SILICA-BASED NANOPARTICLE INFUSED POLYMERIC MICROFIBERS ENHANCE MINERAL DEPOSITION

This application claims the benefit of U.S. Provisional Application No. 63/285,008, filed December 1, 2021, the contents of which is hereby incorporated by reference.

Throughout this application, certain publications are referenced in parentheses. Full citations for these publications may be found immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention relates.

Background of the Invention

Bone fractures remain a global burden on public health. Over 2 million bone graft procedures are performed annually (2nd most frequent tissue transplantation) in the world (J.W. Lee et. al. 2016) (Figure 1). Bone fractures usually result loss of mobility, impaired quality of life and increased healthcare costs. The most common treatments for patients suffering from slow or incomplete bone healing are autologous or allogenic bone grafts. Autologous bone grafts require harvesting large bone volumes from the patient, while allogenic bone grafts carry a high risk of disease transmission (Suchomel et al. 2004). These treatments have inherent limitations including a lack of graft availability, donor site morbidity and disease transmission. Therefore, there is a need for a treatment approach that is more biomimetic and improves material tissue integration.

Tissue-engineered bone grafts using bioactive glasses are a promising alternative because the glasses strongly bind to bone (Gao et al. 2014). Composite materials such as Bioactive Glass nanofibers (Figure 3A), Particle filled/coated Polymer, Bioactive Glass -Hydrogel, Particle filled/coated scaffold, BG nanoparticle-Polymer scaffold, are manufactured to improve mechanical properties and modulate ion release. Traditionally, bioactive glass is fabricated using melt-quenching technique at extreme temperatures and it dissolves in physiological fluid and bonds to native bone. Recently, a ternary system (SiCf-CaO- P2O5) fabricated via Sol-gel processing is developed (Figure 3B) . The Fibrous structure mimic native collagen matrix in bone and osteoblast differentiation can be enhanced using bioactive glass nanoparticles (Figure 4 and Figure 5). BG nanocomposite platform may provide 3D structure template to guide bone healing. However, many bio-glasses are brittle and unable to share load with the bone. Summary of the Invention

According to the present invention, a composition of a nano-bioactive glass (nBG), wherein the nBG comprises SiCh, CaO and P2O5 may be provided, and wherein SiChhas less than 60% mole fraction.

According to the present invention, a composition of a nano-bioactive glass (nBG) may be provided, wherein the nBG comprises S i O 2- CaO and P2O5, and wherein CaO has at least 40% mole fraction.

According to the present invention, a composition of a nano-bioactive glass (nBG) may be provided, wherein the nBG comprises SiO2, CaO and P2O5, and wherein P2O5 has at least 5% mole fraction.

According to the present invention, a composition in the manufacture of a medicament for treating bone fractures on a subject may be provided.

According to the present invention, a method of producing a nano-bioactive glass (nBG) may comprise:

• mixing calcium nitrate solution with Tetraethoxysilane (TEOS) solution;

• adding dropwise of a mixture from step a) to an ammonium dibasic phosphate solution;

• stirring the mixture from step b) for 30-60 hours and permitting the reaction mixture to rest for 20- 30 hours for further precipitation;

• separating a precipitate from step c);

• freezing the precipitate from step d) at -60 to -40 °C for 20-30 hours; and

• heating a sample of the precipitate from step e) at 650 to 800 °C for 2-5 hours and cool down the heated precipitate for 8-14 hours.

A method of stimulating bone regeneration may be performed by treating a subject with the composition disclosed in the present invention.

A method of repairing bone fractures may be performed by treating a subject with the composition disclosed in the present invention.

Brief Description of the Figures

Figure 1. An X-ray showing Bone fracture.

Figure 2. Bone Microarchitecture - (65 wt.% Mineral Phase + 25 wt.% Organic Phase + 10 wt. % Water).

SiO2 enhances osteoblast differentiation and mineralization in vitro. Figure 3A. 45S5 Bioactive Glass (Biogalss®), containing 46.1 mol.% SiCL, 24.4 mol.%Na2O, 26.9 mol. % CaO and 2.6 mol.% P2O5.

Figure 3B. Ternary system (SiO2-CaO-P2O5) via Sol-gel processing.

Figure 4. BG-PLGA Microspheres

Figure 5. BG Gel-PLGA Microfibers,

Figure 6. nBG (55% SiC>2:40% CaO:5% P2O5, all in mol%) Fabrication.

Figure 7. nBG Fiber Fabrication.

Figure 8. Bioactivity Test of nBG and nBG Fiber (37°C, 5% CO2).

Figure 9A. Morphology of nBG.

Figure 9B. FTIR of nBG.

Figure 9C. XDR of nBG.

Figure 10. EDS of nBG.

Figure 11 A. SEM of PLGA (5: 1) PCL Fibers.

Figure 11 B. FTIR and XDR of PLGA (5: 1) PCL Fibers.

Figure 12 A. SEM of nBG Fibers.

Figure 12 B. FTIR and XDR of nBG Fibers.

Figure 13. Bioactivity Test of Plain DMEM, PLGA (5: 1) PCL, nBG, and nBG Fibers.

Figure 14 A. FTIR of nBG after 14 days.

Figure 14 B. Crystallinity change of nBG after 14 days.

Figure 15 A. Bioactivity Test of PLGA (5: 1) PCL.

Figure 15 B. Bioactivity Test of nBG Fibers.

Figure 16. Ion Release (Mechanism of Mineralization, ICP-EOS, n=3/group/timepoint).

Figure 17 A. Cell Viability at Day 1 and Day 7.

Figure 17 B. dsDNA assay for nBG. Detailed Description of the Invention

According to the present invention, a composition of a nano-bioactive glass (nBG) may be provided, wherein the nBG comprises SiCh, CaO and P2O5, and wherein SiO2has less than 60% mole fraction.

In some embodiments, the mole fraction of CaO is at least 40%.

In some embodiments, the mole fraction of P2O5 is at least 5%.

According to the present invention, a composition of a nano-bioactive glass (nBG), wherein the nBG comprises SiO2, CaO and P2O5, wherein CaO has at least 40% mole fraction.

In some embodiments, the mole fraction of SiO2is less than 60%.

In some embodiments, the mole fraction of P2O5 is at least 5%.

According to the present invention, a composition of a nano -bioactive glass (nBG), wherein the nBG comprises SiO2, CaO and P2O5, wherein P2O5has at least 5% mole fraction.

In some embodiments, the mole fraction of SiO2is less than 60%.

In some embodiments, the mole fraction of CaO is at least 40%.

In some embodiments, the mole fraction of SiO2is 30%-60%.

In some embodiments, the mole fraction of SiO2is 40%-55%.

In some embodiments, the mole fraction of CaO is 37%-57%.

In some embodiments, the mole fraction of P2O5 is 5%-10%.

In some embodiments, the mole fraction of SiO2is 55%.

In some embodiments, the mole fraction of CaO is 40%. According to the present invention, a polymer-bioactive glass fiber composite can be provided. The porous structure of the composite allows for controlled release of silica and calcium phosphate ions. The polymer component allows for enhanced flexibility. The composite is biocompatible and enhances bioactivity via mineralization. Such composite may promote cell attachment, and therefore promote bone regeneration, and have dental and orthopedic applications.

The microarchitecture of bone indicates that bone is a biological composite material. It is composed mainly type I collagen fibers and calcium phosphate/hydroxyapatite crystals. Mineralized collagen is aggregated into small fibrils, which further combine to form fibers a few microns in diameter and several mm long. For trabecular bone, the fibers are randomly laid out. For the cortical bone, fibers are wrapped around a hollow core. Since the advent of tissue engineering, many strategies have an aim to mimic the bone microarchitecture in order to enhance and facilitate tissue regeneration. The Hydroxyapatite (HA) nanocrystals are principally arranged with their c-axes parallel to the collagen fibrils and organized in a periodic, staggered arrangement along the fibrils.

This biomineralization process of bone proceeds via a matrix vesicle-mediated mechanism, in which the matrix vesicles are secreted by the outer membranes of bone-forming osteoblasts. The enzyme alkaline phosphatase present in the matrix vesicles cleaves the phosphate esters and acts as the foci for calcium and phosphate deposition (Wei et al. 2007). The self-assembly process of collagen molecules defines the framework and spatial constraints for HA nucleation and propagation

Bioactive glass is a synthetic material which is able to bond to bone via the formation of an apatite layer on their surface (Rezwan et al. 2006). This material was first developed in the 1970s. The composition of the first bioglass contained a main silica network which contained CaO and P2O5, the natural constituents of bone and silica enhancing osteoblast differentiation and mineralization (Yunos et al. 2013, Kumar et al. 2019). Traditionally it is fabricated using a high temperature melt-quenching technique. However, with the improvement of technology, now bioactive glass can be fabricated under ambient conditions, typically using a ternary system (Magazzini et al. 2002). Once bioglass immerses in body fluid, ion exchange reaction occurs allowing release of Ca and PO4 groups from bulk of the material to the surface (Roether JA et al. 2002). In addition silanol bonds are formed in the silica network and produces a rich silica gel layer which allows sites of nucleation to form and crystallization happens when other ions in solution such as fluoride or carbonate groups binds with the amorphous apatite to improve stability (Robert Kane et al. 2013). In some embodiments, the mole fraction of P2O5 is 5%.

In some embodiments, composition of nano-bioactive glass (nBG) further comprising a polymer blend of at least two polymers.

In some embodiments, the polymer blend consists of two polymers,

In some embodiments, the polymer blend consists of three polymers.

In some embodiments, the polymer amongst the polymers comprises aliphatic polyesters, poly( amino acids), copoly (ether-esters), polyalkylenes oxalates, polyamides, tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoe-sters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, polydiglycolates, polylactic-co-glycolic acid (PLGA) and poly(8-caprolactone) (PCL), or any combination thereof.

In some embodiments, the polymer comprises polylactic-co-glycolic acid (PLGA) and poly(s-caprolactonc) (PCL).

In some embodiments, the polymer blend consists of polylactic-co-glycolic acid (PLGA) and poly(s- caprolactone) (PCL).

In some embodiments, the mass ratio between PLGA and PCL is 1: 1 to 10: 1.

In some embodiments, the mass ratio between PLGA and PCL is 3: 1 to 7: 1.

In some embodiments, the mass ratio between PLGA and PCL is 5 : 1.

In some embodiments, the polymer amongst the polymers is a biocompatible polymer.

In some embodiments, the biocompatible polymer has a diameter between 1.50-3.00 pm. In some embodiments, the composition of nano-bioactive glass (nBG) further comprising a therapeutic agent.

In some embodiments, the therapeutic agent is selected from the group consisting of antibiotics, antivirals, adhesion preventative s, contraceptives, and analgesics.

In some embodiments, the composition of nano-bioactive glass (nBG) further comprising an antimicrobial agent.

In some embodiments, the antimicrobial agent is poly chloro phenoxy phenol.

According to the present invention, a method of producing a nano-bioactive glass (nBG) comprises: a) mixing calcium nitrate solution with Tetraethoxysilane (TEOS) solution; b) adding dropwise of a mixture from step a) to an ammonium dibasic phosphate solution; c) stirring the mixture from step b) for 30-60 hours and permitting the reaction mixture to rest for 20-30 hours for further precipitation; d) separating a precipitate from step c); e) freezing the precipitate from step d) at -60 to -40 °C for 20-30 hours; and f) heating a sample of the precipitate from step e) at 650 to 800 °C for 2-5 hours and cool down the heated precipitate for 8-14 hours.

In some embodiments, in step a) the TEOS was dissolved in anhydrous ethanol.

In some embodiments, step a) is conducted at a pH between 1-2 and a reaction mixture is stirred for 20-40 minutes.

In some embodiments, step a) is conducted at a pH between 1-2 and a reaction mixture is stirred for 30 minutes.

In some embodiments, step b) further comprises adding ammonium hydroxide solution to adjust the pH to be between 9-12. In some embodiments, step b) further comprises adding ammonium hydroxide solution to adjust the pH to 11.

In some embodiments, step d) further comprises freezing the precipitate at -20-30 °C for 8-14 hours.

In some embodiments, step d) further comprises freezing the precipitate at-25 °C for 12 hours.

According to the present invention, a method of stimulating bone regeneration by treating a subject with the composition disclosed in the present application.

According to the present invention, a method of repairing bone fractures by treating a subject with the composition disclosed in the present application.

According to the present invention, a composition in the manufacture of a medicament for treating bone fractures on a subject.

In some embodiments, the treatment comprises oral administration or surgical implantation of the composition.

In some embodiments, the subject of treatment is a mammal.

In some embodiments, the subject of treatment is human.

In some embodiments, the subject was treated for dental treatment, or orthopedic surgery.

Unless otherwise specified, "a," "an," "at least one," and "one or more" are used interchangeably. Within the description of this disclosure, where a numeri-cal limit or range is stated, the endpoints are included unless stated otherwise. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

As used herein, the words "substantially the same", "approximately", or "about" may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is± 1 % of the stated value (or range of values), ±2% of the stated value (or range of values), ±5% of the stated value (or range of values), ±10% of the stated value (or range of values), ±15% of the stated value (or range of values), or±20% of the stated value (or range of values).

In some embodiments, the nano-bioactive glass comprises at least one alkali metal such as, for example, lithium, sodium, potassium, rubidium, cesium, francium, or combinations of these metals. In other embodiments, however, the nano-bioactive glass has little to no alkali metal. For example, in certain embodiments, the nano-bioactive glass has 30% or less of alkali metal. In other embodiments, the nanobioactive glass has 25% or less of alkali metal. In yet other embodiments, the nano-bioactive glass has 20% or less of alkali metal. In yet other embodiments, the nano-bioactive glass has 15% or less of alkali metal.

In some embodiments, the nano-bioactive glass has 10% or less of alkali metal. In still other embodiments, the nano-bioactive glass has 5% or less of alkali metal. In yet other embodiments, the nano-bioactive glass has substantially no alkali metal. Without intending to be bound by any particular theory, it is believed that the presence of certain metals may catalyze further polymerization of the biocompatible polymer such as, for example, PLGA or PCL, thereby (1) increasing its molecular weight and/or (2) increasing its degree of cross-linking/cross-link density. Either event increases the viscosity of the polymer and may seize up the equipment used to process the com-posite material. As such, a nano-bioactive glass with a low percentage of alkali metal may be utilized to prevent equip-ment failure and/or to allow a high percentage of nanobioactive glass to be utilized.

In some embodiments of the present invention, the nano-bioactive glass has osteoproductive properties. As used in this application, "osteoproductive" refers to an ability to allow osteoblasts to proliferate, allowing bone to regenerate. "Osteoproductive" may also be defined as conducive to a process in which a bioactive surface is colonized by osteogenic stem cells and which results in more rapid filling of defects than that produced by merely osteoconductive materials. nBG fiber as disclosed in the present invention is an example of an osteoproductive, bioactive material.

Osteoconductivity of a biomaterial is largely dependent on the biocompatibility and bioactivity of the biomaterial's surface [Smith D C, Pilliar R M and Chemecky R 1991 Dental implant materials. I. Some effects of preparative procedures on surface topography J of Biomedical Materials Research 25 1045-68], Factors affecting properties of hydroxyapatite in cell adhesion and cell proliferation include its structure, phase purity, porosity, surface properties and sintering temperature [Khan A, Wong F, McKay I, Whiley R, Rehman I. Structural, mechanical, and biocompatibility analyses of a novel dental restorative nanocomposite. Jour-nal of Applied Polymer Science 2013; 127:439-47, incorpo-rated herein by reference in its entirety] (Stamboulis, 2002). Several methods including wet precipitation [Santos M H, Oliveira M d, Souza L P d F, Mansur H S, Vasconcelos W L. Synthesis control and characterization of hydroxyapatite prepared by wet precipitation process. Materials Research 2004; 7:625-30, incorporated herein by reference in its entirety], chemical precipitation [Monmaturapoj N. Nano-size hydroxyapa-tite powders preparation by wet-chemical precipitation route. Journal of Metals, Materials and Minerals 2017; 18, incorporated herein by reference in its entirety], sol -gel method [Khan A, Ahmed Z, Edirisinghe M, Wong F, Reh-man I. Preparation and characterization of a novel bioactive restorative composite based on covalently coupled polyure-thane-nanohydroxyapatite fibres. Acta Biomaterialia 2008; 4: 1275-87, incorporated herein by reference in its entirety], and microwave irradiation [Nazir R, Khan A S, Ahmed A, Ur-Rehman A, Chaudhry AA, Rehman I U, et al. Synthesis and in-vitro cytotoxicity analysis of microwave irradiated nanoapatites. Ceramics International 2013;39:4339-47, incorporated herein by reference in its entirety] have been developed to prepare synthetic hydroxyapatite. Microwave-assisted synthetic approaches have been recently used for the preparation of composites, polymers, and ceramics [Das S, Mukhopadhyay A, Datta

S, Basu D. Prospects of micro-wave processing: an overview. Bulletin of Materials Science 2009;32: 1-13, incorporated herein by reference in its entirety]. Khan et al., synthesized high purity nano hydroxy-apatite (nHA) and nHA/carbon nanotube in a short amount of time using a microwave-assisted wet precipitation tech-nique [Khan A, Hussain A, Sidra L, Sarfraz Z, Khalid H, Khan M, et al. Fabrication and in vivo evaluation of hydroxyapatite/carbon nanotube electrospun fibers for bio-medical/dental application. Materials Science and Engineer-ing: C 2017, incorporated herein by reference in its entirety].

The water used herein may be tap water, distilled water, bidistilled water, deionized water, deionized distilled water, reverse osmosis water, and/or some other water. In one embodiment, the water is bidistilled to eliminate trace met-als. Preferably the water is bidistilled, deionized, deinonized distilled, or reverse osmosis water and at 25° C. has a conductivity at less than 10 pS cm ¹ , preferably less than 1 pS cm ¹ , a resistivity greater than 0.1 MQ cm, preferably greater than 1 MQ cm, more preferably greater than 10 MQ cm, a total solid concentration less than 5 mg/kg, preferably less than 1 mg/kg, and a total organic carbon concentration less than 1000 pg/L, preferably less than 200 pg/L, more preferably less than 50 pg/L.

In some embodiments, the nBG fiber may comprise up to about 50% of the nano-bioactive glass. In certain embodiments, the nano-bioactive glass is present in an amount of about 5 to 50% by weight of the compounded composite material. In other embodiments, the nano-bioactive glass is present in an amount of about 15 to 30% by weight of the nBG fiber. In yet other embodiments, the nano bioactive glass is present in an amount of about 20 to 30% by weight of the nBG fiber. In embodiments in which a low molecular weight biocompatible polymer is used, bioactive glass may be present in higher weight percentages, such as 60% by weight of the compounded composite material.

In some embodiments of the present invention, a coupling agent is added to the mixture of the biocompatible polymer and the nano-bioactive glass. The coupling agent acts as a bonding agent between the biocompatible polymer and the nano-bioactive glass which translates into increased tensile/flexural strength of the nano-bioactive composite. Non-limiting examples of coupling agents suitable for use in the present invention include, for example, silane, titanium-based and zirconium-based coupling agents, specifically, organotitanate, multifunctional amine compounds such as 4-aminophenyl sulfone, azo compounds such as 4-cyanovaleric acid, and combinations thereof. The preferred coupling agent is one that includes multifunctional groups that are capable of chemically bonding with a functional group of the biocompatible polymer and binding the bioactive glass. The nano-bioactive glass may be coated with the coupling agent prior to being combined/ mixed with the biocompatible polymer. Alternatively, both the nano-bioactive glass and biocompatible polymer may be individually coated with the coupling agent before being combined.

In some embodiments, at least one other agent may be added to the mixture of the biocompatible polymer and nano-bioactive glass. Such agents can comprise, at least partially, reinforcing fibers. Non-limiting examples of other agents include carbon, glass, radiopaque material, barium glass, resorbable material, strontium, strontium nitrate, strontium -calcium-zinc-silicate glasses, silver, calcium apatite, calcium silicate or mixtures of these materials. In certain aspects of the invention, the other agent is barium sulfate, barium- boroaluminosilicate (BBAS) glass, silica or e-glass fibers.

In some embodiments, the other agents include radiopaque markers situated in predetermined locations within the shaped implant to aid in visualizing the implant once in the body. For example, FIGS. Sa and Sb show titanium alloy (Ti-6A1-4V ELI) markers incorporated into the composite shaped body. In certain embodiments, the other agent may comprise calcium phosphate having macro-, mesa-, and microporosity. More preferably, the porosity of the calcium phosphate is interconnected. The preparation of preferred forms of calcium phosphate for use in the present invention is described in U.S. Pat. No. 6,383,519 and U.S. Pat. No. 6,521,246, incorporated into this application by reference in their entireties. An exem-plary calcium phosphate product is Vitoss® Bone Graft Substitute (available from Orthovita, Inc. of Malvern, Pa.). The at least one other agent may be incorporated within the bioactive composite, or in the case of a shaped implant be used to fill cavities of the implant. For instance, when used with a shaped spinal implant, the other agent may be present within the center cavity of the implant to facilitate fusion of the adjacent vertebral bodies.

In addition to other agents, bone augmentation materials or bone cements may be used in conjunction with the bioactive composite (nBG fiber) in applications where additional reinforcement is required. For instance, in certain bone fractures it may first be required that certain portions of the fracture be stabilized with a bone augmentation material prior to placing the bioactive composite implant of the present invention. Alternatively, in certain spine fusion procedures, it may first be desired to prophylactically treat the adjacent vertebrae prior to placing the bioactive spinal implant of the present invention in the disc space between the two vertebrae, if the bone stock appears weakened due to trauma or disease such as osteoporosis. An exemplary bone augmen-tation product is Cortoss® Bone Augmentation Material (available from Orthovita, Inc. of Malvern, Pa.).

In a preferred embodiment of the present invention, a nBG fiber (bioactive composite) is formed upon combining a biocompatible polymer with a nano-bioactive glass as described in the present invention. The biocompatible polymer preferably has a diameter of 1.5 - 3 pm.

In some embodiments, the biocompatible polymer and the nano-bioactive glass (as well as other components, if present) may be dry mixed for a period of time and under conditions sufficient to achieve substantial homogeneity of the mixture. As used herein, the term "dry mixed" refers to mixing the components in a dry state, i.e., in the absence of added liquid water or organic solvent. The dry mixing of the bioactive glass with the biocompatible polymer granules or pellets may be accomplished using any methods known in the art per se, including milling, spinning, tumbling, sonication, vibrating, or shaking.

In some embodiments, the mixture is tumbled on rollers for about one to about two hours. As used herein, the terms "homogeneity" and "homogeneous" describe a composition that is substantially uniform in structure and/or composition throughout. The term "substantially homogeneous" is to be understood within the context of the invention and is not to be taken as an absolute.

In some embodiments, suitable polymers that may be included in the polymer blends of the present invention include: suitable biocompat-ible, biodegradable polymers which may be synthetic or natu-ral polymers. Suitable synthetic biocompatible, biodegrad-able polymers include polymers selected from the group consisting of aliphatic polyesters, poly( amino acids), copoly (ether-esters), polyalkylenes oxalates, polyamides, tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoe-sters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, polydiglycolates, polylactic-co-glycolic acid (PLGA) and poly(s-caprolactonc) (PCL), and combinations thereof. It is to be understood that inclusion of additional suitable polymers is dependent upon obtaining dimensional stability in the fabricated device.

For the purposes of this invention the above optional aliphatic polyesters include, but are not limited to, homopolymers and copolymers of lactide (which include lactic acid, D-, L- and mesa lactide), glycolide (including glycolic acid), epsilon-caprolactone, p-dioxanone (1 ,4-dioxan-2-one ), trimethylene carbonate (l,3-dioxan-2-one), alkyl derivatives of trimethylene carbonate, and blends thereof.

Other Definitions

As used herein, the term “activity” refers to the activation, production, expression, synthesis, intercellular effect, and/or pathological or aberrant effect of the referenced molecule, either inside and/or outside of a cell. Such molecules include, but are not limited to, cytokines, enzymes, growth factors, pro-growth factors, active growth factors, and pro-enzymes. Molecules such as cytokines, enzymes, growth factors, pro-growth factors, active growth factors, and pro-enzymes may be produced, expressed, or synthesized within a cell where they may exert an effect. Such molecules may also be transported outside of the cell to the extracellular matrix where they may induce an effect on the extracellular matrix or on a neighboring cell.

It is understood that activation of inactive cytokines, enzymes and pro-enzymes may occur inside and/or outside of a cell and that both inactive and active forms may be present at any point inside and/or outside of a cell. It is also understood that cells may possess basal levels of such molecules for normal function and that abnormally high or low levels of such active molecules may lead to pathological or aberrant effects that may be corrected by pharmacological intervention.

According to the present invention, it may also provide isotopic variants of the compounds disclosed herein, including wherein the isotopic atom is ² H and/or wherein the isotopic atom ¹³ C. Accordingly, in the compounds provided herein hydrogen can be enriched in the deuterium isotope. It is to be understood that the invention encompasses all such isotopic forms. It is understood that the structures described in the embodiments of the methods hereinabove can be the same as the structures of the compounds described hereinabove.

It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.

Except where otherwise specified, if the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.

The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as ¹² C, ¹³ C, or ¹⁴ C. Furthermore, any compounds containing ¹³ C or ¹⁴ C may specifically have the structure of any of the compounds disclosed herein.

It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as ¹ H, ² H, or ³ H. Furthermore, any compounds containing ² H or ³ H may specifically have the structure of any of the compounds disclosed herein. Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.

In the compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise.

In the compounds used in the method of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.

It is understood that substituents and substitution patterns on the compounds used in the method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.

The compounds used in the method of the present invention may be prepared by techniques well known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.

The compounds used in the method of the present invention may be prepared by techniques described in Vogel’s Textbook of Practical Organic Chemistry, A. I. Vogel, A.R. Tatchell, B.S. Fumis, A. J. Hannaford, P.W.G. Smith, (Prentice Hall) 5 ^th Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5 ^th Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.

Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition. In some embodiments, according to the present invention, a pharmaceutical composition comprising the nBG Fiber disclosed in the present invention and a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically active agent” means any substance or compound suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians’ Desk Reference (PDR Network, LLC; 64th edition; November 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (U.S. Department Of Health And Human Services, 30 ^th edition, 2010), which are hereby incorporated by reference. Pharmaceutically active agents which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a pharmaceutically active agent does not possess a carboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so long as the modification does not interfere with the pharmaceutically active agent’s biological activity or effect.

The compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat an infection or disease caused by a pathogen, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium. The term "pharmaceutically acceptable salt" in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and lauryl sulphonate salts and the like. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", Pharm. Set. 66: 1-19).

As used herein, "treating" means preventing, slowing, halting, or reversing the progression of a disease or infection. Treating may also mean improving one or more symptoms of a disease or infection.

The compounds used in the method of the present invention may be administered in various forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.

As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier.

The dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.

A dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional antibacterial agents. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or onto a site of infection, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. The compounds used in the method of the present invention can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-efferve scent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modem Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein. Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow -inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

The compound/composition used in the method of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue- targeted emulsions.

The compound/composition used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. For oral administration in liquid dosage form, the oral drug components are combined with any oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.

Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

The compound/composition used in the method of the present invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.

Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

The compounds and compositions of the present invention can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by topical administration, injection or other methods, to the afflicted area, such as a wound, including ulcers of the skin, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

Specific examples of pharmaceutical acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U.S. Pat. No. 3,903,297 to Robert, issued Sept. 2, 1975. Techniques and compositions formaking dosage forms useful in the present invention are describedin the following references: 7 Modem Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

The term "prodrug" as used herein refers to any compound that when administered to a biological system generates the compound of the invention, as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), photolysis, and/or metabolic chemical reaction(s). A prodrug is thus a covalently modified analog or latent form of a compound of the invention.

The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, powders, and chewing gum; or in liquid dosage forms, such as elixirs, syrups, and suspensions, including, but not limited to, mouthwash and toothpaste. It can also be administered parentally, in sterile liquid dosage forms.

Solid dosage forms, such as capsules and tablets, may be enteric coated to prevent release of the active ingredient compounds before they reach the small intestine. Materials that may be used as enteric coatings include, but are not limited to, sugars, fatty acids, waxes, shellac, cellulose acetate phthalate (CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), and methyl methacrylate -methacrylic acid copolymers.

The compounds and compositions of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.

The compounds of the present invention can be synthesized according to general Schemes. Variations on the following general synthetic methods will be readily apparent to those of ordinary skill in the art and are deemed to be within the scope of the present invention.

Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

While the invention has been shown and described with reference to certain embodiments of the present invention thereof, it will be understood by those skilled in the art that various changes in from and details may be made therein without departing from the spirit and scope of the present invention and equivalents thereof.

Experimental Details

Synthesis of nano-Bioactive Glass (nBG)/ Bioactive Glass Nanoparticles (BGNPs)

Materials & Methods - nBG (55% SiO2:4Q% CaO:5% P2O5, all in mol%) Fabrication (Figure 6)

Principle of Sol-gel processing fabrication: TEOS, Calcium Nitrate and Ammonium Dibasic Phosphate are first dissolved in ethanol and water to facilitate reaction with each other. Citric acid is used as a catalyst to initiate the hydrolysis reaction with TEOS to form the glass network. Addition of calcium nitrate during the reaction is to act as a network modifier to improve the dissolution of bioactive glass allowing HCA layer deposition. Ammonium dibasic phosphate also provides a glass network to improve dissolution of the bioactive glass. Reaction mixture is aged in the presence of ammonium hydroxide to catalyze the gelation process to form Bioactive Glass Nanoparticles (BGNPs). Drying is completed to prevent aggregation of the discrete particles and lastly sintering stage is done to remove any organic residuals within the BGNPs.

1. Precursor Mixing a. Tetraethyl orthosilicate (TEOS) was mixed with calcium nitrate after dissolution in ethanol and water. b. Ammonium dibasic phosphate was dissolved in deionized water.

2. Hydrolysis and Polycondensation a. IM Citric acid and IM Ammonium hydroxide were used as catalysts.

3. Aging & Washing a. Particles were washed to remove catalysts.

4. Drying and Calcination a. Lyophilized overnight and calcinated at 600°C for 2 hrs.

Materials:

1. Reagents:

1. Calcium Nitrate (Fisher Scientific - AC423530250).

2. Tetraethoxysilane (TEOS) (Fisher Scientific - AC157811000).

3. Ammonium dibasic phosphate (Sigma Aldrich - 215996-100).

4. Citric Acid Monohydrate (Sigma Aldrich - 120M0186V).

5. Ammonium Hydroxide (Fisher Scientific - A669-212).

Preparation of IM Citric Acid: 42.028 g of Citric Acid Monohydrate was weighed and dissolved in 200 ml of diH ₂ O in a beaker and stir at 150 rpm for 5 mins until a uniform solution is formed.

Preparation of Ammonium Dibasic Phosphate Solution:

269.50 mg of ammonium dibasic phosphate was weighed and dissolved in 375mL of diH ₂ O and stir at 250 rpm for 1 minute.

BGNPs Fabrication: Sol-Gel Processing (Acid/Base Co-catalyzed method):

Day 1 (Dissolution and Reaction):

1. Dissolve 1.910 g calcium nitrate in 120 mb of diH ₂ O at room temperature, stir at 200 rpm using a magnetic stir bar for 10 minutes until calcium nitrate has fully dissolved - Solution A.

2. Draw out 2.46 ml of TEOS using a 1 ml pipette and slowly inject into a beaker containing 60 mb of anhydrous ethanol and stir at 200 rpm for 10 minutes - Solution B.

3. Slowly pour Solution A into Solution B and continue to add 1 M citric acid into reaction mixture to adjust the pH to 1-2 measured by a pH probe and continue to stir the reaction mixture for 30 mins.

4. Increase the ammonium dibasic phosphate solution stirring speed to 500 rpm and slowly add the acidic mixture dropwise into ammonium dibasic phosphate solution using a 10 ml pipette and draw up small amounts of the acidic solution and add into a separation flask on top the alkali solution to drip the acid into the alkali. Adjust the pH of the mixture to 11 using ammonium hydroxide solution.

Day 2 & Day 3 (Gelation):

1. Keep stirring the mixture for 48 hours and rest for 24 hours.

Day 4 (Separation):

Pipette reaction mixture into separate centrifuge tubes (50 mb). Separate the precipitate from reaction solution by centrifugation at 44000 rpm for 5 mins and wash the precipitate with 3 times with diH ₂ O.

A. Place sample in drying vessels and freeze from room temperature to -25°C in freezer overnight to ensure entire sample was at the setting temperature before lyophilization.

Day 5 (Drying):

1. Place frozen sample into freeze-dryer at -50°C and High Pressure for 24 hours.

Day 6 (Calcination):

1. After freeze drying, place samples in inert ceramic crucibles and heat in oven at 700°C for 3 hours and cool down the white precipitate over-night in the fume hood.

Recovery and Storage of BGNPs. 1. Using a spatula to transfer the BGNPs into a 20 ml glass vial.

2. Parafilm the sealed glass vial and store inside the desiccator cabinet.

Materials & Methods - nBG Fiber Fabrication (Figure 7)

1. Solution Preparation:

1. PLGA (5: 1) PCL was dissolved in acetic acid.

1. Electrospinning

1. nBG (5wt%) was mixed with polymer solution and pumped through a syringe needle under high voltage.

2. Polymeric fibers were collected on a ground collector and sectioned into flat meshes.

Materials & Methods - nBG & nBG Fibers Characterization

Material Characterization:

Experimental Groups: a. nBG b. PLGA (5: 1) PCL Fibers c. nBG Fibers

Morphology:

1. Scanning Electron Microscopy (SEM) (n=2/group)

2. SEM diameter measurement (n= 100/group)

Structure/Crystallinity:

• X-ray Diffraction Spectroscopy (XRD) (n=l/group)

Composition/Chemistry:

1. Fourier Transform Infrared Spectroscopy - Attenuated total reflection (FTIR-ATR) (n=3/group)

2. Energy-dispersive X-ray spectroscopy (EDS) (n=3/group)

After characterizing the materials, bioactivity of the nBG and nBG fibers were evaluated. The materials were immersed in DMEM media and incubated at 37 °C and 5% CO2 to mimic the physiological environment without changing the media throughout the study. The changes in crystallinity were analyzed using XRD. Composition changes were monitored using FTIR. pH levels of the media were recorded. The supernatant of the DMEM were collected at each timepoint and the ion profiles of Si Ca and P were analyzed using inductive coupled plasma spectroscopy. Bioactivity Test (37°C, 5% CO2) (Figure 8):

Negative Control:

• Plain DMEM (n=5/timepoint)

• PLGA (5: 1) PCL Fibers (Img/mL) (n=5/timepoint) Experimental Groups:

• nBG (Img/mL) (n=5/timepoint)

• nBG Fibers ( Img/mL) (n=5/timepoint)

Time points: Day 0, 1, 7 & 14

Endpoint Analysis:

• XRD (n=l/group/timepoint)

• FTIR (n=3/group/timepoint)

• pH level (n=5/group/timepoint)

• Si, Ca & P (n=3/group/timepoint)

Result

SEM revealed the material to be globular in structure with a diameter of approximately 11 nm (Figure 9A). FTIR was then generated showing the signature peaks at 1100, 810 and 470 which shows the silica network that is responsible for housing the Ca and P2O5 ions (Figure 9B). XRD showed the amorphous nature of the material which is quite important. Since the fabrication process involving calcinating the materials at high temperatures, which could increase the crystallinity of the silica network material but will in turn reduce the bioactivity (Figure 9C).

EDS showed presence of Si, Ca and P (Figure 10). Quantification of these ion was also conducted using EDS. The theoretical mass % three elements were calculated based on the molar ratio and were compared to the actual mass% calculated from EDS, and showed larger proportion of Si and lower Ca and P.

Table 1. Theoretical mass percentage of Si, Ca and P (55% SiC>2:40% CaO:5% P2O5, all in mol%).

Table 2. Actual mass percentage of Si, Ca and P.

Table 3. Increasing the concentration of CaO and P2O5 achieved more similar ratio compared to theoretical ratio.

Larger proportion of Si and lower Ca and P could be due to the lower concentrations of Ca used during synthesis. The washing step using deionized water can also wash away the Ca content and P content before drying. So, a higher concentration was tested and resulted in a more similar ratio compared to the theoretical mass ratio

The polymeric fibers and the nBG fibers were characterized. SEM showed that particles were deposited along the length of the fibers (Figure 11A and Figure 12A). FTIR showed the organic carbon bonds of the polymeric fibers and XRD showed the relative amorphous nature of PLGA PCL mix (Figure 11B). Similarly, for the nanoparticle fibers similar carbon bonds were seen but XRD revealed during the electrospinning process crystallization of the silica network was seen post fabrication as shown by the peaks at 26 and 38 degrees (Figure 12B).

After completing the material characterization, bioactivity test was completed and DMEM was used as negative control. The pH level of the supernatant was measured. DMEM showed constant pH throughout the 14 days (Figure 13). PLGA+PCL showed constant pH until the last timepoint. This could be due to the degradation of the material producing lactic acid and thus lowering the pH of the environment. nBG and nBG fibers also maintained pH throughout the 14 days. From stat analysis, PLGA+PCL at day 14 showed significant difference compared to the rest of the groups. Therefore, it is suggested that nBG incorporation into the polymeric fibers may cause a pH buffering effect. This happens when mineralization occurs usually it drives up the pH in the environment and is not due to the pH buffering of the 5% CO2 on DMEM because polymeric showed significant decrease in pH meaning incubation conditions alone was not sufficient for pH buffering. Therefore, the nBG fibers PH buffering can be attributed to the nBG presence in the material.

The composition and crystallinity changes after the bioactivity study were evaluated. FTIR showed the development of phosphate peaks for the nBG and disruption of the phosphate network within the nBG suggesting possible calcium phosphate formation on the surface (Figure 14A). This was confirmed by the crystalline peaks of calcium phosphate shown on the XRD (Figure 14B).

Development of PCL crystalline peak on the material was observed, this may be due to the degradation of PLGA component of the material (Figure 15A). Lastly nBG fibers also showed growth of calcium phosphate peak after day 14 suggesting mineral deposition on the material which suggests possible ion release of the mineral ions and apatite formation (Figure 15 B).

The ion release profile of the four groups during the duration of the study was evaluated (Figure 16). DMEM and PLGA fibers showed no Si concentration as expected and Ca and P ion concentration also remained relatively constant. nBG showed burst release of silicon into the media after D 1 and remained constant after 2 weeks. However, there was a significant lower Ca and P concentration at Day 7 suggesting unstable CaP precipitation since the level of both ions were increased again at Day 14. nBG fibers showed similar profile as PLGA fibers however the fibers only contained 5wt% of nBG, it may be below the detection level for the changes the be detected. Cell viability indicates that nBG is not cytotoxic and may have support cell attachment (Figure 17A and Figure 17B).

Discussion

Bioactive glass nanoparticles have been used for a wide range of biomedical applications including bone regeneration due to its high specific surface area and reactivity. Inspired by the composite nature of bone (Figure 2), bioactive glass nanoparticles (nBG) derived from a SiCL-CaOJ^CL ternary system may be incorporated into polymer blend (PLGA: PCL) fibers, and this application explores nBG synthesis and evaluates the bioactivity and osteogenic potential of this composite fiber platform. (Antrobus et al. 2020) The development of a Ca-P phase on the polymer-nBG fibers were monitored longitudinally under physiological conditions using FTIR-ATR, XRD and SEM. It is hypothesized that the nanoparticle infused polymeric fibers will be bioactive and provide structural template for mineralization in situ (Grease ly et al. 2009). Here, a coprecipitation sol-gel method was used to fabricate the nBG (55%SiO2-40%CaO-5%P2C>5, wt%). For the fabrication of polymer-nBG fibers, 5 wt% of nBG were added to a polymeric blend of PLGA: PCL (5: 1) and electrospun at 10 kV. To test the bioactivity and mineralization potential of the composite fibers, the samples with corresponding controls were immersed in DMEM (1 mg/ml) and collected at Day 0, 1, 3, 7 and 14.

One-way ANOVA and Tukey-Kramer HSD post-hoc tests were used to study significant differences (p<0.05) of pH levels between different experimental groups. The nBG appeared globular and are 10-20 nm in diameter under SEM. The fibers with or without nBG were 2.21±0.64 pm and 2.24±0.58 pm in diameter, respectively. FTIR analysis reveals a peak at 1100 cm' ¹ representing the Si-0 bond of the silica network. After immersion in DMEM for 14 days, pH level remained similar across all experimental groups except for day 14 where particle-free control measured significantly lower pH compared to all other groups, indicative of polymer degradation. Coupling the polymer fiber with nBG served to neutralize the negative impact on pH and allow maintenance of physiological pH. XRD patterns showed crystallization peaks at 31.8° and 45° suggesting crystallization of phosphate and the early formation of carbonated apatite. This was confirmed by FTIR spectra showing growing peaks at 603 and 568 cm' ¹ and 960 cm' ¹ suggesting dissociation of Si-0 bonds and mineral crystal formation by day 14. These bulk and surface characterization analysis confirm the bioactivity and mineralized in this novel composite system. It also confirms both osteoconductive and osteoinductive potential of these composites for bone regeneration.

Characterization results showed nBG and nBG fibers supported mineral deposition of bioactivity. Silica release indicates formation of silica gel layer, which is important in mineralization (Li. et. al. 2003). It also indicates CaP release and re -precipitation onto the silica gel layer. nBG releases alkaline products which balances acidic degradation products of PLGA which indicates biocompatibility. nBG showed potential to serve as an apatite nucleation site and nBG fibers showed potential to serve as a structural template for mineral deposition. References

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