Hybrid scaffolds comprising collagen-like protein and resorbable polymers for medical applications

Abstract

The invention relates to an electrospun hybrid scaffold for medical applications comprising at least one synthetic polymer selected from poly(caprolactone), polylactide, poly(trimethylene carbonate), poly(lactide-co-caprolactone), poly(lactide-co-trimethylene carbonate), polylactide-b-polyethylene glycol, their copolymers or mixtures thereof and collagen-like protein. The invention also relates to methods of dispersing collagen-like protein into synthetic resorbable polymers and forming compositions suitable for electrospinning.

Description

[0001] 202400117 1

[0002] HYBRID SCAFFOLDS COMPRISING COLLAGEN-LIKE PROTEIN AND RESORBABLE POLYMERS FOR MEDICAL APPLICATIONS

[0003] Field of the Invention

[0004] The invention relates to an electrospun hybrid scaffold for medical applications comprising at least one polymer selected from poly(caprolactone), polylactide, poly(trimethylene carbonate), polylactide-b- polyethylene glycol, their copolymers or mixtures thereof and collagen-like protein (CLP). Furthermore, the invention relates to methods of dispersing CLP into polymers and the resultant composition is suitable for electrospinning.

[0005] Background of the Invention

[0006] Animal derived collagen is currently the main source of collagen used in life sciences applications. However, they present several drawbacks including biological risk contamination and batch-to-batch variability, which can lead to low data reproducibility. The collagen-like protein such as VECOLLAN® developed by Evonik Corporation can be produced under precisely controlled conditions through an established fermentation-based process. Either animal derived or collagen-like protein has shown excellent biodegradation and biocompatibility. However, they generally lack sufficient mechanical strength.

[0007] Synthetic resorbable polymers such as polylactide, polycaprolactone (PCL), poly(lactide-glycolide) and their copolymers have received considerable attention in medical applications such as in repairing damaged tissue [E. Lih et al. (2016) Biomimetic porous PLGA scaffolds incorporating decellularized extracellular matrix for kidney tissue regeneration. ACS Appl. Mater. Interfaces, 8, 21145-21154], These resorbable polymers provide sufficient or tunable mechanical strength to support tissue growth or functions. The relative hydrophobic and bio-inert surface of these polymers; however, makes them undesirable to utilize as implant materials by limiting their ability to promote cell attachment and proliferation [J.F. Zhang, et al. (2017) Cross-linked poly(lactic acid) / dextran nanofibrous scaffolds with tunable hydrophilicity promoting differentiation of embryoid bodies. Materials Today Communications, 13, 306-316],

[0008] Combining collagen with synthetic resorbable polymers can bridge the benefits of individual components. However, options are limited. Collagen is a heat sensitive natural polymer. Temperature higher than body temperature (37°C) normally can denature collagen structure. Synthetic resorbable polymers typically have to be thermally processed at elevated temperatures that are much higher than body temperature; therefore, in order to retain collagen structure and functions, thermal processing collagen and these resorbable polymers are not good options.

[0009] Mixing animal derived collagen with resorbable polymers in organic solvents is then a typical approach. The dissolved collagen and resorbable polymers solution can produce composite fibrous scaffolds for example by electrospinning. Animal derived collagen can be dissolved in organic solvents such as 1 ,1 ,1 ,3,3,3-hexafluoro-2-propanol (HFIP), or acids such as formic acid, or acetic acid. These solvents can 202400117 2 also dissolve synthetic polymers. PCL, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and polyglycolide are examples of synthetic resorbable polymers that have been blended with collagen for engineering of different tissues [US10405963 B2; M.P. Prabhakaran et al. (2013) Electrospun aligned PHBV / collagen nanofibers as substrates for nerve tissue engineering. Biotechnol. Bioeng. 110, 2775-2784;

[0010] S. El-Ghazali et al. (2021) Preparation of a cage-type polyglycolic acid / collagen nanofiber blend with improved surface wettability and handling properties for potential biomedical applications. Polymers, 13, 3458], Such individual or mixed solvents can be prepared for dissolving animal derived collagen and resorbable polymers together to a certain concentration for electrospinning. For example, 5% PCL and type I collagen derived from rat tails at 1 :1 ratio were solubilized overnight in HFIP for electrospinning nanofibrous scaffolds [H.A. Rather, et al. (2022) Polycaprolactone-collagen nanofibers loaded with dexamethasone and simvastatin as an osteoinductive and immunocompatible scaffold for bone regeneration applications. Biomaterials and Biosystems, 8, 100064], 15% w / v PCL and 15% W / v collagen type I were dissolved in a solvent mixture of formic acid : acetone (70:30) v / v for electrospinning [E. F. Ediz, et al. (2024) In vitro assessment of Momordica charantia / Hypericum perforatum oils loaded PCL / collagen fibers: Novel scaffold fortissue engineering. J. Appl. Biomater. Func. Mater. 22],

[0011] Although collagen-like protein, such as VECOLLAN®, has a similar structure to animal derived collagen, it can only be dissolved in water. A number of synthetic resorbable polymers are water insoluble. Thus, the normal process to dissolve animal derived collagen and synthetic resorbable polymers into the cosolvent is impossible in mixing VECOLLAN® and synthetic resorbable polymers.

[0012] The present invention describes a method to disperse collagen-like protein, into synthetic polymer solutions for fabricating hybrid porous scaffolds by electrospinning.

[0013] Summary of the Invention

[0014] One embodiment described herein is an electrospun hybrid scaffold comprising a collagen-like protein and at least one synthetic resorbable polymer.

[0015] Another embodiment described herein is a process of preparing an electrospun hybrid scaffold comprising a collagen-like protein and at least one synthetic resorbable polymer, comprising the steps: (a) dissolving the resorbable polymer and collagen-like protein in a solvent forming a solution, (b) loading the solution onto an electrospinner, and (c) electrospinning the solution until a scaffold is formed.

[0016] Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description, or can be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive. 202400117 3

[0017] Brief Description of the Drawings

[0018] The above and other objects, features and advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0019] FIG. 1 depicts mechanical properties of electrospun scaffolds of polycaprolactone (PCL C212) and PCL C212 with 10% CLP,

[0020] FIG. 2 depicts the Fourier transform infrared (FT-IR) spectra of neat collagen-like protein in its powder form, electrospun scaffold of polycaprolactone (PCL C212) and the electrospun scaffold of PCL C212 with 10% CLP.

[0021] FIG. 3 depicts the Fourier transform infrared (FT-IR) spectra of neat CLP in its powder form, RESOMER® LRP t7046 pellets and the dried casting film of RESOMER® LRP t7046 with 10wt% CLP and low molecular weight PEG,

[0022] FIG. 4 depicts morphology of electrospun scaffolds of polycaprolactone (PCL C212),

[0023] FIG. 5A depicts morphology of electrospun scaffolds of polycaprolactone (PCL C212) with 2wt% CLP,

[0024] FIG. 5B depicts morphology of electrospun scaffolds of polycaprolactone (PCL C212) with 2wt% CLP at different area,

[0025] FIG. 6 depicts morphology of electrospun scaffolds of polycaprolactone (PCL C212) with 5wt% CLP and low molecular weight PEG,

[0026] FIG. 7 depicts morphology of electrospun scaffolds of polycaprolactone (PCL C212) with 10wt% CLP and low molecular weight PEG,

[0027] FIG. 8 depicts morphology of electrospun scaffolds of RESOMER LRP t7046 (poly(D,L-lactide)-b-PEG-b- poly(D,L-lactide) with 10wt% CLP and low molecular weight PEG,

[0028] FIG. 9 depicts morphology of electrospun scaffolds of RESOMER LRP t7046 (poly(D,L-lactide)-b-PEG-b- poly(D,L-lactide) with 20wt% CLP and low molecular weight PEG,

[0029] Detailed Description of the Invention

[0030] Before the present hybrid scaffolds and processes are disclosed and described, it is to be understood that the aspects described herein are not limited to specific processes, compounds, synthetic methods, articles, devices, or uses as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0031] Described herein are hybrid scaffolds comprising collagen-like protein and synthetic resorbable polymer. 202400117 4

[0032] Described herein are also methods to disperse collagen-like protein into synthetic resorbable polymers to form hybrid scaffolds by electrospinning. More specifically, certain embodiments of the present invention are related to scaffolds comprising at least one synthetic resorbable polymer and collagen-like protein, where the synthetic resorbable polymer(s) provide mechanical support and integrity of the scaffold and collagen-like protein enhances biocompatibility.

[0033] Definition of Terms

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice ortesting of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0035] The terms “comprise^)”, “include(s)”, “having”, “has”, “can”, “contain(s)”, and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising”, “consisting of’ and “consisting essentially of’, the embodiments or elements presented herein, whether explicitly set forth or not.

[0036] The conjunctive term “or” includes any and all combinations of one or more listed elements associated by the conjunctive term. For example, the phrase “an apparatus comprising A or B” may refer to an apparatus including A where B is not present, an apparatus including B where A is not present, or an apparatus where both A and B are present. The phrases “at least one of A, B, . . . and N” or “at least one of A, B, . . . N, or combinations thereof’ are defined in the broadest sense to mean one or more elements selected from the group comprising A, B, . . . and N, that is to say, any combination of one or more of the elements A, B, . . . or N including any one element alone or in combination with one or more of the other elements which may also include, in combination, additional elements not listed.

[0037] The modifier “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4”. The term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9-1 .1 . Other meanings of “about” may be apparent from the context, such as rounding off, so, for example “about 1” may also mean from 0.5 to 1.4.

[0038] The term “wt. %” means weight percent.

[0039] The term “w / w” means weight per weight. 202400117 5

[0040] The term “resorbable” means able to be resorbed. Resorb means absorb again.

[0041] Ther term “hybrid” means a mixture of two things from different resources.

[0042] The term “hybrid scaffold” in current application means a scaffold that has compositions from different sources.

[0043] The term “collagen-like protein” is referred to as “CLP” as well. The collagen-like protein is from a fermentation process involving living microorganisms and the synthetic resorbable polymer is an artificial material from synthesis rather than occurring naturally.

[0044] In general, all collagen-like proteins are suitable for this invention.

[0045] In a preferred embodiment of the present invention the collagen-like protein is a collagen-like protein from Streptococcus pyogenes, which is preferably the Scl2 protein from Streptococcus pyogenes.

[0046] Expression of collagen-like proteins have been attempted in several systems, including Escherichia coli and Saccharomyces cerevisiae. In one embodiment the at least one collagen-like protein is a bacterial collagen-like protein, preferably produced by fermentation in Pichia, Brevibacillus, Bacillus, Escherichia or Corynebacterium, preferably Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum.

[0047] In a preferred embodiment the collagen-like proteins may be expressed in Corynebacterium, preferably in Corynebacterium glutamicum.

[0048] One particularly suitable collagen-like protein is derivable from following polynucleotide.

[0049] A polynucleotide encoding an amino acid sequence that is at least > 60%, identical to the amino acid sequence of SEQ ID NO:1 , wherein the polynucleotide is a replicable polynucleotide encoding a collagen-like protein and wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N- terminus of the amino acid sequence of SEQ ID NO:1 .

[0050] It is preferred, when the amino acid sequence comprises a deletion of between 38 and 74 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1 . This includes a complete deletion of the N- terminal V-domain (comprising 74 amino acids) and different truncations of the V-domain of at least 38 amino acids.

[0051] In a preferred embodiment, the amino acid sequence that is at least > 60%, identical to the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0052] In a further configuration, the amino acid sequence that is at least > 65%, or > 70%, or > 75%, or > 80%, or > 85% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0053] In a preferred configuration, the polynucleotide encodes an amino acid sequence that is at least > 90%, > 92%, > 94%, > 96%, > 97%, > 98%, > 99% or 100%, preferably > 97%, particularly preferably > 98%, very particularly preferably > 99%, and extremely preferably 100%, identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. 202400117 6

[0054] In a preferred embodiment of the present invention the polynucleotide is a replicable nucleotide sequence encoding the collagen-like protein from Streptococcus pyogenes.

[0055] Polynucleotide and nucleic acid molecules comprising such sequences and encoding polypeptide variants of SEQ ID NO:1 to 4, which contain one or more insertion(s) or deletion(s) are suitable as well. Preferably, the polypeptide contains a maximum of 5, a maximum of 4, a maximum of 3, or a maximum of 2, insertions or deletions of amino acids.

[0056] Mixture of polypeptides comprising one of the polypeptide variants of SEQ ID NO:1 to 4 and on or more of the truncated variants of the collagen-like protein of SEQ ID NO:5 to 12 can be used as well.

[0057] Plasmids and vectors that comprise the nucleotide sequences according to the invention and optionally replicate in microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia are suitable. In a preferred configuration, the vector comprising the nucleotide sequences according to the present invention is suitable for replication in yeast of the genus Pichia pastoris.

[0058] Microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia that comprise the polynucleotides, vectors and polypeptides according to the invention are suitable as well. Preferred microorganisms are Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum.

[0059] Microorganisms of the species P. pastoris, E. coli, P. putida or C. glutamicum comprising any of the nucleotide sequences according to the present invention, any of the polypeptides or any of the vectors according to the present invention are suitable.

[0060] The microorganism may be a microorganism in which the nucleotide sequence is present in overexpressed form.

[0061] Overexpression according to the invention means, generally, an increase in the intracellular concentration or activity of a ribonucleic acid, a protein (polypeptide) or an enzyme, compared with the starting strain (parent strain) or wild-type strain, if this is the starting strain. A starting strain (parent strain) is taken to mean the strain on which the measure leading to the overexpression was carried out.

[0062] In the overexpression, the methods of recombinant overexpression are preferred. These include all methods in which a microorganism is produced using a DNA molecule provided in vitro. Such DNA molecules comprise, for example, promoters, expression cassettes, genes, alleles, encoding regions etc. These are converted into the desired microorganism by methods of transformation, conjugation, transduction or the like methods.

[0063] The extent of the expression or overexpression can be established by measuring the amount of the mRNA transcribed by the gene, by determining the amount of the polypeptide, and by determining the enzyme activity.

[0064] The bacterial collagen-like protein can be obtained in a fermentative process comprising the following steps: 202400117 7 a) fermentation of a microorganism according to the present invention in a medium, b) accumulation of the bacterial collagen-like protein in the medium, wherein a fermentation broth is obtained.

[0065] The culture medium or fermentation medium that is to be used must appropriately satisfy the demands of the respective strains. Descriptions of culture media of various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981). The terms culture medium and fermentation medium or medium are mutually exchangeable.

[0066] As carbon source, sugars and carbohydrates can be used, such as, e.g., glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing solutions from beet sugar or sugar cane processing, starch, starch hydrolysate and cellulose, oils and fats, such as, for example, soybean oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerol, methanol and ethanol, and organic acids, such as, for example, acetic acid or lactic acid.

[0067] As nitrogen source, organic nitrogen compounds such as peptones, yeast extract, meat extract, malt extract, corn-steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used. The nitrogen sources can be used individually or as a mixture.

[0068] As phosphorus source, phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used.

[0069] The culture medium must, in addition, contain salts, for example in the form of chlorides or sulphates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth. Finally, essential growth substances such as amino acids, for example homoserine and vitamins, for example thiamine, biotin or pantothenic acid, can be used in addition to the above-mentioned substances.

[0070] Said starting materials can be added to the culture in the form of a single batch or supplied in a suitable manner during the culturing.

[0071] Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds such as phosphoric acid or sulphuric acid, are used in a suitable manner for pH control of the culture. The pH is generally adjusted to 6.0 to 8.5, preferably 6.5 to 8. For control of foam development, antifoams can be used, such as, for example, polyglycol esters of fatty acids. For maintaining the stability of plasmids, suitable selectively acting substances such as, for example, antibiotics, can be added to the medium. The fermentation is preferably carried out under aerobic conditions. In order to maintain said aerobic conditions, oxygen or oxygen-containing gas mixtures such as, for example, air, are introduced into the culture. The use of liquids that are enriched with hydrogen peroxide is likewise possible. Optionally, the fermentation is carried out at superatmospheric pressure, for example at a superatmospheric pressure of 202400117 8

[0072] 0.03 to 0.2 MPa. The temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40°C, particularly preferably 30°C to 37°C. In the case of batch or fed-batch processes, the culturing is preferably continued until an amount sufficient for the measure of obtaining the desired organic chemical compound has formed. This goal is usually reached within 10 hours to 160 hours. In continuous processes, longer culture times are possible. Due to the activity of the microorganisms, enrichment (accumulation) of the fine chemicals in the fermentation medium and / or in the cells of the microorganisms occurs.

[0073] Examples of suitable fermentation media may be found, inter alia, in patent documents US 5,770,409, US 5,990,350, US 5,275,940, WO 2007 / 012078, US 5,827,698, WO 2009 / 043803, US 5,756,345 or US 7,138,266; appropriate modifications may optionally be carried out to the requirements of the strains used.

[0074] The process may be characterized by a process which is selected from the group consisting of batch process, fed-batch process, repetitive fed-batch process and continuous process.

[0075] The process may be further characterized by a fine chemical, or a liquid, or a solid fine chemicalcontaining product obtained from the fine chemical-containing fermentation broth.

[0076] The performance of the processes or fermentation processes according to the invention with respect to one or more of the parameters selected from the group of concentration (compound formed per volume), yield (compound formed per carbon source consumed), volumetric productivity (compound formed per volume and time) and biomass-specific productivity (compound formed per cell dry mass or bio dry mass and time or compound formed per cell protein and time) or other process parameters and combinations thereof, is increased by at least 0.5%, at least 1 %, at least 1 .5% or at least 2%, based on processes or fermentation processes with microorganisms in which the promoter variant according to the invention is present.

[0077] Owing to the measures of the fermentation, a fermentation broth is obtained which contains the desired collagen-like protein, and preferably amino acid or organic acid.

[0078] Then, a product in liquid or solid form that contains the collagen-like protein is provided or produced or obtained.

[0079] A fermentation broth means, in a preferred embodiment, a fermentation medium or nutrient medium in which a microorganism was cultured for a certain time and at a certain temperature. The fermentation medium, or the media used during the fermentation, contains / contain all substances or components that ensure production of the desired collagen-like protein and typically ensure growth and / or viability.

[0080] On completion of the fermentation, the resultant fermentation broth accordingly contains a) the biomass (cell mass) of the microorganism resulting from growth of the cells of the microorganism, b) the desired collagen-like protein formed in the course of the fermentation, c) the organic by-products possibly formed in the course of the fermentation, and 202400117 9 d) the components of the fermentation medium used, or of the starting materials, that are not consumed by the fermentation, such as, for example, vitamins such as biotin, or salts such as magnesium sulphate.

[0081] The organic by-products include substances which are generated in addition to the respective desired compound by the microorganisms used in the fermentation and are possibly secreted.

[0082] The fermentation broth is withdrawn from the culture vessel or the fermentation container, optionally collected, and used for providing a product in liquid or solid form containing the collagen-like protein. The expression "obtaining the collagen-like protein-containing product" is also used therefor. In the simplest case, the collagen-like protein-containing fermentation broth withdrawn from the fermentation container is itself the product obtained.

[0083] By way of one or more of the measures selected from the group a) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%) removal of the water, b) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%) removal of the biomass, wherein this is optionally inactivated before the removal, c) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, > 99.3%, > 99.7%) removal of the organic by-products formed in the course of the fermentation, and d) partial (> 0%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, > 99.3%, > 99.7%) removal of the components of the fermentation medium used or the starting materials that are not consumed by the fermentation, a concentration or purification of the desired collagen-like protein is achieved from the fermentation broth. In this manner, products are isolated that have a desired content of the compound.

[0084] The partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80% to < 100%) removal of the water (measure a)) is also termed drying.

[0085] In a variant of the process, by complete or virtually complete removal of the water, the biomass, the organic by-products and the non-consumed components of the fermentation medium used, pure (> 80% by weight, > 90% by weight) or high-purity (> 95% by weight, > 97% by weight, > 99% by weight) product forms of the desired collagen-like protein, preferably bacterial collagen-like protein, are successfully arrived at. For the measures according to a), b), c) or d), a great variety of technical instructions are available in the prior art.

[0086] In the case of processes for producing bacterial collagen-like protein processes are preferred in which products are obtained that do not contain any components of the fermentation broth. These products are used, in particular, in human medicine, in the pharmaceuticals industry, and in the food industry. 202400117 10

[0087] The characterization of the different samples can be done on standard test machines. Scanning electron microscope can be performed on a desktop machine (commercially available from the company Hitachi). Mechanical data can be performed on a dynamic mechanical analyzer commercially available from TA Instruments.

[0088] Porous scaffolds can be electrospun on an electrospinner (commercially available from the company Elmarco). Electrospinning is a voltage-driven technology which uses an electrohydrodynamic process, during which a liquid droplet is electrified to generate a jet, followed by stretching and elongation to generate fibers [J. Xue et al. (2019) Electrospinning and electrospun nanofibers: methods, materials, and applications. Chem. Rev., 119, 5298- 5415],

[0089] One embodiment described herein is a method to disperse water soluble CLP into synthetic resorbable polymers. The CLP used is free from animal- derived contaminants and can be engineered to have specific properties [A. Fertala. (2020) Three decades of research on recombinant collagens: reinventing the wheel or developing new biomedical products? Bioengineering, 7(4), 155],

[0090] Another embodiment described herein is use of synthetic resorbable polymers to help improve or retain mechanical properties and morphology of the scaffolds.

[0091] Another embodiment described herein are the solutions comprising synthetic resorbable polymers and dispersed CLP, which enables electrospinning to form hybrid scaffolds.

[0092] Suitable synthetic resorbable polymers of the invention may include, but are not limited to poly(caprolactone), polylactide, poly(lactide-co-caprolactone), poly(lactide-co-trimethylene carbonate), poly(lactide-co-glycolic acid) (PLGA), poly(trimethylene carbonate), polylactide-b-polyethylene glycol, and or mixtures thereof.

[0093] In one embodiment of this invention, polyethylene glycol) (PEG) polymers have been used. Examples of PEG containing polymers may include, but are not limited to PEG, polyethylene glycol) diol, poly(ethylene glycol) methyl ether (mPEG), multi armed PEG, poly(lactic acid-b-polyethylene glycol) (PLA- b-PEG), PCL-PEG, or mixtures thereof.

[0094] In one embodiment of this invention, the PEG containing polymers can be soluble in water and organic solvents. In another embodiment, some part of the PEG containing polymers can be soluble in water, and the other part of the PEG containing polymers can be soluble in organic solvents.

[0095] Suitable organic solvents of the invention may include, but are not limited to acetone, dimethyl sulfoxide, dimethylformamide, chloroform, dichloromethane, tetra hydrofuran, hexafluoroisopropanol, or their mixtures thereof.

[0096] In one embodiment of the invention, hybrid scaffolds can be formed by electrospinning through a single solution in which the CLP is dispersed.

[0097] Certain embodiments of the present invention are related to synthetic resorbable polymers which can be used to form scaffolds by the electrospinning process. The electrospinning process may comprise batch process or continuous process. In a further embodiment, the electrospinning process may comprise single spinneret, multiple spinneret, needle type spinneret, or needle-free spinneret. The scaffolds of the 202400117 11 invention may comprise one or more synthetic resorbable polymers, such as PCL, PLA, PLA-b-PEG, PDLLA-b-PEG, or their copolymers. In another embodiment, the scaffolds of the invention comprise CLP. In the electrospinning process, a synthetic resorbable polymer or a polymer blend or a polymer mixture of more than two synthetic resorbable polymers is dissolved in an appropriate solvent. The CLP is dispersed or dissolved in the same solvent. The electrospinning process may comprise CLP in a single polymer solution, a polymer blend solutions, or multiple polymer solutions. The polymer solution may be fed into a single electrospinning spinneret or multiple spinneret to improve production rate. In a further embodiment, the CLP solution may be fed into a separate spinneret and electrospun with synthetic resorbable polymer solutions simultaneously with blend of synthetic resorbable polymer fibers and CLP fibers. In a further embodiment, the CLP can be further crosslinked to reduce its solubility in wet condition, for example, in physiological conditions.

[0098] Another embodiment of the invention is related to dispersing CLP into synthetic resorbable polymer solution for electrospinning. The CLP is only soluble in water up to 10% by weight. Synthetic resorbable polymers, however, are water insoluble and are only soluble in organic solvent(s). Direct mixing of CLP, or CLP aqueous solution with synthetic resorbable polymer solutions results in up to 2% by weight of CLP in the organic solvent. Higher than 2% resulted in large CLP aggregation and synthetic polymer precipitation from organic solvents, which prevented electrospinning. The dispersion is made from a method where the CLP is introduced to the synthetic resorbable polymer solution thus forming a relatively stable suspension of CLP without forming large aggregation or synthetic resorbable polymer precipitation. The dispersion of CLP is made using the addition of PEG, which is soluble in the solvent that can dissolve the synthetic resorbable polymer(s). Polyvinyl alcohol-graft-PEG could enhance the fibrillation kinetics, wetting properties and stability of collagen scaffolds [R. Hatwell etal. Polyvinyl alcohol- graft-polyethylene glycol hydrogels improve utility and biofunctionality of injection collagen biomaterials. Biomed. Mater., 2016, 11 , 035013], Furthermore, PEG alone has been utilized to precipitate type I, II, and II collagens at neutral pH [J.A.M. Ramshaw, J.F. Bateman, W.G. Cole. Precipitation of collagens by polyethylene glycols. Analytical Biochemistry, 1984, 141 , 361-365], In this invention, PEG was introduced to CLP to assist disperse into synthetic resorbable polymer solution. So far no one has reported using PEG to assist collagen dispersion with other synthetic resorbable polymers in organic solvents.

[0099] EXAMPLES

[0100] Example 1. Electrospinning of polycaprolactone

[0101] 6 g of RESOMER® C212 (polycaprolactone, Evonik commercial product) was dissolved in 30 mL of chloroform and 10 mL of acetone mixture to make a 15 wt% solution. The solution was loaded onto an electrospinner (NS LAB, Elmarco s.r.o., Czech Republic). The solution was electrsopun at 30 kV with a distance between collector and wire collecting electrode of 14 cm and carriage speed of 50 mm / s. After electrospinning the scaffold was peeled off for further characterization. 202400117 12

[0102] Example 2. Electrospinning of polycaprolactone with CLP

[0103] After preparing the same concentration of the above solution stated in Example 1 , dry collagen-like protein, namely CLP was weighed on the basis of dissolved PCL amount and then added to the solution. With aggressive stirring, only up to 2.0 wt% CLP could be added to the solution in order to immerse it into the solution. The solution with CLP suspension was loaded to the electrospinner cartridge for electrospinning. The same electrospinning condition was used as in Example 1 for electrospinning. Upon analyzing electrospun fiber morphology using scanning electron microscope, individual CLP flakes at micro-scale was visible.

[0104] Example 3. Electrospinning of polycaprolactone with high ratio of CLP

[0105] Direct mixing of more than 2 wt% CLP into PCL solution was not possible. The dried collagen-like protein, namely CLP was fluffy and had low density. It was not able to be wetted by either chloroform or acetone. A modified procedure was utilized to introduce more than 2 wt% CLP into polymer solution. 5 wt% of CLP was weighed and then low amounts of polyethylene glycol) methy ether (mPEG, Mn = 550, commercial product from Sigma-Aldrich) was added dropwise to the CLP in a glass vial and was vigorously mixed on a vortex for about 1 min. The CLP and mPEG was then transferred to 15 wt% RESOMER C212 solution in chloroform and acetone (3:1 by volume). The solution was transferred to an electrospinner for electrospinning using the same electospinning condition as Example 1 . Using the same mixing method, a RESOMER C212 solution (15 wt%) with 10 wt% CLP was also prepared and electrospun. Morphology analysis on SEM indicated fibrous structure.

[0106] Example 4. Electrospinning of poly(D,L-lactide-PEG) copolymer

[0107] 4 g of RESOMER LRP t7046 (poly(D,L-lactide)-b-PEG-b-poly(D,L-lactide), Evonik commercial product) was dissolved in 30 mL of chloroform and 10 mL of acetone to make 10 wt% polymer solution. The solution was loaded onto an electrospinner (NS LAB, Elmarco s.r.o., Czech Republic). The solution was electrsopun at 30 kV with a distant between collector and wire collecting electrode of 14 cm and carriage speed of 50 mm / s. After electrospinning the scaffold was peeled off for further characterization.

[0108] Example 5. Electrospinning of poly(D,L-lactide-PEG) copolymer with CLP

[0109] 20 wt% of CLP was weighed and added to a glass vial. Then a low amount of polyethylene glycol) methy ether (mPEG, Mn = 550, commercial product from Sigma-Aldrich) was added dropwise in the glass vial and vigorously mixed on a vortex for about 1 min, then transferred the CLP and mPEG to 10 wt% RESOMER LRP t7046 solution in chloroform and acetone (3:1 by volume). The solution was transferred to an electrospinner for electrospinning using the same electospinning conditions as in Example 4.

[0110] Example 6. Solution preparation with hexafluoroisopropanol

[0111] RESOMER® C212 was dissolved in hexafluoroisopropanol (HFIP) at 12 w / v%. However, unlike animal derived collagen, CLP is insoluble in HFIP, which is soluble in water at up to 10 wt%. 10 wt% CLP was weighed and dissolved in water. Then the CLP water solution was transferred to RESOMER® C212 solution in HFIP. 202400117 13

[0112] When less than 10% water was added to HFIP the solution remained clear. However, after extended mixing time the solution become greenish and was not used for electrospinning. When more water was added to HFIP the solution became cloudy, and after extended mixing time the solution become greenish and was not used for electrospinning.

[0113] The tensile test on electrospun PCL, and PCL with CLP scaffolds were performed on a dynamic mechanical analyzer (DMA, Q800, TA Instruments) at 22°C. The electrospun PCL and CLP scaffolds were cut into strip and mounted on the DMA tensile clamp and tested at a rate of 1 mm / min to 25 mm.

[0114] The analysis of the chemical composition of the CLP loaded electrospun fibers was performed on Fourier transform infrared spectroscopy (FT-IR, Thermo Scientific Nicolet iS50) in an ATR mode, with a scanning range from 4000 to 550 cm-1at a speed of 4 cm Vs and with an average of 128 measurements in the final spectrum. Wavenumbers of 1560 cm-1and 1655 cm-1associated with the amide groups from collagen were used to determine whether there was CLP in the sample.

[0115] Results

[0116] FIG. 1 depicts mechanical properties of electrospun scaffolds of polycaprolactone (PCL C212) (dotted lines in the graph) and PCL C212 with 10% CLP (solid lines in the graph). All scaffolds show high strain at break in a range of 120-170%, and tensile strength in a range of 1 .5 - 2.0 MPa. No dramatic difference is observed among these scaffolds, which indicates that CLP doesn’t negatively reduce the overall mechanical properties of electrospun scaffolds compared to electrospun neat PCL scaffolds.

[0117] FIG. 2 depicts the Fourier transform infrared (FT-IR) spectra of neat CLP in its powder form, electrospun scaffold of polycaprolactone (PCL C212) and the electrospun scaffold of PCL C212 with 10% CLP. The CLP presents two unique peaks at wavenumber of 1560 cm-1and 1655 cm-1corresponding to its amide groups. At these locations there is no peak on the PCL spectrum as seen from the dotted line in the graph. As arrows indicated for the solid line in the graph, there are two peaks at 1560 cm-1and 1655 cm-1showing in the spectra for the electrospun scaffold of PCL 212 with 10% CLP. Then these two peaks confirmed the presence of CLP in the electrospun scaffold.

[0118] FIG. 3 depicts the Fourier transform infrared (FT-IR) spectra of neat CLP in its powder form, RESOMER® LRP t7046 pellets and the dried casting film of RESOMER® LRP t7046 with 10wt% CLP and low molecular weight mPEG. The CLP presents two unique peaks at wavenumber of 1560 cm-1and 1655 cm-1corresponding its amide groups. At this location there are no peaks on the RESOMER® LRP t7046 spectrum. As arrows indicated for the solid line in the graph, there are two peaks at 1560 cm-1and 1655 cm-1showing in the spectra for the RESOMER® LRP t7046 with 10 wt% CLP. Then these two peaks confirmed the presence of CLP in the casting film.

[0119] FIG. 4 depicts morphology of electrospun scaffolds of polycaprolactone (PCL C212). The scaffold is made of fibers with micro-scale diameter.

[0120] FIG. 5 A depicts morphology of electrospun scaffolds of polycaprolactone (PCL C212) with 2 wt% CLP. The CLP were added to PCL C212 solution in a mixed solvent of chloroform and acetone. This was the 202400117 14 maximum amount of CLP that could be directly dispersed into the solution. The electrospun scaffold is made of largely microfibers. At different areas micro-flakes of CLP were visible.

[0121] FIG. 5 B depicts morphology of electrospun scaffolds of polycaprolactone (PCL C212) with 2 wt% CLP at different area. The CLP were added to PCL C212 solution in a mixed solvent of chloroform and acetone. This was the maximum amount of CLP that could be directly dispersed into the solution. Large CLP flake was visible on the scaffold.

[0122] FIG. 6 depicts morphology of electrospun scaffolds of polycaprolactone (PCL C212) with 5 wt% CLP and low molecular weight mPEG. With the assistance of mPEG, the CLP was uniformly dispersed in the solution as microfibrils. These CLP microfibrils were well distributed into the PCL and the overall scaffolds had no visible CLP flakes.

[0123] FIG. 7 depicts morphology of electrospun scaffolds of polycaprolactone (PCL C212) with 10 wt% CLP and low molecular weight mPEG. With the assistance of mPEG, the CLP was uniformly dispersed in the solution as microfibrils. These CLP microfibrils were well distributed into the PCL and the overall scaffolds had no visible CLP flakes.

[0124] FIG. 8 depicts morphology of electrospun scaffolds of RESOMER LRP t7046 (poly(D,L-lactide)-b-PEG- b-poly(D.L-lactide) with 10 wt% CLP and low molecular weight mPEG. With the assistance of mPEG, the CLP was uniformly dispersed in the solution as microfibrils. These CLP microfibrils were well distributed into the PCL and the overall scaffolds had no visible CLP flakes.

[0125] FIG. 9 depicts morphology of electrospun scaffolds of RESOMER LRP t7046 (poly(D,L-lactide)-b-PEG- b-poly(D,L-lactide) with 20 wt% CLP and low molecular weight mPEG. After CLP was dispersed into the RESOMER LRP t7046 solution with the assistance of low molecular weight mPEG, the suspension stability without stirring was evaluated. The solution was left without stirring at room temperature for overnight then the solution was fed into the electrospinner for electrospinning. The scaffolds were largely made of microfibers, and a few CLP flakes were visible.

[0126] Item 1 is an electrospun hybrid scaffold comprising a collagen-like protein, at least one synthetic resorbable polymer, and optionally comprising polyethylene glycol.

[0127] Item 2 is the electrospun hybrid scaffold of item 1 , wherein the collagen-like protein is produced by fermentation.

[0128] Item 3 is the electrospun hybrid scaffold of items 1 or 2, wherein the at least one synthetic resorbable polymer is selected from poly(caprolactone), polylactide, poly(trimethylene carbonate), poly(lactide-co- caprolactone), poly(lactide-co-trimethylene carbonate), polylactide-b-polyethylene glycol, with molecular weight in a range of 10,000 to 500,000 g / mol, and mixtures thereof. 202400117 15

[0129] Item 4 is a process of preparing the electrospun hybrid scaffold of any of claims 1 to 3, comprising the steps: (a) dissolving the resorbable polymer in a solvent forming a solution, (b) dispersing collagen-like protein in the solvent forming a suspension solution, (c) loading the solution onto an electrospinner, and (d) electrospinning the solution until a scaffold is formed.

Claims

202400117 16CLAIMS:1 . An electrospun hybrid scaffold comprising a collagen-like protein, at least one synthetic resorbable polymer, and optionally comprising polyethylene glycol.

2. The electrospun hybrid scaffold of claim 1 , wherein the collagen-like protein is produced by fermentation.

3. The electrospun hybrid scaffold of claims 1 or 2, wherein the at least one synthetic resorbable polymer is selected from poly(caprolactone), polylactide, poly(trimethylene carbonate), poly(lactide- co-caprolactone), poly(lactide-co-trimethylene carbonate), polylactide-b-polyethylene glycol, with molecular weight in a range of 10,000 to 500,000 g / mol, and mixtures thereof.

4. A process of preparing the electrospun hybrid scaffold of any of claims 1 to 3, comprising the steps: (a) dissolving the resorbable polymer in a solvent forming a solution, (b) dispersing collagen-like protein in the solvent forming a suspension solution, (c) loading the solution onto an electrospinner, and (d) electrospinning the solution until a scaffold is formed.

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