition 870170048142, of 10/07/2017, p. 60/102
53/68 [0142] According to Figure 12, the relationship between reduced viscosity and concentration is q _sp / c = 0.00487 + 0.0000476c. In comparison with equation 5, it is possible to achieve:
[rç] = 0.00487 L / g [rç] = 0.00487 L / g σ _Ν = 0.00024 L / g σ _{[ί |} = 0.00024 L / g [η] _x = (0.00487 ± 0.00024) L / g [η] _x = (0.00487 ± 0.00024) L / g [0143] Similarly, the relationship between intrinsic viscosity and concentration is lnqr / c = 0.00571+ 0.00000921c, and compared to equation 6, obtained:
[rç] ₂ = (0.00571 ± 0.000109) L / g [η] ₂ = (θ, 00571 ± 0.000109) L / g [0144]
The average of [η] is treated as the final result:
| _{THF =} lnHnL ₌ 0.00487.0.00571 _{= 0 00529} ffl „l = 1 Vo, 00024 ² + 0.000109 ² = 0.00013
I n I ™ 2 | _{t [| f =} | 01. ^ Í ₌ 0.00487.0.00571 _{= 0 00529} σ _[π1 = - V0,00024 ² + 0.000109 ² = 0.00013
I n I ™ 2 [η] = 0.00529 ± 0.00013 L / g
Example 8
Intrinsic Viscosity of PPF Polymer (M _n = = 2450 Da)
Experimental Materials and Equipment [0145] The Materials and Equipment and Preparation used in this Example were the same as those set out in Example 4, above.
[0146] Measure. The capillary viscometer was taken to be rinsed with pure THF in the first place, which was then filled with pure THF to an appropriate level by a filter. The thermostatic bath was heated to maintain a temperature of 35 ° C. O
Petition 870170048142, of 10/07/2017, p. 61/102
54/68 capillary viscometer was maintained in the thermostatic bath for at least 15 minutes to establish thermal equilibrium. An injector was used to make the liquid fill up to more than 1/3 of the upper ball of the capillary viscometer and then the liquid was allowed to flow downwards. A stopwatch was used to record the time when the liquid flowed upwards from the first line on the capillary viscometer and stopped recording when the liquid passed from the second line on the capillary viscometer. The time for this period has been recorded. The flow time was measured at least 5 times to take 3 times At among which none exceeded 0.2 s. Then the THF in the capillary viscometer was spilled out. The capillary viscometer was refilled by a filter with 5 mL of the prepared solution of PPF and THF. The capillary viscometer was placed back in the thermostatic bath. The flow was measured and recorded at least 3 times, according to the procedures above. Then, 5 mL, 3 mL and additionally 2 mL (dependent results) of pure THF solvent were added to the capillary viscometer using a filter respectively, and the corresponding flow time was measured and recorded at least 3 times each, as the above procedures.
Results and Discussion [0147] Flow times. The flow times of the solutions with different concentrations (c ₀ , Ci, c ₂ , c ₃ , c ₄ ) were obtained in the experiment. The mean values of the flow times and errors were calculated. Representative 2450 Da PPF data is shown in Table 16.
Table 16
Flow times of 2450 Da PPF solutions with different concentrations.
PPF of2450 Da THF Ci c ₂ c ₃ c ₄ C (g / L) 0.00 50.00 25.00 19.23 16.67 t (s) you 123.56 178.47 146.75 141.22 137.97 t2 123.66 178.34 146.69 141.16 137.97 t ₃ 123.46 178.31 146.84 141.17 138.12maybe 123.56 178.37 146.76 141.18 138.02at) 0.00 50.00 25.00 19.23 16.67
Petition 870170048142, of 10/07/2017, p. 62/102
55/68 [0148] Based on the data obtained from the experiment, a series of quantities were calculated using the following equations:
Hi ti q _r = - = - no t ₀ ns _P = nr ~ ¹ lnq _r 1 _n ! = - (1) (2) (3) (4)
- where: q _r is the relative viscosity, q _sp is the specific viscosity, q _in h is the inherent viscosity, q _r d is the reduced specific viscosity, q is the solution viscosity and q ₀ is the solvent viscosity; t, is the flow time of the solution and t ₀ is the flow time of the solvent; and c is the concentration of the solution. The results are shown in Table 17.
Table 17
Calculation results
solutions key (S) Hr = t / to Ιηη _Γ (Ιηη _Γ ) / ο nsp = (Hr-1) Hsp / c solvent 123.56 AT AT AT AT AT Cl 178.37 1.443617 0.3667152 0.007343 0.443617 0.008872 C ₂ 146.76 1.187763 0.172072 0.006883 0.187763 0.007511 C ₄ 138.02 1.117028 0.110672 0.006640 0.117028 0.007022
[0149] The intrinsic viscosity ([q]) can then be obtained by the Huggins equation and the Kraemer equation where [q] is the intrinsic viscosity and k ', k are constant.
The Huggins Equation:
q _sp / c = [q] + k '[q] ² c (5)
The Kraemer Equation:
Petition 870170048142, of 10/07/2017, p. 63/102
56/68
In (n _r ) / c = [n] + k ”[n] ² c (6)
- q _sp / c and In (q _r ) / c were both plotted versus c, as shown in Figure 13 by the origin 8.0. (See also Table 18, below). For the adjusted line of q _sp / c versus c in the
Figure 13, the linear adjustment was obtained by the origin 8.0.
Table 18
Linear adjustment values for 2450 Da PPF solutions
(lnr] r) / c ~ cValue Standard Error Action intercept 0.00633 7.20E-05 Slope 2.05E-05 2.14E-06 R-SquareAdjusted 0.97839 Value Standard Error Osp / C-C Action intercept 0.00611 2.82E-05 Slope 5.53E-05 8.37E-07 R-SquareAdjusted 0.99954
[0150] According to Figure 13, the relationship between reduced viscosity and concentration is q _sp / c = 0.00611 + 0.000000553c. In comparison with equation 5, it is possible to achieve:
[rç] = 0.00611 L / g σ _Ν = 0.0000282 L / g [r7] ₁ = (0.00611 ± 0.0000282) L / g [0151] Similarly, the relationship between intrinsic viscosity and
Petition 870170048142, of 10/07/2017, p. 64/102
57/68 concentration is lnq _r / c = 0.00633+ 0.0000205c, and compared to equation 6, obtained:
[η] ₂ = (0.00633 ± 0.000072) L / g [0152] The mean of [η] is treated as the final result:
_{| ιΒί =} ΙηΙΗηί ₌ 0.00611.0, OO633 _{= ooo622,,} = - Vo, 0000282 ² +0.000072 ² = 0.000055 wIthf 2 [η] = 0.00622 ± 0.00006 L / g
Example 9
Intrinsic Viscosity of PPF Polymer (M _n = 3160 Da)
Experimental Materials and Equipment [0153] The Materials and Equipment and Preparation used in this Example were the same as those set out in Example 4, above.
[0154] Measure. The capillary viscometer was taken to be rinsed with pure THF first, which was then filled with pure THF to an appropriate level by a filter. The thermostatic bath was heated to maintain a temperature of 35 ° C. The capillary viscometer was kept in the thermostatic bath for at least 15 minutes to establish thermal balance. An injector was used to make the liquid fill up to more than 1/3 of the upper ball of the capillary viscometer and then the liquid was allowed to flow downwards. A stopwatch was used to record the time when the liquid flowed upwards from the first line on the capillary viscometer and stopped recording when the liquid passed from the second line on the capillary viscometer. The time for this period has been recorded. The flow time was measured at least 5 times to take 3 times At which none exceeded 0.2 s. Then the THF in the capillary viscometer was spilled out. The capillary viscometer was refilled by a filter with 5 mL of the prepared solution of PPF and THF. The capillary viscometer was placed back in the thermostatic bath. The flow was measured and recorded at least 3 times, according to the procedures above. Then, 5 mL, 3 mL and additionally 2 mL (dependent results) of pure THF solvent were added to the capillary viscometer using a filter respectively, and the flow time
Petition 870170048142, of 10/07/2017, p. 65/102
Corresponding 58/68 was measured and recorded at least 3 times each, according to the procedures above.
Results and Discussion [0155] Flow times. The flow times of the solutions with different concentrations (c ₀ , Ci, c ₂ , c ₃ , c ₄ ) were obtained in the experiment. The mean values of the flow times and errors were calculated. Representative PPF data for 3160 Da is shown in Table 19.
Table 19
Flow times of 3160 Da PPF solutions with different concentrations
PPF of3160Gives THF Ci c ₂ c ₃ C (g / L) 0.00 107.30 53.65 41.27 t (s) you 123.56 323.32 195.81 176.50 ^ 2 123.66 323.32 195.94 176.66 t ₃ 123.46 323.50 195.97 176.60maybe 123.56 323.38 195.91 176.59at) 0.10 0.10 0.09 0.08
[0156] Based on the data obtained from the experiment, a series of quantities were calculated using the following equations:
H, ti n _r = - = -
Ho t ₀ ^r ls _P = ^r lr- ¹ lnq _r Hi _n h ⁼ ~ ^ ~ (1) (2) (3) (4)
Petition 870170048142, of 10/07/2017, p. 66/102
59/68
- where: q _r is the relative viscosity, q _sp is the specific viscosity, q _in h is the inherent viscosity, q _r d is the reduced specific viscosity, q is the solution viscosity and q ₀ is the solvent viscosity; t, is the flow time of the solution and t ₀ is the flow time of the solvent; and c is the concentration of the solution. The results are shown in Table 20.
Table 20
Calculation results
solutions key (S) Hr = t / to Ιηη _Γ (lnr] _r ) / c ns _P = (nr-i) Hsp / C solvent 123.56 AT AT AT AT AT Ci 323.38 2.617190 0.962101 0.008966 1.617190 0.015072 c ₂ 195.91 1.585519 0.460911 0.008591 0.585519 0.010914 c ₃ 176.59 1.429157 0.357085 0.008653 0.429157 0.010399
[0157] The intrinsic viscosity ([q]) can then be obtained by the Huggins equation and the Kraemer equation where [q] is the intrinsic viscosity and k ', k are constant.
Huggins equation:
q _sp / c = [q] + k '[q] ² c (5)
The Kraemer Equation:
ln (q _r ) / c = [q] + k ”[q] ² c (6)
- q _sp / c and ln (q _r ) / c were both plotted versus c, as shown in Figure 14 by origin 8.0. (See also Table 21, below). For the adjusted line of q _sp / c versus c in Figure 14, the linear adjustment was obtained by the origin 8.0.
Table 21
Linear adjustment values of 3160 Da PPF solutions
(lnqr) / c ~ cValue Standard Error Action intercept 0.00837 1.36E-04 Slope 5.43E-06 1.86E-06
Petition 870170048142, of 10/07/2017, p. 67/102
60/68
R-SquareAdjusted 0.78926 Value Standard Error qsp / c-c Action intercept 0.00722 4.10E-04 Slope 7.28E-05 5.59E-06 R-SquareAdjusted 0.98826
[0158] According to Figure 14, the relationship between reduced viscosity and concentration is q _sp / c = 0.00722 + 0.0000728c. In comparison with equation 5, it is possible to achieve:
[rj] = 0.00722 L / g σ _Ν = 0.00041 L / g [^ = (0.00722 ± 0.00041jL / g [0159] Similarly, the relationship between intrinsic viscosity and concentration is lnq _r / c = 0.00837 + 0.00000543 c, and compared to equation 6, obtained:
[rj] ₂ = (0.00837 ± 0.000136) L / g [0160] The mean of [η] is treated as the final result:
_{| τΗρ =} Ιπί4πΙ _{ί =} 0.00722 + 0.00837 _{= 0 007795} σ, l = 5,0,0004Γ + 0.000 Γ * 6 '= 0.00022 in I ™ 2 [η] = 0.00780 ± 0.00022 L / g
Petition 870170048142, of 10/07/2017, p. 68/102
61/68
Example 10
Impression Resin Formulation [0161] Polypropylene fumarate (PPF) with a molecular mass (M _n ) of 1496 Da was used for the printing tests, diethyl fumarate (DEF) (Sigma-Aldrich, St. Louis, MO) was added to PPF in a 1: 3 mass ratio in order to reduce the viscosity of the polymer. DEF was used as a solvent, along with heating, to dissolve the photoinitiators and oxybenzone before their addition to the resin at a 3: 1 mass ratio between PPF and DEF. The mixture was stirred and heated to 200 ° F in an exhaust fan. A resin suitable for photoreticulation was then created from the mixture of 3: 1 PPF: DEF by adding the photoinitiators Irgacure 819 and Irgacure 784 (BASF, Ludwigshafen, Germany), as well as oxybenzone (Sigma-Aldrich) and additional DEF was added to achieve a 1: 1 PPF: DEF mass ratio. The final resin formulation had a 1: 1 mass ratio between PPF and DEF and contained 3% Irgacure 819, 0.4% Irgacure 784 and 0.7% oxybenzone, by weight of PPF and DEF.
Example 11
Polypropylene Fumarate Curing Tests [0162] The EnvisionTEC Perfactory ™ 3 Mini Multi Lens (Dearborn, Ml) was used to perform curing tests on PPF resin. Curing tests were conducted to measure the potential of PPF to successfully print a 3D mold. Replication tests (n = 4) were conducted at exposure times of 30 seconds, 60 seconds and 90 seconds. The exposure time is related to the time it would take to print a layer of a 3D mold. Before the start of curing tests, the thickness of two microscope slides was measured using a material thickness gauge (MTG) (Checkline Electromatic, Cedarhurst, NY). Actory Perfactory 3 ™ has been calibrated to generate a square UV mask with a target radiation of 350 mW dm ^-2 . The thickness of a glass slide was taken into account during calibration. The aforementioned resin was heated and stirred in an exhaust fan at a temperature close to 200 ° F to ensure homogeneity. To start the curing tests, a pipette was used to place 5-7 drops of resin on the center of the microscope slide that was used for calibration. The time of
Petition 870170048142, of 10/07/2017, p. 69/102
62/68 exposure has been adjusted in Perfactory ™ 3 to reflect the appropriate test time. The slide was placed on the calibration plate in Perfactory ™ 3, above a square mask of UV light, and the curing test was started. Upon completion of the curing test, the slide was removed from the printer. The slide was turned so that the top of the slide containing the resin was stained. This was done to ensure that any excess liquid resin was removed from the slide and that only the cured square of resin remained. Another slide, the one that was measured before the test, was placed on the slide that contained the cured material. This stack of slides was measured using MTG. The thickness of the two sheets with the material cured between them was compared with the thickness of the two sheets stacked on top of each other without any material between them. The difference was taken between these two measures to obtain the thickness of the cured material. This process was repeated for each cure test (n = 4).
Example 12
Photochemical 3D Printing (350 pm Pore Size) [0163] To start printing 3D molds, an EnvisionTEC Perfactory ™ 3 3D printer was calibrated to generate a UV mask with a nominal irradiation of 350 mW dm ^-2 . A mold geometry was chosen and the design files, which were previously created using SolidWorks software (Dassault Systèmes SolidWorks Corp., Waltham, MA), were obtained. The chosen mold geometry was a helical sleeve design, with 350 pm square pores and supports at the bottom. (See Figure 15) 50 mL of resin was poured into the base plate of the 3D Perfactory ™ 3 printer. The construction file was sent from the computer to the printer using Perfactory ™ Software Suite 2.6 (EnvisionTEC, Dearborn, Ml) . The 3D Perfactory ™ printer was operated using a 75 mm focal length lens. This enabled a native resolution of 42 pm on the XY plane. The advanced resolution module (ERM), which allows a native resolution of 21 pm on the XY plane, was not used for this study. The print job was completed in 4 hours and 11 minutes. As soon as the molds were completed, the building plate containing the attached molds was removed from the printer. The molds were washed, first with 70% acetone, to remove any uncured resin from within the pores of the molds. The molds were then
Petition 870170048142, of 10/07/2017, p. 70/102
63/68 briefly rinsed with 70% EtOH followed by a rinse with dH ₂ O. Compressed air was used to gently dry the molds. The molds were then removed from the construction board using a razor blade (a plastic card or a scraper can also be used). The molds were placed on microscope slides, in an upright position, and placed inside the UV chamber for an additional 8 hours to complete the additional crosslinking.
Example 13
Photochemical 3D Printing [0164] To ensure that the resorbable smoked polypropylene (PPF) that was synthesized with the ring-opening method could be printed in 3D, a material with a molecular weight of 1496 Da was tested for 3D printing tests on a device based on photoreticulation EnvisionTEC (Dearborn, Ml) Perfactory P3. diethyl fumarate (DEF) (Sigma-Aldrich, St. Louis, MO) was added to the PPF in a mass ratio of 1: 3 in order to reduce the viscosity of the polymer. The mixture was then stirred and heated to 200 ° F in an exhaust fan. A resin suitable for photoreticulation was then created from the mixture of 1: 3 PPF: DEF by adding the photoinitiators Irgacure 819 and Irgacure 784 (BASF, Ludwigshafen, Germany), as well as oxybenzone (Sigma-Aldrich) and additional DEF to bring the final resin composition to 1: 1 DEF: PPF, 3% Irgacure 819, 0.4% Irgacure 784 and 0.7% oxybenzone. DEF was used as the solvent, along with heating, to dissolve the photoinitiators and oxybenzone prior to their addition to the resin at a mass ratio of 3: 1 PPF: DEF.
[0165] A porous, cylindrical mold CAD file using the minimally periodic triple-pore Schoen gyro surface geometry with 125 pm support thickness, 600 pm pore diameter, and 93.5% porosity was created in SolidWorks (Dassault Systèmes, Waltham, MA). The CAD file was printed in 3D using the PPF-containing resin previously described using an EnvisionTEC (Dearborn, Ml) Perfactory P3 3D printer (See Figures 16A-C). No morphometric analysis of the molds has been performed (these comparisons are currently underway), but it has been found that the accuracy of 3D printing, in quick inspection with a calibrator, is identical for molds using PPF synthesized by the step growth method.
Petition 870170048142, of 10/07/2017, p. 71/102
64/68
Example 14
Mold imaging [0166] The molds were imagined using a Stereoscope
Olympus (Center Valley, PA) to represent mold characteristics and individually cured layers in greater detail. (See Figure 15).
Example 15
PPF Thin Films [0167] The resins of Example 3 above were heated to ensure homogeneity before being used to create the thin films. To create the thin films, a transfer pipette was used to place 5-7 drops of resin in the middle of a glass slide, in the longitudinal direction. A second glass slide was slowly placed over the first slide, ensuring that no air bubbles were formed while the resin was spread evenly between the two slides. The slides were placed in a UV chamber (3D Systems, Rock Hill, SC) for 30 minutes. After this time, the blades were removed and a razor blade was used to peel the thin films of the partially cross-linked PPF resin from the blades. The films were cut into squares measuring 1 cm along each edge. The cut squares were “sandwiched” between two slides to avoid curling, and placed back in the UV chamber for 7.5 hours to complete additional crosslinking.
Example 16
Washing / Sterilization [0168] Before starting the contact test, thin films were washed and sterilized. The wash protocol started with a 15 minute wash in Dulbecco phosphate buffered saline (DPBS) (Life Technologies, Carlsbad, CA) to remove surface debris introduced during production. This was followed by three washes separated in 70% acetone for durations of 30 minutes, 20 minutes and 10 minutes. Between acetone washes, the films were soaked in DPBS to remove excess acetone from the films and to prevent them from drying out. The protocol is terminated by the completion of two more washes in DPBS, each one of
Petition 870170048142, of 10/07/2017, p. 72/102
65/68 minutes. This entire process was repeated, so that thin films passed through the washing protocol twice. After washing, the thin films were soaked in DPBS for 72 h in an incubator at 37 ° C, 5% CO ₂ .
Example 17
Cell Culture [0169] Murine fibroblasts, L929 cell line (Sigma-Aldrich, St. Louis, MO) were used for in vitro cytotoxicity analysis in accordance with ISO 10993-5, which describes standards for direct contact assays. L929 cells were cultured with Minimum Essential Medium (MEM) (Sigma-Aldrich, St. Louis, MO) containing 10% horse serum (Sigma-Aldrich, St. Louis, MO) and 1% Penicillin Streptomycin (Life Technologies, Carlsbad , CA), as described by the manufacturer. The cells were plated at 75,000 cells per well in a 24-well polystyrene cell culture plate (Corning Life Sciences, Corning, NY). The cells were cultured at -80% confluence in the coverslips before the start of the direct contact assay. Coverslips were used so that they could be removed when stained and mounted on a microscope slide for examination under a fluorescence microscope.
Example 18
Cytotoxicity Assay [0170] A direct contact test was conducted in accordance with Standard ISO 10993-5 using the cell culture of Example 17, above. Cytotoxicity was assessed at 24, 48 and 72 h. To start the test, the medium was aspirated from wells containing cells. Then, a thin PPF film was placed over the cell monolayers in each well. About 150 pL of medium was then added back to each well - enough to cover the well, but preventing the thin film from floating above the cell monolayer. The cells and thin films were then incubated at 37 ° C and 5% CO ₂ for 24, 48 or 72 h. Subsequently, the cytotoxicity of the material was assessed using a fluorescence microscope and analyzed as described below. (See Examples 19 and 20) The cells were incubated with live / dead solutions, which make dead cells red and living cells green under fluorescence. Imaging was performed and results were evaluated qualitatively
Petition 870170048142, of 10/07/2017, p. 73/102
66/68 in images where green = living cells and red = dead cells. From these evaluations, it was determined that the tested PPF polymers were not toxic.
Example 19
Microscope [0171] The molds were imagined using an Olympus Stereoscope (Center Valley, PA) to represent the mold characteristics and the individually cured layers in greater detail. (See Figure 15). As stated above, live / dead staining was performed to assess the cytotoxicity of PPF. A solution containing 2 μΜ of AM calcein and 4 μΜ of homodimer ethidium-1 (EthD-1) was prepared in DPBS using a cytotoxicity kit (Life Technologies, Carlsbad, CA). Wells containing thin films, as well as those serving as controls, were incubated with 150 pL of live / dead solution at room temperature for 30 minutes in the dark. Cells that were cultured as mentioned above and then incubated in 70% methanol for 30 minutes before incubation in a live / dead solution were used as a positive cytotoxic control. As a negative non-cytotoxic control cells were cultured under normal conditions in polystyrene culture plates prior to live / dead staining and received no other treatment. After incubation with the live / dead solution, images were taken with an Olympus CKX41 fluorescence microscope equipped with a 12.8 MP digital camera (Olympus, Center Valley, PA), as set out in Example 20, below.
Example 20
Fluorescence imaging [0172] Live / dead staining was performed on the coverslips to assess the cytotoxicity of PPF in the cell culture of Example 17. A solution containing 2 μΜ of AM calcein and 4 μΜ of ethidium homodimer-1 (EthD-1 ) was prepared in DPBS using a cytotoxicity kit (Life Technologies, Carlsbad, CA). The coverslips with attached cells were incubated with the live / dead solution at room temperature for 30 minutes in the dark. Cells that were incubated with 70% methanol for 30 minutes were used as a positive cytotoxic control. As a negative non-cytotoxic control, cells were grown under
Petition 870170048142, of 10/07/2017, p. 74/102
67/68 normal on HDPE culture plates. After incubation with the live / dead solution, coverslips were removed from the well plate and mounted on microscope slides for imaging. Images were taken with an inverted Diaphot-TMD microscope (Nikon, Chiyoda, Tokyo, Tokyo) equipped with an epifluorescent kit (Nikon, Chiyoda, Tokyo, Tokyo) and a 5.0 MP CCD digital camera. The results were evaluated qualitatively from images in which live cells fluoresce green and dead cells fluoresce dead. From these evaluations, it was determined that the tested PPF polymers were not toxic.
Example 21
Degradation of 3D Printed Porous PPF Molds [0173] 3D porous molds were printed using the procedure set forth in Example 12 above with the use of a PPF polymer according to an embodiment of the present invention. The PPF polymer had an average numerical molecular mass M _n of 1260 Daltons and a D _m of 1.5 and was synthesized as described in Examples 1-3 above. The resin used to produce the 3D porous molds shown in Figures 17A-E was made using a solution that has a 1: 1 mass ratio between PPF and DEF polymer and that contains 30.0 mg / g (DEF + PPF ) of lrgacure-819 (BAPO) (BASF, Germany) as a photoinitiator, 4.0 m / g (DEF + PPF) of I-784 (BASF, Germany) as a photoinitiator, and 7.0 mg / g (DEF + PPF) of 2-hydroxy-4-methoxybenzophenone (also known as oxybenzone or HMB) (Sigma-Aldrich Co., St. Louis, MO) as a light-absorbing dye. The 3D printed molds were generally cylindrical with a porous Schoen gyro architecture (See Figures 17A-D). They are 88.2% porous with a support diameter of approximately 200 pm and a pore diameter of approximately 700 pm. See Figure 17E. The 3D printed molds had a height of approximately 5 mm and a diameter of approximately 10 mm and the shrinkage when cured was XY = 17.16 ± 0.26%; Z = 13.96 ± 0.32% (actual size after shrinkage: h = 4.30 ± 0.02; 0 = 8.28 ± 0.03). Five 3D printed molds were weighed and then immersed for 7 days in a solution of 0.1 M NaOH (13.0 pH) under static conditions at 37 ° C (n = 5). A second set of five 3D printed (KO) molds were weighed and then immersed for 14 days in a 0.1 M NaOH solution (13.0 pH) under static conditions at 37 ° C. The treated samples (AE) and (KO) were weighed and the
Petition 870170048142, of 10/07/2017, p. 75/102
68/68 degradation rate determined and plotted in Figure 18. Five other non-degraded samples (C-C ₅ ) were used as a control.
Example 22
Dynamic Mechanical Analysis [0174] The samples C1-C5 (control: non-degraded), AE (treated), and (KO) (described in Example 21 above) were dried and their mechanical dynamic characteristics (loss module, storage, Tan Δ) were analyzed using a BOSE ElectroForce 3230 machine equipped with a 450 N load cell. The results are shown in Figures 19-22.
Example 23
Break Compression [0175] The C1-C5 (control: non-degraded) and AE (treated) samples (described in Example 21 above) were then compressed to failure using the same BOSE ElectroForce 3230 machine and load cell 450 N at a strain rate of 1.0% / sec and its pressure and strain characteristics were analyzed. The Young's Mean Modulus was estimated at 26 MPa The yield pressure (the _y ) for the control samples (C-C ₅ ) was 1.27 ± 0.06 MPa. For samples treated for 7 days (AE) Oy was 0.69 ± 0.04 Mpa and for samples treated for 14 days (KO) _y was 0.51 ± 0.12. The results are shown in Figures 23 and 24.
[0176] In light of the above, it should be understood that the present invention significantly advances the technique by providing a PPF polymer (and related method for producing a PPF polymer) that is structurally and functionally improved in several ways. While particular embodiments of the invention have been disclosed in detail in the present, it should be understood that the invention is not limited to the same or by the same, as variations on the present invention will be quickly appreciated by those of ordinary skill in the art. The scope of the invention must be analyzed from the claims as follows.

权利要求:
Claims (30)
[1]
1) “DEGRADABLE POLYPROPYLENE FUMARATE POLYMER AND
WELL DEFINED ”characterized by the fact that it is a polymer for use in 3D printing that has a number of average molecular weight (M _n ) of approximately 450 daltons to approximately 3500 daltons and a molecular mass distribution (D _m ) of 1.0 to 2.0.
[2]
2) “DEGRADABLE POLYPROPYLENE FUMARATE POLYMER AND
WELL DEFINED ”according to Claim 1, characterized in that said number of average molecular weight (M _n ) is from approximately 700 Daltons to approximately 3200 Daltons.
[3]
3) “DEGRADABLE POLYPROPYLENE FUMARATE POLYMER AND
WELL DEFINED ”according to Claim 1, characterized in that it has a glass transition temperature (T _g ) of approximately -25 ° C to approximately 12 ° C.
[4]
4) “DEGRADABLE POLYPROPYLENE FUMARATE POLYMER AND
WELL DEFINED ”according to Claim 1, characterized in that it has a maximum number of average molecular weight from approximately 980 Daltons to approximately 5900 Daltons.
[5]
5) “DEGRADABLE POLYPROPYLENE FUMARATE POLYMER AND
WELL DEFINED ”according to Claim 1, characterized in that it has an intrinsic viscosity from approximately 0.025 dL / g to approximately 0.078 dL / g.
[6]
6) “DEGRADABLE POLYPROPYLENE FUMARATE POLYMER AND
WELL DEFINED ”according to Claim 1, characterized in that said polypropylene fumarate polymer contains less than 1% w / w of poly (propylene oxide-maleic coanhydride) chains.
[7]
7) “DEGRADABLE POLYPROPYLENE FUMARATE POLYMER AND
WELL DEFINED ”according to Claim 1, characterized by the fact that said polypropylene fumarate polymer does not contain
Petition 870170048142, of 10/07/2017, p. 77/102
2/7 chains of poly (propylene oxide-maleic co-anhydride) polymer.
[8]
8) “DEGRADABLE POLYPROPYLENE FUMARATE POLYMER AND
WELL DEFINED ”according to Claim 1, characterized by the fact that it has the formula:

- where n is an integer from 3 to 30.
[9]
9) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” characterized by understanding the steps of:
- Step A: the dissolution of maleic anhydride and propylene oxide in a suitable solvent under an inert atmosphere;
- Step B: adding a suitable initiator to the solution in Step A;
- Step C: heating the mixture from Step B to a temperature of approximately 60 ° C to approximately 120 ° C for a period of approximately 0.5 hours to approximately 100 hours to produce a poly (propylene oxide-co- maleic anhydride);
- Stage D: the collection and purification of the poly (propylene oxide-maleic co-anhydride) polymer;
- Step E: dissolving the poly (propylene oxide-maleic co-anhydride) in a suitable solvent and adding a catalyst;
- Step F: heating the mixture from Step E to a temperature of approximately 5 ° C to approximately 80 ° C for a period of approximately 5 hours to approximately 100 hours to produce a poly (propylene oxide-maleic co-anhydride) polymer ).
[10]
10) “METHOD TO PRODUCE A POLYPROPYLENE POLYMER
Petition 870170048142, of 10/07/2017, p. 78/102
3/7
FUMARATE FOR USE IN 3D PRINTING ”according to
Claim 9, characterized by the fact that the Step A solvent is selected from the group consisting of toluene, tetrahydrofuran (THF), dioxane and combinations thereof.
[11]
11) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” according to
Claim 9, characterized by the fact that the solvent in Step A is toluene.
[12]
12) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” according to
Claim 9, characterized by the fact that the initiator of Step B is magnesium ethoxide (Mg (OEt) ₂ ).
[13]
13) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” according to
Claim 9, characterized by the fact that the molar ratio between maleic anhydride or propylene oxide from Step A and the initiator from Step B is from approximately 3: 1 to approximately 400: 1.
[14]
14) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” according to
Claim 9, characterized by the fact that Step D additionally comprises:
- Step G: cooling the mixture from Step C under an inert atmosphere;
- Step H: evaporation of volatile compounds from the mixture in Step G;
- Step I: the addition of chloroform or dichloromethane to the mixture of Step H;
- Step J: washing the solution from Step I with an aqueous solution, thereby forming an organic layer and an aqueous layer;
- Step K: collecting the organic layer from Step J and adding the
Petition 870170048142, of 10/07/2017, p. 79/102
4/7 same in a non-polar organic solvent to cause the precipitation of the poly (propylene oxide-maleic co-anhydride) polymer;
- Stage L: collection of the poly (propylene oxide-maleic co-anhydride) polymer;
- Step M: the dissolution of the poly (propylene oxide-maleic coanhydride) polymer in a small amount of a suitable solvent and the concentration of the solution by evaporation;
- Step N: drying the concentrated solution of Step M under vacuum, to produce a purified poly (propylene oxide-maleic anhydride) polymer.
[15]
15) "METHOD FOR PRODUCING A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING" according to Claim 14, characterized by the fact that the inert atmosphere comprises nitrogen.
[16]
16) "METHOD FOR PRODUCING A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING" according to Claim 14, characterized in that the volatile compounds are evaporated from the mixture of Step G by distillation or reduced pressure.
[17]
17) "METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING" according to Claim 14, characterized by the fact that the poly (propylene oxide-maleic anhydride) polymer from Step L is collected by funnel separation.
[18]
18) "METHOD FOR PRODUCING A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING" according to Claim 14, characterized by the fact that the appropriate solvent from Step M comprises chloroform or dichloromethane.
[19]
19) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” according to
Petition 870170048142, of 10/07/2017, p. 80/102
5/7
Claim 9, characterized by the fact that the Step E solvent is selected from the group consisting of chloroform, tetrahydrofuran (THF), dioxane and combinations thereof.
[20]
20) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” according to
Claim 9, characterized by the fact that the solvent in Step E is chloroform.
[21]
21) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” according to
Claim 9, characterized by the fact that the catalyst for Step E is diethylamine.
[22]
22) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” according to
Claim 9, characterized by the fact that Step C occurs at room temperature.
[23]
23) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” according to
Claim 9, characterized by the fact that it additionally comprises the collection and purification of the polypropylene fumarate polymer.
[24]
24) "METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING" according to Claim 23, characterized by the fact that it comprises:
- Stage O: the concentration of the mixture from Stage F by evaporation;
- Step P: washing the mixture from Step O with an aqueous buffer solution to remove the catalyst, thereby forming an organic layer and an aqueous layer;
- Stage Q: the collection of the organic layer;
- Step R: the concentration of the organic layer by evaporation;
- Step S: the addition of an inorganic drying agent, acid proton
Petition 870170048142, of 10/07/2017, p. 81/102
6/7 or molecular sieve to remove remaining water;
- Step T: filtering the mixture from Step S to remove the inorganic drying agent, acidic proton or molecular sieve;
- Step U: the addition of the Step T mixture in the organic non-polar solvent to cause precipitation of the polypropylene fumarate polymer;
- Stage V: the collection of the polypropylene fumarate polymer from Stage U;
- Step W: drying the step V polypropylene fumarate polymer under vacuum to produce a purified polypropylene fumarate polymer.
[25]
25) "METHOD FOR PRODUCING A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING" according to Claim 24, characterized by the fact that the Step O mixture is concentrated by rotary evaporation or reduced pressure.
[26]
26) "METHOD FOR PRODUCING A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING" according to Claim 24, characterized in that the aqueous buffer solution of Step P comprises a phosphate buffered saline solution.
[27]
27) "METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING" according to Claim 24, characterized by the fact that the organic layer of Step Q is collected by a separating funnel.
[28]
28) "METHOD FOR PRODUCING A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING" according to Claim 24, characterized by the fact that the organic layer of Step R is concentrated by distillation, rotary evaporation or reduced pressure.
[29]
29) "METHOD FOR PRODUCING A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING" according to Claim 24, characterized by the fact that sodium sulfate is
Petition 870170048142, of 10/07/2017, p. 82/102
7/7 added to the mixture from Step R to remove remaining water.
[30]
30) “METHOD TO PRODUCE A POLYPROPYLENE FUMARATE POLYMER FOR USE IN 3D PRINTING” according to
Claim 24, characterized in that the organic non-polar solvent from Step U comprises hexane.

类似技术:

---

公开号 | 公开日 | 专利标题

---

BR112017010057A2|2019-11-05|well-defined degradable polypropylene fumarate polymers and scalable methods for their synthesis

---

Wang et al.2015|A simple and high-resolution stereolithography-based 3D bioprinting system using visible light crosslinkable bioinks

---

Melchels et al.2010|A review on stereolithography and its applications in biomedical engineering

---

Ji et al.2019|3D bioprinting of complex channels within cell-laden hydrogels

---

Elomaa et al.2011|Preparation of poly |-based tissue engineering scaffolds by stereolithography

---

Kumar et al.2020|Stereolithography 3D bioprinting

---

Le Fer et al.2019|Poly | stars, using architecture to reduce the viscosity of 3D printable resins

---

Xu et al.2020|Effects of Irgacure 2959 and lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate on cell viability, physical properties, and microstructure in 3D bioprinting of vascular-like constructs

---

Krishnamoorthy et al.2020|Investigation of gelatin methacrylate working curves in dynamic optical projection stereolithography of vascular-like constructs

---

Felipe-Mendes et al.2020|Biomaterials obtained by photopolymerization: from UV to two photon

---

Yin et al.2017|Material design and photo-regulated hydrolytic degradation behavior of tissue engineering scaffolds fabricated via 3D fiber deposition

---

Wilts et al.2020|Vat photopolymerization of liquid, biodegradable PLGA-based oligomers as tissue scaffolds

---

Rizzo et al.2021|Optimized Photoclick | Resins for Fast Volumetric Bioprinting

---

Jain et al.2019|Role of pendant side-chain length in determining polymer 3D printability

---

KR20160021741A|2016-02-26|Absorbant and reflecting biocompatible dyes for highly accurate medical implants

---

JP2021536515A|2021-12-27|Star-shaped poly | copolymer for 3D printing applications

---

US20200291253A1|2020-09-17|Ink composition for bioprinting and hydrogel formed from the same

---

Concellón et al.2017|Photopolymers based on ethynyl-functionalized degradable polylactides by thiol-yne ‘Click Chemistry’

---

US20210069986A1|2021-03-11|Location-Based Mode| For Biasing Provisioning Of Content When An Automated Assistant Is Responding To Condensed Natural Language Inputs

---

Lin et al.2020|Development of polycaprolactone/hydroxyapatite composite resin for 405 nm digital light projection 3D printing

---

JP2020530055A|2020-10-15|Synthesis and characterization of well-defined poly | and poly | block copolymers

---

Melchels2010|Preparation of advanced porous structures by stereolithography for application in tissue engineering

---

KR20210110120A|2021-09-07|Photocuring GelMA Hydrogel Bioink Manufacturing Method

---

Guzzi et al.2021|Hierarchical biomaterials via photopatterning-enhanced direct ink writing

---

Luo2015|Synthesis and Characterization of Poly | Oligomers via a Chain-Growth Mechanism for Continuous Digital Light Processing |

同族专利:

---

公开号 | 公开日

---

WO2016081587A9|2017-07-27|

---

CN107207714A|2017-09-26|

---

JP2018500450A|2018-01-11|

---

WO2016081587A1|2016-05-26|

---

AU2015349988A1|2017-05-25|

---

JP6826990B2|2021-02-10|

---

IL252288D0|2017-07-31|

---

AU2015349988B2|2019-08-01|

---

US10465044B2|2019-11-05|

---

CN107207714B|2021-02-05|

---

KR20170107430A|2017-09-25|

---

CA2967949A1|2016-05-26|

---

US20170355815A1|2017-12-14|

---

EP3221379A1|2017-09-27|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

GB1225803A|1967-06-02|1971-03-24|

---

US5733951A|1994-04-28|1998-03-31|Yaszemski; Michael J.|Poly|

---

WO1999052469A1|1998-04-10|1999-10-21|Wm. Marsh Rice University|Synthesis of poly by acylation of propylene glycol in the presence of a proton scavenger|

---

US6849223B2|2001-04-19|2005-02-01|Case Western Reserve University|Fabrication of a polymeric prosthetic implant|

---

JP5178520B2|2005-09-22|2013-04-10|メディバスエルエルシー|Solid polymer delivery compositions and methods of use thereof|

---

EP1926780B1|2005-09-22|2013-08-14|Medivas, LLC|Bis--diol-diester-containing poly and poly compositions and methods of use|

---

CA2666866A1|2005-10-21|2007-05-03|Medivas, Llc|Poly polymers and methods of use|

---

US8974815B2|2005-12-16|2015-03-10|Cornell University|Fibrous membrane for biomedical application based on polys|

---

WO2008121895A1|2007-03-30|2008-10-09|Medivas, Llc|Bioabsorbable elastomeric polymer networks, cross-linkers and methods of use|

---

US8809212B1|2009-11-10|2014-08-19|Stc.Unm|Electrospun fiber mats from polymers having a low Tm, Tg, or molecular weight|CN106674503B|2016-12-12|2018-07-13|暨南大学|A kind of poly- fumaric acid pinacol ester material and preparation method and application|

---

US20190359766A1|2017-02-02|2019-11-28|Matthew Becker|Functionalized polypolymers made by ring opening polymerization using magnesium catalysts|

---

KR20200035195A|2017-05-03|2020-04-02|더 유니버시티 오브 아크론|Block copolymer of lactone and poly |

---

WO2018204611A1|2017-05-03|2018-11-08|The University Of Akron|Post-3d printing functionalization of polymer scaffolds for enhanced bioactivity|

---

IT201700050248A1|2017-05-09|2018-11-09|Graf Synergy Srl|PROCEDURE FOR THREE-DIMENSIONAL PRINTING, PARTICULARLY FOR THE IMPLEMENTATION OF WINDOWS, ELASTIC ELEMENTS FOR FLOORS OR SIMILAR|

---

JP2020530055A|2017-08-07|2020-10-15|アクロン大学|Synthesis and characterization of well-defined polyand polyblock copolymers|

---

CN108309517B|2018-01-25|2019-08-30|中国人民解放军新疆军区总医院|A kind of absorbable cervical fusion cage and preparation method thereof|

---

USD882782S1|2018-02-21|2020-04-28|Ethicon Llc|Three dimensional adjunct|

---

US10779817B2|2018-02-21|2020-09-22|Ethicon Llc|Three dimensional adjuncts|

---

US10966713B2|2018-02-21|2021-04-06|Ethicon Llc|Three dimensional adjuncts|

---

US10183442B1|2018-03-02|2019-01-22|Additive Device, Inc.|Medical devices and methods for producing the same|

---

USD870890S1|2018-03-02|2019-12-24|Restor3D, Inc.|Spiral airway stent|

---

USD870888S1|2018-03-02|2019-12-24|Restor3D, Inc.|Accordion airway stent|

---

USD870889S1|2018-03-02|2019-12-24|Restor3D, Inc.|Cutout airway stent|

---

USD871577S1|2018-03-02|2019-12-31|Restor3D, Inc.|Studded airway stent|

---

CA3106265A1|2018-07-13|2020-01-16|The University Of Akron|Poly-based copolymers for 3d printing applications|

---

US20220041804A1|2018-09-10|2022-02-10|The University Of Akron|Star-shaped poly copolymers for 3d printing applications|

---

US10889053B1|2019-03-25|2021-01-12|Restor3D, Inc.|Custom surgical devices and method for manufacturing the same|

---

US20210077094A1|2019-09-16|2021-03-18|Ethicon Llc|Compressible Non-Fibrous Adjuncts|

---

EP3791807A1|2019-09-16|2021-03-17|Ethicon LLC|Compressible non-fibrous adjuncts|

---

WO2021055458A1|2019-09-16|2021-03-25|Carbon, Inc.|Bio absorbable resin for additive manufacturing|

---

EP3791800A1|2019-09-16|2021-03-17|Ethicon LLC|Compressible non-fibrous adjuncts|

---

EP3791806A1|2019-09-16|2021-03-17|Ethicon LLC|Compressible non-fibrous adjuncts|

---

EP3791810A1|2019-09-16|2021-03-17|Ethicon LLC|Compressible non-fibrous adjuncts|

---

EP3791809A1|2019-09-16|2021-03-17|Ethicon LLC|Compressible non-fibrous adjuncts|

---

EP3791799A1|2019-09-16|2021-03-17|Ethicon LLC|Compressible non-fibrous adjuncts|

---

EP3791804A3|2019-09-16|2021-04-07|Ethicon LLC|Compressible non-fibrous adjuncts|

---

US20210077104A1|2019-09-16|2021-03-18|Ethicon Llc|Compressible Non-Fibrous Adjuncts|

---

EP3791808A1|2019-09-16|2021-03-17|Ethicon LLC|Compressible non-fibrous adjuncts|

---

USD920515S1|2020-01-08|2021-05-25|Restor3D, Inc.|Spinal implant|

---

US10772732B1|2020-01-08|2020-09-15|Restor3D, Inc.|Sheet based triply periodic minimal surface implants for promoting osseointegration and methods for producing same|

---

USD920516S1|2020-01-08|2021-05-25|Restor3D, Inc.|Osteotomy wedge|

---

USD920517S1|2020-01-08|2021-05-25|Restor3D, Inc.|Osteotomy wedge|

法律状态:
2019-11-26| B15I| Others concerning applications: loss of priority|Free format text: PERDA DAS PRIORIDADES REQUERIDAS US 62/081,219 DE 18.11.2014 E US 62/139,196 DE 27.03.2015, POIS POSSUEM DEPOSITANTE DIFERENTE DO INFORMADO NA ENTRADA NA FASE NACIONAL E SUAS RESPECTIVAS CESSOES NAO FORAM APRESENTADAS, MOTIVO PELO QUAL SERA DADA PERDA DESTAS PRIORIDADES, CONFORME AS DISPOSICOES PREVISTAS NA LEI 9.279 DE 14/05/1996 (LPI) ART. 167O. |

---

2020-02-11| B12F| Other appeals [chapter 12.6 patent gazette]|

---

2021-10-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|

---

2021-11-09| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

优先权:

---

申请号 | 申请日 | 专利标题

---

US201462081219P| true| 2014-11-18|2014-11-18|

---

US201562139196P| true| 2015-03-27|2015-03-27|

---

PCT/US2015/061314|WO2016081587A1|2014-11-18|2015-11-18|Well-defined degradable poly polymers and scalable methods for the synthesis thereof|

---