Drug-loaded 3-dimensional scaffold for accelerating bone repair

Abstract

The invention relates to a 3D tissue scaffold containing the active pharmaceutical ingredients, memantine hydrochloride and donepezil hydrochloride, and a method of production of this tissue scaffold. Said scaffold accelerates bone repair thanks to the active pharmaceutical ingredients contained therein and a 3D porous structure.

Description

[0001] DRUG-LOADED 3-DIMENSIONAL SCAFFOLD FOR ACCELERATING BONE REPAIR

[0002] Field of the Invention

[0003] The present invention relates to a three-dimensional tissue scaffold containing the active pharmaceutical ingredients, memantine hydrochloride and donepezil hydrochloride, and a method of production of said tissue scaffold.

[0004] State of the Art of the Invention (Prior Art)

[0005] Disruption of bone integrity, bone fractures and damage, which are defined as a clinical condition that may occur after a physical injury or spontaneously and require urgent intervention, are among the injuries caused by trauma. Healing of bone tissue takes place between 4 and 6 weeks and may require physical therapy in the long term. Accelerating the tissue healing and enabling patients to return to their daily life early are the basic rationales of the treatment. Studies on effective tissue engineering approaches and new options on mechanisms and pathways for the treatment of bone fractures are ongoing. For this purpose, there are many approaches such as bone grafts, growth factors, and the application of osteoconductive scaffolds for the treatment of non-healing fractures.

[0006] The most common method currently used in the treatment of bone fractures is autogenous grafts. However, various problems such as donor site morbidity and limited resources in the supply of autogenous grafts limit their use in treatment. For this reason, research continues in tissue engineering.

[0007] Nanofiber technology is one of the applications of tissue engineering. The application of nanofiber mats prepared conventionally by electrospinning method in bone defects is limited due to their formal structure. Due to the preparation methods, the thickness of the nanofiber structures is limited. Therefore, it becomes difficult to obtain the desired matrix structure in clinical situations such as bone defects where a 3-dimensional scaffold structure is needed. For this reason, 3-dimensional nanofiber scaffolds obtained by additional methods are needed. Studies investigating the effectiveness of 3 -dimensional nanofiber scaffolds in the treatment of bone injury, which combine nanotechnology-based drug delivery systems and tissue engineering applications, are ongoing. Among these methods, the thermally induced selfagglomeration (TISA) method stands out compared to other methods due to its similarity to the natural extracellular matrix and ease of production, and the availability of equipment. Thanks to the TISA method, which entered the literature in 2015, 2-dimensional nanofiber mats can be converted into 3 -dimensional nanofiber scaffolds. There are some studies conducted with this method. In some of these studies, different polymer mixtures were investigated and their effectiveness was examined. In a few of them, scaffolds loaded with active substances have been developed.

[0008] The most important deficiency in the production of 3 -dimensional scaffolds, which have been studied before, is that they are far from the nanofiber structure. The nanofiber structure is the most similar structure to the natural extracellular matrix, so it provides an advantage in tissue engineering applications. In other methods that provide 3-dimensional scaffold production from nanofiber membranes, there are disadvantages such as the use of extra binding agent and the provision of equipment is difficult and expensive. For this reason, 3-dimensional scaffolds produced by the TISA method come to the fore. This method, which enables the production of 3-dimensional scaffolds from nanofiber membranes, entered the literature in 2015, but drug-loaded scaffolds produced by this method are limited in the literature.

[0009] In the publication of Xu et al., in 2015, liquid nitrogen and a mortar / pestle were used to obtain small membrane particles from nanofiber membranes. This method is both time-consuming and manpower-intensive. The last publication on TISA was published in 2022. In this study, a hand blender was used to fragment the membranes in size. This method is fast and does not require power. However, in this study, no drug loading was performed, and the effectiveness of polymer mixtures was evaluated.

[0010] To date, studies have been carried out on the production of drug-loaded scaffolds using the TISA method (Hu et al., 2018; Miszuk et al., 2021), however, the efficacy of these drug molecules loaded on scaffolds on bone has already been proven in previous scientific research.

[0011] Previously, no studies were conducted on a scaffold containing the active ingredients memantine hydrochloride (memantine HC1) and donepezil hydrochloride (donepezil HC1) for bone regeneration. A scaffold study containing the active ingredient donepezil HC1 alone has been conducted earlier (Poormoghadam et al., 2021), but additional chemicals have been used in the method used here, the resulting scaffold is free of a nanofiber structure. Therefore, it is a disadvantageous method in terms of similarity to the natural extracellular matrix.

[0012] Summary and Objectives of the Invention

[0013] An object of the present invention is to obtain a three-dimensional alternative tissue scaffold with biocompatibility that accelerates the healing of bone fractures and also provides ease of application by being formed easily.

[0014] The present invention relates to a three-dimensional (3D) tissue scaffold containing the active ingredients, memantine hydrochloride and the active ingredient donepezil hydrochloride, the efficacy of which on bone has not previously been demonstrated by experimental studies.

[0015] The invention includes a three-dimensional tissue scaffold obtained by a thermally induced self-agglomeration (TISA) method, containing the active ingredients, memantine hydrochloride and donepezil hydrochloride, which have different indications but have been found to have positive effects on bone fractures (Figures 1-6).

[0016] Definition of the Drawings of the Invention

[0017] Fig- 1 : Micro-CT images of the three-dimensional tissue scaffold loaded with an active pharmaceutical ingredient. A) a horizontal cross-section B) a vertical cross-section C) an entire scaffold.

[0018] Fig- 2 : A 50x scanning electron microscope image of a three-dimensional tissue scaffold loaded with an active pharmaceutical ingredient.

[0019] Fig- 3 : A lOOx scanning electron microscope image of a three-dimensional tissue scaffold loaded with an active pharmaceutical ingredient.

[0020] Fig. 4 : A 500x scanning electron microscope image of a three-dimensional tissue scaffold loaded with an active pharmaceutical ingredient. Fig- 5 : A lOOOx scanning electron microscope image of a three-dimensional tissue scaffold loaded with an active pharmaceutical ingredient.

[0021] Fig- 6 : A 2500x scanning electron microscope image of a three-dimensional tissue scaffold loaded with an active pharmaceutical ingredient.

[0022] Fig- 7 : Alizarin red intensity after 21 days of osteogenic induction. The data are presented as mean ± SD (n = 3) (D: Donepezil (10 pm), M:Memantine (10 pm), DM: Donepezil+ Memantine (10 pm)).

[0023] Detailed Description of the Invention

[0024] The three-dimensional tissue scaffold of the invention contains the active pharmaceutical ingredients, memantine hydrochloride and donepezil hydrochloride, which have not been combined before.

[0025] Thanks to the mechanisms of action of the combined use of active pharmaceutical ingredients, the healing process of bone fractures is accelerated by providing a bidirectional, synergistic effect with a decrease in osteoclastic activity and an increase in osteoblastic activity.

[0026] Within the scope of the invention, Alizarin Red staining was performed on a human osteosarcoma cell line (SAOS-2 cells) and the results were evaluated both in order to demonstrate the aforementioned effect of the memantine HC1 active pharmaceutical ingredient alone, which has not previously been shown to be effective on bone by experimental studies, and in order to demonstrate the above-mentioned bidirectional and synergistic effect of the 3-dimensional scaffold loaded with memantine HC1 and donepezil HC1 together. The details of this study are described below.

[0027] SAOS-2 cells were plated in flasks of 75 cm2using DMEM growth medium containing a high concentration of glucose. DMEM growth medium was prepared to contain 1% Pen-strep, 100 nM dexamethasone, 200 pM ascorbic acid, 10 mM B-glycerophosphate. Cells were incubated at 37°C, 5% CO2 ambient conditions. In order to evaluate osteogenic differentiation, cell suspension was plated in 24-well plates as 2.5xl04cells / ml. Drug solutions containing Memantine (MEM-10 pM), Donepezil (DON-10 pM), Memantine / Donepezil (MEM / DON-IO pM) were added to 24-well plates (n=3). The cells were fed with fresh growth medium every other day. Samples taken on days 7, 14 and 21 were evaluated by staining with Alizarin Red without removing the surface-bound cells to determine stem cell differentiation. Growth medium was used as control.

[0028] At the end of the time points, the amount of calcium in the plates was analyzed by Alizarin red staining. After the growth medium was removed from the wells, the cells were washed with PBS buffer at pH 7.4 and treated with a formalin solution (10%) sufficient to coat the cell surface for 30 min. Then the formalin solution was removed and the cells were washed with distilled water. The washed cells were treated with Alizarin red solution for 45 minutes in the dark at room temperature. After staining, the Alizarin red solution was removed and the cells were washed with distilled water. The intensity of the obtained red color was measured with Image J software (n=3).

[0029] As a result of the analyzes carried out to examine the effects of active pharmaceutical ingredients on the differentiation of bone cells, the presence of Alizarin red and an increase in the density of red color were detected in the images taken with a light microscopy in the wells to which Memantine (10 pm), Donepezil (10 pm) and Memantine / Donepezil (10 pm) were added. Osteogenic differentiation and calcium mineralization were found to be higher in these groups compared to the control group (p<0.05). The maximum red density was obtained at the end of the 21st day. The results obtained are presented in the graph (Figure 7) and support the accelerating effect of the active pharmaceutical ingredients on bone fractures by providing a bidirectional effect with a decrease in osteoclastic activity and an increase in osteoblastic activity.

[0030] The three-dimensional scaffold structure to which the active pharmaceutical ingredients are loaded provides cell growth and a mechanical support. The last structure of the invention is a porous, flexible 3D scaffold structure that can be easily shaped according to the bone area to be used. SEM and micro CT images of the tissue scaffold obtained within the scope of the invention are shown in Figure 1- Figure 6. The release of drugs from the porous structure is controlled and slow. In this way, it is an ideal structure for a condition that requires a longterm drug release, such as bone regeneration. In a preferred embodiment of the invention, the 3-dimensional (3D) tissue scaffolds containing memantine hydrochloride and donepezil hydrochloride as active pharmaceutical ingredients are obtained by the thermally induced self-agglomeration (TISA) method. Compared to the scaffolds obtained by other three-dimensional scaffold production methods, the scaffolds obtained by the TISA method stand out with their similarity to the natural extracellular matrix and their flexible structure that provides high porosity. Scaffolds loaded with the active pharmaceutical ingredients, memantine hydrochloride and donepezil hydrochloride, prepared by thermally induced self-agglomeration (TISA) method provide ease of application thanks to their easy shaping during a surgical application and positively affect bone regeneration with long-term release of the active pharmaceutical ingredients. After the surgical application, the biocompatible scaffolds degrade on their own and allow the bone to take the place of the scaffold thanks to their porous structure, thus eliminating the need for a secondary surgical operation. At the same time, thanks to their biocompatible structure, they do not cause any immune response.

[0031] The method of producing a three-dimensional tissue scaffold containing memantine hydrochloride and donepezil hydrochloride as active pharmaceutical ingredients comprises the steps of firstly preparing a polymer solution containing the active pharmaceutical ingredients, memantine HC1 and donepezil HC1, then producing a nanofiber membrane by subjecting the prepared solution to an electrospinning method, obtaining a nanofiber membrane suspension in a solvent by physically reducing the size of the nanofiber membrane obtained, obtaining three-dimensional scaffolds loaded with memantine HC1 and donepezil HC1 by thermally induced self-agglomeration (TISA) method from the nanofiber membrane suspension obtained.

[0032] In a preferred embodiment of the invention, polycaprolactone (PCL) is used as a polymer. Again, preferably a concentration of 14% is used for polycaprolactone. The reason why PCL is preferred is that its melting point is low and there is no need to use extra binding agents by acting as a binding agent.

[0033] In alternative embodiments of the invention, other polymers may be used alone, or a combination of PCL with other polymers may also be used. Synthetic polymers that can be used as alternatives may be: polylactide acid, cellulose acetate, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polyurethane, poly(vinyl pyrrolidone), or poly(ethyleneoxide). Natural polymers that can be used as an alternative may be: Chitosan, collagen, hyaluronic acid, elastin, alginate, fibrinogen, cellulose, or gelatin.

[0034] In a preferred embodiment of the invention, considering the active pharmaceutical ingredient loading capacity of nanofibers, the active pharmaceutical ingredient, namely memantine HC1 and donepezil HC1, is loaded in the range of 5-15% by weight compared to the polymer.

[0035] For the production of nanofiber membranes by electrospinning method, firstly all solid materials, i.e., active pharmaceutical ingredients and polymer, preferably acetic acid: formic acid: dimethylformamide (AA: FA: DMF) are taken into a solvent system, and a polymer solution containing the active pharmaceutical ingredient is prepared. The period for stirring of the solution varies depending on the polymer and solvent used. In a preferred embodiment of the invention, stirring is carried out between 8 and 16 hours, depending on the use of polycaprolactone as a polymer and AA:FA:DMF as a solvent. The preferred period here is 12 hours. The solution is preferably stirred on a magnetic stirrer for 12 hours at a temperature in the range of 22-26°C. In alternative embodiments of the invention, dichloromethane: dimethylformamide; chloroform: dimethylformamide; only chloroform; acetic acid: formic acid solutions can be used as solvent systems.

[0036] The solution prepared is taken into a 5 ml syringe and then connected to the electrospinning device with a needle and pump system. In a preferred embodiment of the invention, the distance between the collector and the needle is set at 14 cm, and the rotation speed is set at 200 rpm. However, in alternative embodiments, the distance between the collector and the needle can vary between 10-20 cm. Again, in alternative embodiments, the voltage can vary between 15-25 kV and the flow rate can vary between 0.3-1.5 ml / h. In a preferred embodiment of the invention, the voltage is in the range of 20-23 kV and flow rate is in the range of 0.5-1.0 ml / h. These values are preferred as they provide the best results in terms of the morphology of the nanofibers obtained and the stability of the taylor cone.

[0037] In order to obtain three-dimensional tissue scaffolds, firstly, the drug-loaded nanofiber membranes are produced by the electrospinning method, and then the nanofiber membranes produced by the electrospinning method are fragmented and suspended to obtain three- dimensional scaffolds by the TISA method. Within the scope of the invention, nanofiber membranes produced by the electrospinning method are physically fragmented to preferably about 0.5*0.5 cm2, preferably by means of scissors. In alternative embodiments, the sizes between 0.25 - 1.0 cm2can also be used.

[0038] Then, the fragmented nanofiber membranes are preferably taken into a deionized water: ethanol mixture and ground using a blender to obtain a nanofiber membrane suspension. Preferably, a deionized waterethanol mixture at a ratio of 95:5 w / w is used. In alternative embodiments of the invention, the alternative ratios for deionized water: ethanol mixture may be 80:20 or 60:40. In addition, gelatin solution can be added to this solution. The period for blender can vary from 2 to 10 minutes, but in the preferred embodiment of the invention, the grinding process is carried out for 10 minutes. In alternative embodiments, grinding is carried out by liquid nitrogen and a mortar instead of a blender, or grinding is carried out by a homogenizer. In order to save time and power in the invention, grinding by a blender is preferred.

[0039] Preferably a glass bottle containing homogeneous nanofiber membrane suspension is taken into a water bath and subjected to heat treatment based on the TISA method. After the heat treatment, the fibers clump themselves, and a three-dimensional scaffold structure is obtained. Here, the temperature for the heat treatment is decided based on the melting point of the polymer used. The preferred temperature should be below the melting point of the polymer. In fact, it should preferably be below 5°C. This value will not melt the polymer, but will soften it. Therefore, based on the polymer preferred in the invention and the melting temperature of this polymer, the glass bottle containing the homogeneous suspension is subjected to heat treatment between 54-57°C, preferably in a water bath of 55°C.

[0040] In a preferred embodiment of the invention, after the heat treatment, which continues for about 80 to 150 seconds, the glass bottle is taken into an ice bath to prevent further shrinkage. The period for the heat treatment herein is affected by factors such as the amount of nanofiber used, the thickness and width of the glass bottle used. Glass bottles can be selected according to the size and thickness of the scaffold to be produced. As the amount of nanofiber material increases, the exposure period of the heat treatment also increases. Finally, after the heat treatment, the structures which have become fibers, that is, the three- dimensional scaffolds loaded with the active pharmaceutical ingredients obtained, memantine hydrochloride and donepezil hydrochloride, are dried. In a preferred embodiment of the invention, the three-dimensional scaffolds are dried by a lyophilization process.

[0041] The three-dimensional scaffold containing the active pharmaceutical ingredients, memantine HC1 and donepezil HC1, which is produced by the TISA method, provides a drug release profile with slow and controlled release. In the meantime, the polymeric structure provides a mechanical support and preferably the scaffold remains in the environment during the healing period of the bone tissue, thanks to the polymeric structure with a slow degradation rate such as polycaprolactone. Thanks to the porous structure, the cells can reproduce in the scaffold and the scaffold begins to degrade at the same rate as the bone tissue renews itself.

Claims

CLAIMS1. A three-dimensional tissue scaffold, characterized in that it comprises memantine hydrochloride and donepezil hydrochloride as active pharmaceutical ingredients.

2. A tissue scaffold according to Claim 1, characterized in that it comprises active pharmaceutical ingredients in a range of 5-15% by weight.

3. A production method for tissue scaffold according to Claim 1, characterized in that it comprises the process steps of: i. preparing a polymer solution containing the active pharmaceutical ingredients, memantine hydrochloride and donepezil hydrochloride, ii. producing a nanofiber membrane by subjecting the prepared solution to an electrospinning method, iii. obtaining a nanofiber membrane suspension in a solvent by fragmenting the size of the nanofiber membranes obtained, iv. obtaining three-dimensional scaffolds loaded with memantine hydrochloride and donepezil hydrochloride by thermally induced self-agglomeration (TISA) method from the obtained nanofiber membrane suspension.

4. The method according to Claim 3, characterized in that the solution prepared in step i contains an active pharmaceutical ingredient in a range of 5-15% by weight based on the polymer.

5. The method according to Claim 3, characterized in that the polymer solution prepared in step i contains polycaprolactone.

6. The method according to Claim 3, characterized in that the polymer solution is prepared by a mixture of acetic acid: formic acid: dimethylformamide solvent.

7. The method according to Claim 5, characterized in that it comprises polycaprolactone at a concentration of 14%.

8. The method according to Claim 3, characterized in that in step ii, the distance between the collector and the needle during the electrospinning method is in a range of 10 to 20 cm, the voltage is in a range of 15 to 25 kV, and the flow rate is in a range of 0.3 to 1.5 ml / h.

9. The method according to Claim 8, characterized in that the distance between the collector and the needle is 14 cm, the voltage is in a range of 20 to 23 kV, and the flow rate is in a range of 0.5 to 1.0 ml / h.

10. The method according to Claim 3, characterized in that in step iii, the nanofiber membranes are fragmented so as to be 0.5*0.5 cm2in size.

11. The method according to Claim 3, characterized in that in step iv, the nanofiber membranes are subjected to thermal heat in a range of 54-57°C.

12. The method according to Claim 11, characterized in that the nanofiber membranes are subjected to thermal heat of 55°C.

13. The method according to Claim 3, characterized in that it also comprises a step of drying the three-dimensional scaffolds loaded with memantine HC1 and donepezil HC1.

14. The method according to Claim 13, characterized in that the three-dimensional scaffolds are dried by a lyophilization process.

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