Generation of nanoparticles with acellular porcine bone for orthopedic regeneration and imaging

Abstract

A composition is disclosed for orthopedic regeneration. The composition comprises bone extracellular matrix particles, wherein the particles have an average particle size in a range of 1 nanometer to 500 nanometers. The composition can further comprise a fluorescent dye associated with each particle of at least a portion of the particles. The composition can further comprise one or more bioactive agents associated with each particle of at least a portion of the particles. Also disclosed are methods of making the compositions.

Description

Generation of Nanoparticles with Acellular Porcine Bone for Orthopedic Regeneration and ImagingCross-Reference To Related Applications

[0001] The present application is based on, claims priority to, and incorporates herein by reference in its entirety for all purposes, United States Provisional Application Serial no. 63 / 619,634, filed January 10, 2024.Statement Regarding Federally Sponsored Research

[0002] Not applicable.Technical Field

[0003] The invention relates to nanoparticles, and in particular, to a composition for orthopedic regeneration, wherein the composition comprises: nanoparticles comprising bone extracellular matrix; and a fluorescent dye associated with each nanoparticle of at least a portion of the nanoparticles.Background

[0004] Bone diseases and physiological conditions, such as osteoporosis. Paget's disease, osteosarcomas, osteoarthritis, metastatic bone cancer, hormonal status and chronic diseases, and traumatic bone injury, affect millions of people worldwide and impose an increasingly heavy burden on an aging society [Ref. 1-5], Although bone is a living tissue that is capable of continuous regeneration, disturbances in bone homeostasis lead to impaired bone healing [Ref. 6, 7], Pharmacologic therapy is the primary clinical treatment for such bone diseases [Ref. 8]; However, unlike soft organs such as the liver or kidney, bone is highly mineralized and less vascularized, which ultimately decreases drug delivery efficiency to bone [Ref. 9, 10], Another limitation of current pharmacologic therapy is that many drugs have a short circulation halflife, resulting in poor assimilation and low bioavailability in diseased bone [Ref. 11, 12], Thus, drugs are normally administered at high dosage levels at an increased frequency. This often leads to side effects resulting from systemic toxicity due to the large amount of drug accumulation in other unintended organs [Ref. 13, 14],

[0005] Bone is a heterogeneous composite tissue which is composed of a dynamic ECM network consisting of 50-70% inorganic minerals, 20-40% organic matrix, and 5-10% water [Ref. 6, 7], The inorganic minerals, primarily consisting of calcium phosphate (CaP), specifically hydroxyapatite (HA), are the source for hardness and strength of the bone, while the organic matrix, including collagens and non-collagenous proteins, provide the tissue with flexibility and a template to regulate new bone formation [Ref. 15. 16], Collagens, especiallycollagen type I, are the most abundant proteins found in bone, playing a critical role in their structure, tissue organization, and mechanical support [Ref. 17, 18], Non-collagenous proteins, such as proteoglycans, glycoproteins, y Carboxy glutamic acid-containing proteins, and other serum-derived proteins, are associated in regulating fundamental cellular processes (such as attachment, proliferation, differentiation, and migration), tissue mineralization, and driving functions related to tissue regeneration [Ref. 19. 20],

[0006] Decellularized bone matrix (DBM) is a widely used graft material in clinical orthopedic treatment, as DBM is naturally derived from bone tissues that have undergone a meticulous decellularization process, which removes cellular and antigen components while preserving the extracellular matrix (ECM) components (collagens, proteoglycans, glycoproteins, y Carboxy glutamic acid-containing proteins, serum-derived proteins, etc.) and mechanical and structural properties [Ref. 21-24], There is a wide diversify of DBMs that have been approved by the U.S. Food and Drug Administration (FDA) for clinical use [Ref. 23, 25-28], DBM offers numerous advantages over other bone graft materials, such as bone cement, bone glass, hydroxyapatite (HA), and synthetic polymers, due to its preservation of natural signaling cues necessary for enhancing cell activity, osteoinduction, osteoconduction, and osteogenesis [Ref. 29-32], Previous work has established a decellularization protocol for porcine bone and demonstrated a near-to-complete cellular and porcine antigen removal of the obtained DBM. The DBM was further digested with pepsin and desalted with acetone precipitation to obtain a gellable DBM. The gellable DBM demonstrated excellent biocompatibilify and osteogenesis [Ref. 33],

[0007] Image-guided interventions are playing an increasing role in diagnosis and treatment for bone diseases, as these strategies can provide a less invasive and more accurate way not only for detecting and monitoring of disease but also for improving treatment outcome. Radiographic imaging, such as plain X-ray, Computed Tomography (CT), and Dual Energy X-ray Absorptiometry (DEXA), are the most common methods for clinical bone diagnosis, but many studies find that radiography alone offers an incomplete assessment of bone healing and is unreliable in determining the healing stages of bone [Ref. 14-18], Other drawbacks of radiographic imaging include frequent radiation exposure and high cost [Ref. 19], which can be especially detrimental for immune-compromised or ill patients, children, and pregnant women. Recent efforts have focused on developing image-guided therapies that can monitor host-material interactions, quantitatively assess bone healing, and evaluate the performance of drugs or grafts in a real-time, non-invasive. high-sensitive, and cost-effective way. One such method, Near-Infrared Spectroscopy imaging (NIR), has been used to study the in vitroand in vivo degradation of biomaterials that have been doped with fluorescent markers. Indocyanine Green (ICG) is an FDA approved image contrast dye that is often viewed in the near-IR (NIR) or NIR-I window (700-900nm) for a wide variety of preclinical and clinical applications. It has been found that extending fluorescence imaging into shortwave IR, also known as SWIR or NIR-II (950-1700nm) enhances the advantages of NIR imaging due to lower tissue autofluorescence and increased sensitivity, leading to increased tissue penetration depth and decreased scattering [Ref. 50-54],

[0008] Therefore, there is no satisfactory image-guided diagnosis and treatment approach at the present time for bone diseases. Accordingly, there is a need for an improved image- guided method and system for the diagnosis and treatment of bone diseases.Summary

[0009] The present disclosure provides systems and methods that overcome the aforementioned drawbacks by presenting compositions and methods of making bone-derived nanoparticles (NPs) for bone grafting. The bone-derived NPs may further encapsulate dyes for image-guided diagnosis and treatment. Throughout the disclosure, NPs and bone particles (BPs) may be used interchangeably unless stated otherwise.

[0010] In accordance with the present disclosure, a composition is described. The composition comprises bone extracellular matrix particles, wherein the particles have an average particle size in a range of 1 nanometer to 500 nanometers.

[0011] In accordance with the present disclosure, a composition is described. The composition comprises bone extracellular matrix particles, wherein the particles have an average particle size in a range of 1 nanometer to 500 nanometers and a fluorescent dye associated with each particle of at least a portion of the particles.

[0012] In accordance with the present disclosure, a method of making particles is described. The method comprises (a) providing a solution including particles comprising bone extracellular matrix, wherein the particles have an average particle size in a range of 1 nanometer to 500 nanometers; and (b) adding a fluorescent dye to the solution to associate an amount of the dye with each particle of at least a portion of the particles.

[0013] In accordance with the present disclosure, a method of making particles is described. The method comprises (a) providing a solution including particles comprising bone extracellular matrix, wherein the particles have an average particle size in a range of 1 nanometer to 500 nanometers, and (b) adding one or more bioactive agents to the solution to associate an amount of the bioactive agent with each particle of at least a portion of the particles.

[0014] In accordance with the present disclosure, a method of treating an orthopedic defect is described. The method comprises filling the orthopedic defect with the any of the compositions described herein.

[0015] These aspects are nonlimiting. Other aspects and features of the systems and methods described herein will be provided below.Brief Description of the Drawings

[0016] The foregoing features of embodiments will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:

[0017] FIG. 1 A is a flow chart showing the processing of porcine bone into extracellular matrix stock (ECM), according to aspects of the present disclosure.

[0018] FIG. IB is a diagram of fabricating indocyanine green (ICG) / bone particles (BPs), according to aspects of the present disclosure.

[0019] FIG. 2A is a flow chart of the method of making particles from bone extracellular matrix, according to aspects of the present disclosure.

[0020] FIG. 2B is a flowchart of the of the method step of providing a solution comprising bone extracellular matrix, according to aspects of the present disclosure.

[0021] FIG. 3 is a flowchart of the method of treating an orthopedic defect, according to aspects of the present disclosure.

[0022] FIG. 4A is a flowchart showing the resuspension and lyophilization of the ICG / BPs, according to aspects of the present disclosure.

[0023] FIG. 4B is a diagram showing the in vitro cellular uptake of ICG / BPs by bone marrow stem cells (BMSCs), according to aspects of the present disclosure.

[0024] FIG. 4C is a diagram of localized implantation (left) and systemic administration (right) of the ICG / BPs, according to aspects of the present disclosure.

[0025] FIG. 5A is a transmission electron microscopy (TEM) image of the ICG / BPs including zoomed in images of individual ICG / BPs.

[0026] FIG. 5B is a plot of ICG / BP particles size distribution.

[0027] FIG. 5C is a plot of the average ICG / BP particle size using dynamic light scattering.

[0028] FIG. 5D is a plot of ICG / BP zeta potential measurement.

[0029] FIG. 5E is a plot of the protein characterization of the ICG / BPs my mass spectrometry.

[0030] FIG. 6A is a photograph (top) and fluorescence image (bottom) of ICG / BPs post- svnthesis.

[0031] FIG. 6B is plot (bottom) of loading efficiency (LE) of the ICG / BPs using different initial concentrations of ICG during synthesis, with a decreased loading efficiency consistent with a greener supernatant in the photographs (top). One way ANOVA followed by group post hoc comparison was performed on select data, statistical significance is quantified as: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05, ns = no significance.

[0032] FIG. 6C is a plot of absorption measurements of ICG (5 pM) and the ICG / BPs (10 pM encapsulated ICG), showing two characteristic peaks in the NIR region represented by the blue bar (-720 nm) and red bar (-780 nm), with the ICG / BPs displaying a slight red shift of -1 fl- 20 nm.

[0033] FIG. 6D is a plot of fluorescence intensity of equivalent concentrations of ICG / BPs and free ICG solutions.

[0034] FIG. 6E is a plot (bottom) of the long-term photostability of the ICG / BPs when either constantly exposed (top left) or sheltered (bottom right) from light. One way ANOVA followed by group post hoc comparison was performed on select data, statistical significance is quantified as: ****p < 0.000 E ***p < 0.001; **p < 0.01; *p < 0.05, ns = no significance.

[0035] FIG. 6F is a plot of the degradation of the ICG / BPs as measured by cumulative protein loss (mg) over 42 days.

[0036] FIG. 6G is a plot of ICG release from the ICG / BPs at different timepoints over the course of 5 days.

[0037] FIG. 7A show confocal laser scanning microscope images of BMSCs co-cultured with ICG / BPs (bottom) and a control (top).

[0038] FIG. 7B show transmission electron microscope images of BMSCs co-cultured with ICG / BPs (bottom) and a control (top) at vary ing magnifications.

[0039] FIG. 7C is a series of flow cytometry plots of cellular uptake of control (left), ICG / BPs at 24 h (middle), and ICG / BPs at 48 h (right).

[0040] FIG. 7D shows images of ICG / BP cytotoxicity' after 4-week cell culture ofBM-MSCs. The cytotoxicity of the ICG / BPs was determined by Live / Dead staining (top row) and osteogenic differentiation was evaluated with RUNX2 (middle top row) and OCN (middle bottom row) staining, and calcium deposition (bottom row).

[0041] FIG. 8 A is a diagram of surgically created tibial defect filled with ICG / BP powder, according to aspects of the present disclosure.

[0042] FIG. 8B shows computed tomography (CT, first column), three-dimensional CT (3D- CT, second column), hematoxylin and eosin (H&E. third column), and Goldner’s staining (fourth column) images of the in vivo biocompatibility of ICG / BPs in a tibial defect.

[0043] FIG. 8C shows in vivo near infrared (NIR) imaging of ICG / BPs and associated CT images of a tibial defect over an 8-week time period. One way ANOVA followed by group post hoc comparison was performed on select data, statistical significance is quantified as: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05, ns = no significance.

[0044] FIG. 8D is a series of NIR images of a tibial defect with implanted ICG / BPs postsurgery (top) over the course of 8 weeks, and the corresponding CT of the bone (bottom).

[0045] FIG. 8E is a plot of mean collected data of NIR intensity over 8 weeks.

[0046] FIG. 9A shows the dynamic in situ monitoring of ICG / BPs and biodistribution. (A) White light images (column 1) were used to subtract background fluorescence to obtain the pure NIR-II spectrum image both pre- (columns 2) and post-IV administration (columns 3). PCA (column 4) was used to over timely quantify segment anatomical regions of interest, in this case the liver.

[0047] FIG. 9B is a series of ex vivo fluorescence imaging of the heart, lungs, liver, and kidneys to determine the biodistribution of the ICG / BPs up to 72 hours post-administration.

[0048] FIG. 9C is a plot of the measured normalized intensity of both ICG and ICG / BPs collected from PCA analysis, with a noticeably later peak of intensity in the liver for the ICG / BPs than ICG.

[0049] FIG. 9D is a plot of the mean measured fluorescence of intensity in the heart collected from (B).

[0050] FIG. 9E is a plot of the mean measured fluorescence of intensity in the lungs collected from (B).

[0051] FIG. 9F is a plot of the mean measured fluorescence of intensity in the liver collected from (B).

[0052] FIG. 9G is a plot of the mean measured fluorescence of intensity in the kidney collected from (B).

[0053] FIG. 10 is a series of H&E staining of organs after 15 days of administration. Images of lung (column 1), liver (column 2), kidney (column 3), and spleen (column 4) in different experimental groups didn’t show any obvious adverse effects on organs. (Br = Bronchiole, BV = Blood Vessel. PV = Portal Vein, BD = Bile Duct. GL = Glomerulus, BC = Bowman’s Capsule, DCT = Distal Convoluted Tubule, PCT = Proximal Convoluted Tubule, CA = Central Arteriole).

[0054] FIG. 11 shows images of an exposed bone (left), a defect in the bone prior to implantation (middle left), the defect filled with ICG / bone morphogenic protein 2 (BMP2) / BPs (middle right), and sutures closing the w ound (right).

[0055] FIG. 12A is a series of fluorescence images over time of a cranial defect filled with ICG / BPs (top) or ICG / BMP2 / BPs (bottom).

[0056] FIG. 12B is a plot of the fluorescence intensity of the images of FIG. 12A of the cranial defect filled with ICG / BPs or ICG / BMP2 / BPs.

[0057] FIG. 13 is a series of images (top) and CT images (bottom) showing cranial defect repair in control, BPs only, and BMP2 / BPs after 8 weeks.

[0058] FIG. 14A is an SEM image of crosslinked (X) BPs, according to aspects of the present disclosure.

[0059] FIG. 14B is an SEM image of uncrosslinked (UnX) BPs, according to aspects of the present disclosure.

[0060] FIG. 15 is a methodology of implantation of either X ICG / BPs or UnX ICG / BPs for alveolar cleft palate defect repair.

[0061] FIG. 16A is a time series of fluorescence images of X ICG / BPs in the alveolar cleft palate defect repair methodology of FIG. 15. Top row: fluorescence (FL); Bottom row: spectral unmixing of fluorescence signals (SPUM).

[0062] FIG. 16B is a time series of fluorescence images of UnX ICG / BPs in the alveolar cleft palate defect repair methodology of FIG. 15. Top row: fluorescence (FL); Bottom row: spectral unmixing of fluorescence signals (SPUM).

[0063] FIG. 17 is a plot of fluorescence over time of the fluorescence images of FIGS. 16A- 16B.

[0064] FIG. 18 A is a series of images of the alveolar cleft palate defect and repair using either UnX ICG / BPs or X ICG / BPs after 8 weeks.

[0065] FIG. 18B is a series of CT images (top) and 3D-CT images (bottom) of the alveolar cleft palate defect and repair using either UnX ICG / BPs or X ICG / BPs after 8 weeks.Detailed Description

[0066] According to aspects of the present disclosure, a composition for orthopedic regeneration is provided (FIGS. 1A-1B). In a non-limiting example, the term “orthopedic” may refer to bones, ligaments, tendons, muscles, and nerves. In a preferred embodiment, the composition herein is specific to bone. The composition comprises nanoparticles comprising bone extracellular matrix (ECM). As used here, nanoparticle refers to a particle ranging in diameter from 1 to 500 nm. Alternatively, the nanoparticles may have a size in a range of 10 to 250 nm. In another non-limiting example, the nanoparticles may have a size range of 100 to 400 nm. In one non-limiting example, the ICG / BPs in aqueous suspension range in diameter from 50 to 200 nm.

[0067] Referring back to FIG. 1 A. a non-limiting example of generating bone ECM from porcine tibiae is shown. As will be described in further detail below, the bone is demineralized with one or more acids and decellularized with at least one of a detergent and a surfactant to form bone ECM. Thereafter, the bone ECM may be lyophilized and milled into a powder of bone ECM particles. The resulting powder may then be reconstituted in one or more acids, digested with one or more proteolytic enzymes, and desalted to produce the ECM stock solution for further processing.

[0068] The composition further includes a fluorescent dye associated with each nanoparticle or at least a portion of the nanoparticles. As used herein, a dye is '’associated" with a particle if the dye is directly or indirectly, physically or chemically bound to the particle. Nonlimiting examples of chemical bonds include covalent bonds, ionic bonds, coordinate bonds, and hydrogen bonds. Indirect bonding can include the use of a group of atoms (i.e. , a linker) that chemically links the dye and the particle. Non-limiting examples of physical bonding include adsorption, absorption, and encapsulation. The term "encapsulation" refers to the confinement of a dye within a particle.

[0069] In other non-limiting examples, the nanoparticles may be associated with, but not limited to, a “bioactive agent”, which as used herein includes, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body. A bioactive agent is a substance used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness, or a substance which affects the structure or function of the body, or which becomes biologically active or more active after it has been placed in a predetermined physiological environment. Bioactive agents include, without limitation, enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, allograft bone, gene constructs, and therapeutics. Exemplary7bioactive agents for use with the present invention include bone morphogenetic proteins (“BMPs”) and demineralized bone matrix (“DBM”) as these agents promote the growth of bone, thereby aiding the fixation process. For example, the nanoparticles may encapsulate bone morphogenic protein 2. In another example, peptides may be bound to the surface of the nanoparticles for targeting bone tissue. In further embodiments any combination of fluorescent dyes and therapeutic agents may be associated with the nanoparticles utilizing multiple encapsulation and grafting strategies.

[0070] In a non-limiting example embodiment, the fluorescent dye is a cyanine dye. For example, the fluorescent dye is indocyanine green (ICG). Alternatively, the fluorescent dye may be, but is not limited to, coumarin, rhodamine, xanthene dyes, or derivatives thereof.

[0071] FIG. IB shows a non-limiting example of ECM stock processing by forming bone ECM particles. For example, the fluorescent dye particles, such as ICG is dissolved in the ECM stock solution. Alternatively or additionally, one or more bioactive agents may be added to the ECM stock solution. Acetone may then be added to a mixture of ICG and the ECM stock solution and stirred to form un-crosslinked (UnX) ICG / BPs. Alternatively or additionally, a crosslinking agent may be added to the mixture and stirred to form crosslinked (X) ICG / BPs. In a non-limiting example, the crosslinking agent may be glutaraldehyde.

[0072] In a non-limiting example embodiment, an amount of dye is encapsulated by each nanoparticle of the portion of the nanoparticles. In a non-limiting example embodiment, the fluorescent dye is encapsulated within the nanoparticles, wherein a ratio of the dye to the nanoparticles has an encapsulation efficiency in the range of 13-15 % weight / weight (w / w) based on total weight of the nanoparticles. In a non-limiting example, the encapsulation efficiency may be smaller or greater. As described herein, “encapsulation efficiency7’ refers to the percentage fluorescent dye that is successfully entrapped within the nanoparticles. Encapsulation efficiency is calculated by(total fluorescent dye - non-entrapped fluorescent dye) total fluorecent dye

[0073] In a non-limiting example embodiment, the composition has a photostability of 45% intensity or greater over a 12-week period. In another non-limiting example, the composition has a photostability of 30% intensity or greater over a 1-year period. As used here, the term “photostability” refers to the response of the fluorescent dye to the exposure of electromagnetic radiation, such as visible light, that leads to a physical or chemical change.

[0074] In a non-limiting example embodiment, the bone extracellular matrix comprises demineralized and decellularized bone. The method of demineralizing and decellularizing bone is described in further detail below.

[0075] In a non-limiting example embodiment, the bone extracellular matrix comprises an entire extracellular matrix of mammalian bone. Further, the bone extracellular matrix may comprise an entire extracellular matrix of acellular porcine bone. For example, the nanoparticles comprise at least two of collagens I. II, V, VI, XI, XII, XIV, and XXII. osteomodulin (OMD), fibronectin, aggrecan, biglycan, decorin, chondroadherin,fibromodulin, calcium-binding proteins (CBPs), and albumin, and grow th factors, such as transforming growth factor-beta (TGF-0), beta-enolase (ENO3), phosphoglycerate kinase (PGK), peptidyl-prolyl cis-trans isomerase (PPIA), and pigment epithelium-derived factor (PEDF).

[0076] According to aspects of the present disclosure, a method of making particles for orthopedic regeneration is described and shown in FIG. 2A. In a non-limiting example embodiment, the method 200 comprises providing a solution including particles comprising bone extracellular matrix at step 202 and adding fluorescent dye to the solution at step 204 to associate an amount of the dye with each particle of at least a portion of the particles. The nanoparticles may be those described by the composition described above.

[0077] As shown in a non-limiting example embodiment of FIG. 2B, providing the solution including particles comprising bone extracellular matrix comprises the steps of demineralizing a bone sample with one or more acids at step 206, decellularizing the bone sample with at least one of a detergent and a surfactant at step 208, lyophilizing the demineralized and decellularized bone sample at step 210, and milling the lyophilized bone sample into a powder of the nanoparticles at step 212. In one example, the acid for demineralizing bone includes, but is not limited to, hydrochloric acid (HO), ethylenediamine-tetraacetic acid (EDTA), formic acid (CH2O2), and mixtures thereof. In one example the at least one detergent and surfactant includes, but is not limited to, the ionic detergent sodium dodecyl sulfate (SDS) and non-ionic detergent Triton X-100. Alternatively, other chemical decellularization methods may be used such as the use of acids, bases, and chelating agents. For example, acetic acid, HCL, sulfuric acid, and peracetic acid (PAA) are acids that may be used for bone decellularization. In another example, bases such as sodium hydroxide, sodium sulfide, and calcium hydroxide may be used. Chelating agents for bone decellularization may include EDTA and ethylene glycol tetraacetic acid (EGTA). Alternatively, enzy matic or physical techniques may be employed for bone demineralization. For example, proteases such as trypsin and nucleases such as RNases and DNases may be used in the decellularization process. Physical techniques include repeated freeze-thawing, supercritical carbon dioxide, and high hydrostatic pressure application.

[0078] In a non-limiting example embodiment, providing the solution including particles comprising bone extracellular matrix further comprises the steps reconstituting the powder in one or more acids at step 214, digesting the reconstituted bone sample with one or more proteolytic enzymes at step 216. and desalting the digestion at step 218 to produce the solution. Further details of steps 214-218 are described in the example below.

[0079] In a non-limiting example embodiment, adding a fluorescent dye to the solution to associate an amount of the dye with each particle of at least a portion of the particles further comprises adding the fluorescent dye to the solution in the range of 5-20% w / w based on total weight of the solution. As described previously, the fluorescent dye may be ICG.

[0080] According to another aspect of the present disclosure as shown in FIG. 3, a method 300 of treating an orthopedic defect is provided. The method comprises lyophilizing the composition described above at step 302, filling the orthopedic defect with the lyophilized composition at step 304, and monitoring the orthopedic defect over a duration using a medical imaging technique 306.

[0081] In a non-limiting example embodiment, the medical imaging technique includes at least one of, but is not limited to, fluorescence imaging and fluoroscopy imaging. Fluoroscopic imaging may include X-ray fluoroscopy or CT.

[0082] In a non-limiting example embodiment, the orthopedic defect comprises a bone defect.

[0083] The following Examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present invention and is not to be construed as limiting the scope of the invention. The statements provided in the Example are presented without being bound by theory'.Example 1

[0084] The compositions developed herein provide a new type of nanoparticles using the decellularized bone ECM (bone-based particles, BPs). The following examples described the systematic characterization of the particle properties, including particle size, surface property', protein content, degradation rate, cellular uptake, and their potential use as a therapeutic strategy’ for bone treatment either through local or systemic administration. To better detect the in vivo distribution, degradation, and systemic clearance of these particles, Indocyanine Green (ICG) was encapsulated with BPs (ICG / BPs) for non-invasive, fluorescence image- guided treatment. This is believed to be the first instance of developing protein-based NPs using the entire complex of ECM components when derived directly from the acellular porcine bone.Overview of the Example

[0085] Recent effort focuses on developing image-guided therapies that can monitor hostmaterial interactions, quantitatively assess bone healing, and evaluate the performance of drugs or grafts in a real-time, non-invasive, high-sensitive, and cost-effective way. Nanomedicine is a fast-developing field in medical science that is focused on developingnanoscale materials with valuable applications for diagnosis, prevention, and treatment of a number of diseases. As an important tool of nanomedicine, NPs have been extensively studied in such areas as drug and gene delivery, vaccine adjuvants, disease diagnosis and therapy, and biomedical imaging, due to their extraordinary advantages including low toxicity , enlarged drug-loading ability, tailorable characteristics, controllable physical stability’, intracellular uptake, and targeted and on-demand drug release [Ref. 15-18], Unfortunately, there are no FDA approved NP options for clinical orthopedic treatment currently due to some potential risks, such as elevated risks of low biocompatibility and delivery’ efficiency, potential toxicity’ and side effects from excessive accumulation in healthy organs, probable platelet aggregation, and induced inflammation and tissue damage [Ref. 19- 24], Much more efforts are needed to develop new NPs for improving safety and therapeutic efficiency for hard-to-treat bone diseases [Ref. 25, 26], Numerous types of inorganic (such as hydroxyapatite, silica, metals, calcium phosphate, etc.) and organic materials (such as synthetic polymers, chitosan, liposomes, proteins, etc.) have been formulated into NPs [Ref. 27-30], Among these particles, protein-based NPs are attracting increased attention because of their abundance in natural sources, excellent biocompatibility and cellular uptake, biodegradability, cell-binding ability’, amenability for surface modification and drug loading, self-assembly ability7, reproducibility’, as well as controllable size and physical properties [Ref. 31-34], Moreover, protein-based NPs can elongate the half-life of small proteins and peptides in the body and should possess longer circulation time in the bloodstream, which will largely improve their stability and therapeutic efficacy following systemic administration [Ref. 34, 35], Up to date, multiple ty pes of proteins that are either derived from animals (albumin, collagen, gelatin, lipid, milk, egg white, etc.) [Ref. 36-46] or plants (gliadin, soy protein, zein, legumin, etc.) [Ref. 47-51] have been developed into NPs with desired properties for a wide range of applications in food, industry, and medicine. Extracellular matrix (ECM) proteins, produced from decellularization of animal or human tissues, have attracted significant interest as biomaterials in the field of tissue engineering and regenerative medicine [Ref. 52, 53], After decellularization, the majority of the cellular components are removed but a network of macromolecules, such as collagen, elastin, fibronectin, laminin, and specific growth factors, can be preserved in the ECM [Ref. 54-57], Although ECM derived from different tissue types share major structural and componential similarities, many studies have demonstrated that the construction and composition of biomarkers in ECM vary from tissue to tissue, thus making them unique for applications particular to its original tissue ty pe [Ref. 58-61], Bone is a heterogeneous composite tissue which is composed of a dynamicECM network consisting of 50-70% inorganic minerals, 20-40% organic matrix, and 5-10% water [Ref. 6, 7], The inorganic minerals, mainly consisting of calcium phosphate (CaP), specifically hydroxyapatite (HA), are the source for the hardness and strength of the bone, while the organic matrix, including collagens and non-collagenous proteins, provide the tissue with flexibility and a template to regulate the formation of new bone [Ref. 62, 63], Collagens, especially collagen type I, are the most abundant proteins found in bones, which play a critical role in the structure, tissue organization, and mechanical support of the bones [Ref. 64, 65], Non-collagenous proteins, such as proteoglycans, glycoproteins, y Carboxy glutamic acid-containing proteins, and other serum-derived proteins, are associated in regulating fundamental cellular processes (such as attachment, proliferation, differentiation, and migration), tissue mineralization and regeneration, and mechanical properties [Ref. 66, 67], The inventors have previously developed a gellable bone ECM from the demineralized and decellularized porcine bone and demonstrated its preservation of the collagenous and non-collagenous proteins from the native bone, solubility, biodegradability, low toxicity and immunogenicity, and enhanced osteointegration in repairing large-size bone defects [Ref. 68J. The flexibility of the bone ECM allows it to be processed into various forms for a variety of applications, such as complete scaffold, particles, powders, segments, bio-ink, and hydrogels, as a promising biomaterial for bone-related research and treatment [Ref. 69, 70], These promising results are the inspiration behind developing a new nanostructured biomaterial based on the bone ECM with the common aim of providing a new strategy for bone treatment and diagnosis.1. Materials and Methods

[0086] Bone decellularization and preparation of bone ECM "‘stock” solution: Fresh porcine tibias were collected from approved Medical College of Wisconsin (MCW) vendors and decellularized with the same protocol as previously described [Ref. 68], in which the tibias were sectioned into small pieces and fully demineralized with 0.5 M hydrochloric acid (HCL, Sigma-Aldrich, St. Louis, MO) for ~3 days and decellularized with 0.5% sodium dodecyl sulfate (SDS) and 1% Triton X-100 mixed solution (Sigma- Aldrich, St. Louis, MO) for another ~5 days under agitation at room temperature until all the bone pieces became pale white and porous appearance (FIG. 1 A). After entirely washing with distilled water, the bone ECMs w ere freeze-dried at -80 °C (Cole-Parmer, Vernon Hills, IL) and milled into a fine powder using a Thomas Wiley® Mini Cutting Mill (Thomas Scientific, Swedesboro. NJ).

[0087] The ECM powders were pre-treated with HCL solution (1 gram of ECM powder / 100 ml of IM HCL) under constant stirring for 48 hours at 4 °C and then the pH of such solutionwas adjusted to 2.5 by adding 50% sodium hydroxide (NaOH, Sigma- Aldrich, St. Louis, MO) solution. After stirring for another 15 minutes at 4 °C. the whole solution was moved to a hot stir plate with a temperature setting at 45 °C. Pepsin (Sigma- Aldrich, St. Louis, MO) was then added into the whole solution at 15% w / w and left to stir for 48 hours at 45 °C. A final adjustment to the pH was made using NaOH, bring the final pH = ~7. The digestion is then vacuum filtered using a 140pM nylon net filter. After filtration, the final bone ECM digestion was additionally desalted by adding acetone dropwise to the solution at a 1: 1 v / v ratio following by centrifugation at 8,000 RPM (6,000 x g) for 20 minutes. The resulting pellet was collected and resuspended in distilled water by vertexing and immersion blending, hereby making the ECM ‘stock’ solution (FIG. 1A). The protein concentration of the ‘stock' solution was measured using the Pierce™ bicinchoninic acid assay (BCA assay, ThermoFisher Scientific, Waltham, MA) and further adjusted to a final concentration of 330 ug / ml. The final stock solution could be stored at -20 °C until needed.

[0088] Fabrication of ICG / BPs: As indicated in FIG. IB, ICG dye (Sigma-Aldrich, St. Louis, MO) was dissolved in the ECM ‘stock’ solution at 10% w / w based on the total protein amount (mg) of the ECM ‘stock’ solution. Acetone was then added dropwise into the ICG and ECM mixed solution (the volume ratio of acetone to ICG / ECM solution was 3: 1) at a rate of 1 ml / min with a syringe pump under constant stirring at 500 RPM at room temperature. 50% glutaraldehyde solution was then added into the above solution, with a ratio of 66 ul of glutaraldehyde per 1 ml of the initial solution volume (pre-desolvation) under constant stirring at 500 RPM for 30 minutes at room temperature for particle crosslinking. The final ICG / BPs were washed with distilled water three times by centrifugation, and the resulting pellet w as redispersed into distilled water by sonication using a Branson® Sonifier 150 (Brookfield, CT).

[0089] We also prepared non-ICG-encapsulated BPs (BPs) for cell culture and immunofluorescence staining. The BPs were prepared with the same protocol as making the ICG / BPs excluding the ICG loading step. The obtained ICG / BPs or BPs were freeze-dried at -80 °C for experimental characterization or long-term storage (FIG. 1C).

[0090] Morphological characterization of ICG / BPs: For transmission electron microscopy (TEM), the lyophilized ICG / BPs were dissolved in 0.1 M sodium phosphate buffer (PBS, pH = 7.4) by Bonification and fixed in 1% Osmium tetroxide (Sigma- Aldrich, St. Louis, MO) for 2 hours at room temperature. After fixation, the particles were washed with PBS buffer following by another washing with distilled water. The final particle samples were depositedonto a 400 mesh formvar-carbon-coated copper grid, air-dried at room temperature, and imaged with a JEOL 2100 LaB6 microscope (200 kV).

[0091] A Malvern Zetasizer 3000 dynamic light scattering (DLS) system was used to measure the hydrodynamic size distribution of the fabricated ICG / BPs. For these measurements, pellets of ICG / BPs were resuspended in ImL of Milli-Q water and sonicated for 3 minutes on ice. DLS measurements were taken at 25°C. The approximate net charge on the surface of the ICG / BPs was calculated as the Zeta (Q potential. Zeta potential was calculated using the Smoluchowski approximation, and parameters optimized for protein in solution were chosen for particle characterization. Raw data was exported and analyzed using Microsoft Excel spreadsheet. For reproducibility', five measurements (n=5) of each sample were collected.

[0092] Mass Spectrometry (MS): The MS followed the protocol introduced by Moriya Slavin et al. [Ref. 71] to identify the protein components in the ICG / BPs. The ICG / BP samples were resuspended in 20 pL of 8 M urea with 10 mM DL-Dithiothreitol (DTT) by sonication, lodoacetamide was then added to a final concentration of 25 mM and the alkylation reaction was proceeded for 30 min. The samples were further digested with trypsin at a 1 : 100 protease-to-protein ratio overnight at 37°C under agitation. Following digestion, the samples were cleaned using the PreOmics Phoenix kit according to the manufacturer’s directions. Samples were then dissolved in 2% acetonitrile 0.1% formic acid and analyzed on a Thermo Scientific Orbitrap Fusion Lumos MS. MS data was analyzed using the Proteome Discoverer 2.4 (Thermo Scientific, Waltham, MA) platform and protein identifications were filtered to include only those proteins identified by two or more unique peptides identified and ranked as high confidence.

[0093] Thermogravimetric Analysis (TGA): The thermal behaviors of the ICG / BPs were characterized by TGA, including water loss, phase changes, and decomposition. Weighed amounts of lyophilized ICG / BPs were placed into a pan and inserted into a Thermal Gravimetric Analyzer SDT650 (TGA, TA Instruments, DE). A ramp up temperature of 10°C per minute was selected, starting at room temperature, and recording to 800°C. The starting and ending weight of the sample was recorded and stored, and all relevant data exported into Excel spreadsheet for analysis.

[0094] Determination of loading efficiency: For Loading efficiency (LE) measurements of ICG within the ICG / BPs, the absorbance of the samples at wavelength -780 nm was measured using a Gen5® plate reader. The absorbance of each sample was compared with the standard calibration curve (SI. 1) of ICG to determine the approximate ICG concentrationwithin each sample. ICG was dissolved in the ECM ‘stock’ solution at different concentrations (5, 10, 15, 20% w / w) based on the total protein amount (mg) of the ECM ‘stock’ solution, and ICG / BPs were synthesized as described as section 2.2. LE was used to estimate the loaded amount of ICG within the BPs, using the following equation (1):LE (ICG) (%) = [1 - (cone, of ICG in supernatant after ICG / BPs forming) / (total cone, of ICG in the ICG / ECM mixed solution)] * 100%.

[0095] In vitro Degradation Rate: The lyophilized ICG / BPs were weighed and fully immersed into 2 ml of PBS solution and incubated at 37°C for 6 weeks. Each week during the particle incubation (n = 4 at each time point), 100 pL of the top solution was collected and the protein concentration in the collected solution was measured by a BCA assay according to the manufacturer protocol. In vitro degradation rate of the ICG / BPs was calculated using the following equation (2):In vitro degradation rate (%) = [total protein loss at each time point (mg) / initial dry weight of the sample (mg)] *100%.

[0096] In vitro ICG Release: To investigate the in vitro ICG release rate of ICG / BPs, 10 mg of the lyophilized ICG / BPs was dissolved in 1 ml of plasma isolated from rat blood in an eppendorf tube and kept at 37 °C for 5 days. At pre-determined time intervals (0.5. 1, 2, 3, 4. 8, and 16 hours and 1, 2, 3, 4, and 5 days), the tubes were centrifuged, and the supernatant was removed and replaced with the same amount of the fresh plasma. The particles in each tube were re-suspended and placed back into 37 °C for incubation. The absorbance of the supernatant at each time point was measured at 780 nm for ICG using a Gen5™ Microplate Reader and Imager Software package (BioTek, Winooski, VT). The ICG release rate of ICG / BPs at each time point was calculated using the equation (3):ICG release rate (%) = (At / Ao) * 100% where At was the absorbance of the amount of ICG measured in the supernatant at each time point and Ao is the absorbance of the amount of ICG in the ICG / ECM mixed solution before ICG / BPs formation. The release study was performed in triplicate for each time point (n=3), with all samples kept in black test tubes. An effort was made to prevent samples from any unnecessary light exposure during sample handling.

[0097] Fluorescent Intensity: The fluorescent intensity of the ICG / BPs was evaluated as both lyophilized powder and liquid forms by an IVIS Spectrum CT Fluorescent Imager (PerkinElmer, MA, USA). To prepare the liquid ICG / BP solution, the lyophilized ICG / BPswere resuspended in distilled water at different concentrations: 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1 mg / mL, respectively, based on the dry’ weight of the ICG / BPs (The corresponding molar concentrations of the encapsulated ICG in these solutions were calculated based on the initial ICG concentration used to fabricate the ICG / BPs: 17.0, 22.7, 28.4, 34. 1, 39.7, 45.4, 51.1, 56.8 uM, respectively). As a fluorescence standard, free ICG powder was dissolved in both distilled water and 10% Bovine Serum Albumin (BSA) solution at different molar concentrations: 3.1, 6.3, 12.5, 25, 50, 100. 200, and 500 uM, respectively.

[0098] Fluorescence measurements and photostability': The SWIR imaging system used for static and dynamic fluorescence imaging was comprised of an 808nm diode laser (Diomed, DI 5 Plus) used for illumination, an InGaAs focal plane array (Princeton Instruments, NIRvana 640ST) with a 25mm lens (Navitar, SWIR-25), in conjunction with 808nm notch (Semrock, StopLine®, NF03-808E-50,) and 950nm long pass (Semrock, EdgeBasic™, BLP01-980R-50) filters. For static fluorescence imaging of bone regeneration, an 808nm power density' of approximately 5mW / cm2was used. Dynamic contrast enhanced imaging was performed with approximately 5-25mW / cm2for free ICG and ICG / BPs respectively, with a temporal resolution of 100ms.

[0099] An IVIS Spectrum CT fluorescent animal imager (PerkinElmer, MA, USA) was additionally used for fluorescence analysis. Samples were placed into the imaging chamber and presets were chosen using the Livingimage® software package specific to ICG (Excitation / Emission = 745 / 840 nm). Images were analyzed using the Livingimage® softw are, and the quantified fluorescent intensity was represented by the average radiant efficiency [(p / s / cm2 / sr) / (pW / cm2)] after background subtraction.

[0100] Free ICG dye was mixed into Milli-Q water at a starting concentration of 1000 pM and diluted into various concentrations to create a standard reference curve. To better simulate physiological conditions of ICG in blood plasma, a standard reference curve was also created using free ICG dye diluted in 10% BSA solution. All references were aliquoted into black-bottomed well plates to prevent fluorescent bleed-through.

[0101] A theoretical loading efficiency of ICG at 100% was used to create the scale for a concentration curve of ICG / BPs. The fluorescence intensity of Aliquots of ICG / BPs (lOOuL) that are equivalent to 40, 20, 15, 10, and 5 pM of ICG were measured and compared to the ICG standard reference curves.

[0102] Long-term ICG Stability: To investigate the long-term fluorescence stability of ICG / BPs, ICG / BP powders (lOmg) were placed in Eppendorf tubes and kept either exposedto light constantly or in the dark at room temperature. The fluorescent intensity was measured weekly for up to 12 weeks.

[0103] Cellular uptake of ICG / BPs and cellular compatibility: Human bone marrow-derived mesenchymal stem cells (BMSCs) were purchased from Lonza Inc. (Morristown, NJ). The cells were resuspended and seeded in 75 mm flasks at a density of 103cells / cm2with Mesenchymal Stem Cell Growth Medium (Lonza Inc.) at 37 °C and 5% CO2 atmosphere. The third passage of the BMSCs were seeded at a density of 103cells / cm2in each well of the 24-well cell culture plate. After the cells reached 80% confluence, the cells in plate were randomly divided into the following groups: 1) BMSCs co-cultured with ICG / BPs: Lyophilized ICG / BPs were dispersed in the osteogenic differentiation medium [DMEM. 10% FBS, 1% penicillin, streptomycin and dexamethasone (0.1 uM). b- glycerophosphate (10 mM), and ascorbic acid (50 uM), Lonza Inc.] at the concentration of 50 pg / ml by Bonification and 0.5 ml of ICG / BPs mixed medium was added into each well of the cell culture plate. The ICG / BPs and BMSCs co-culturing was used for intracellular uptake testing. To better characterize the cell viability’ and osteogenic differentiation of the BMSCs. BPs (non-ICG encapsulated) were co-cultured with BMSCs using the same method as the ICG / BPs. After 24 hours’ co-culturing, the medium was changed to the osteogenic differentiation medium (no ICG / BPs or BPs in the medium). 2) Differentiation control group: BMSCs were cultured with 0.5 ml of osteogenic differentiation medium in the cell culture plate (no particle co-culturing). 3) Control group: BMSCs were cultured with 0.5 ml of Mesenchymal Stem Cell Grow th Medium in the cell culture plate (no particle co-culturing). All the cells from the above groups were cultured at 37 °C in a 5% CO2 atmosphere for 4 w eeks with the medium change twice a week.

[0104] Intracellular Uptake: After BMSCs were co-cultured with ICG / BPs for 24 hours, the cells were washed with PBS three times and incubated with Rhodamine Phalloidin (abeam, Waltham, MA) for 20 minutes at room temperature for actin filament staining. The cell nucleus was double stained with 4',6-diamidino-2-phenylindole (DAPI, abeam, Waltham, MA) at 1 ug / ml. The cells were observed using a Nikon Al Laser Scanning Confocal Microscope. The cells with no ICG / BPs co-culturing were used as controls.

[0105] TEM was used to identify the intracellular location of ICG / BPs. After the BMSCs were co-cultured with ICG / BPs for 24 hours, the cells were fixed in 2.5% glutaraldehyde at 4 °C overnight, washed with PBS, and fixated with 1% osmium tetroxide at 4 °C for 4 hours. After fixation, the cells were stained with 2% uranyl acetate, dehydrated in a graded ethanolacetone series, and then embedded in pure Epon resin. After slicing with a ultramicrotome(Leica, Wetzlar, Germany), the final cell sections were mounted on 200 mesh copper grids and examined with a JEOL 2100 LaB6 microscope operated at an accelerating voltage of 100 kV. The cells with no ICG / BPs co-culturing were used as controls.

[0106] Flow cytometry was used to quantify the intracellular uptake of ICG / BPs. After the BMSCs were co-cultured with ICG / BPs for 24 hours and 48 hours, respectively, the cells were washed with PBS solution for three times, detached with TrypLE Reagent (Fisher scientific, Hampton, NH), and collected by centrifugation at 2000 rpm. followed with two times of PBS washing. The cell suspension was filtered through 400-mesh sieves and subjected to a BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences, Franklin Lakes, NJ). The cells with no ICG / BPs co-culturing were used as controls.

[0107] Cell Viability: After 4 weeks of co-culture, cells from different groups were stained with the Live / Dead Cell Viability Assay (ThermoFisher Scientific Inc., Waltham, MA) to determine cell viability, and the cell nuclei were double stained with 1 ug / ml DAPI.

[0108] Osteogenic differentiation: To determine the osteogenic differentiation after 4 weeks of co-culture, cells from different groups were fixed with 4% paraformaldehyde for 30 minutes and blocked with 5% normal serum / 0.3% Triton X-100 solution for 60 minutes. The cells w ere then incubated with primary antibodies [(Monoclonal Anti- osteocalcin antibody (1 : 100 dilution, abeam, Cambridge, MA) and the Anti-RUNX2 antibody (1: 100 dilution, abeam, Cambridge, MA)] overnight at 4 °C. After that, cells were washed three times with PBS and stained with the secondary antibody. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (1 :200 dilution, abeam, Cambridge, MA), for 1 hour at room temperature. Negative control samples w ere not subj ected to primary' antibody incubation. After washing three times with PBS, the nuclei were double stained with 1 ug / ml DAPI. The cells were observed using a Nikon Al Laser Scanning Confocal Microscope. To measure calcium deposition, cells from different groups were fixed with 10% formalin for 30 minutes followed with three times of PBS w ashing and three times of w ashing with distilled water. The cells were then stained with 1 x alizarin red S solution (Millipore, Billerica, MA) for 30 minutes, and washed four times with distilled water. Cell images were then captured with a light microscope.

[0109] Alizarin Red S Staining: Cells from different groups were fixed with 10% formalin for 30 minutes following with three times of PBS w ashing and three times of washing with distillated water. The cells were then stained with 1 x alizarin red S solution (Millipore, Billerica, MA) for 30 min, and w ashed four times with distilled w ater. Cell images w ere then captured with a light microscope.

[0110] Confocal Laser Scanning Microscope: After 24 hours of co-culturing with BMSCs and ICG / BPs, the cells were washed with PBS three times and incubated with Rhodamine Phalloidin (abeam, Waltham, MA) for 20 minutes at room temperature for actin filament staining. The cell nucleus was double stained with 4',6-diamidino-2-phenylindole (DAPI) at 1 ug / ml. The cells were observed using a Nikon Al Laser Scanning Confocal Microscope. The cells in the control group were used as the control cells.

[0111] TEM: TEM was used to identify the intracellular location of ICG / BPs. After 24 hours of co-culturing the ICG / BPs with the BMSCs, the cells were fixed in 2.5% glutaraldehyde at 4°C overnight, washed with PBS, and fixated with 1% osmium tetroxide at 4°C for 4 h. After fixation, the cells were stained with 2% uranyl acetate, dehydrated in a graded ethanolacetone series, and then embedded in pure epoxy resin. After slicing with a ultramicrotome (Leica, Wetzlar, Germany), the final cell sections were mounted on 200 mesh copper grids and examined with a JEOL 2100 LaB6 microscope operated at an accelerating voltage of 100 kV. The cells in the control group were used as the control cells.

[0112] In vivo evaluation of ICG / BPs for tibial defect repair: Sprague Dawley (SD) rats (Charles Rivers, Inc., 8-10-week-old, males and females) were used and split into 3 groups: Tibial receiving no defect (control group, n=4), tibial defect without particle implantation (defect group, n=4), and tibia defect implanted with ICG / BPs (ICG / BP group, n=4). The rats in the control group didn’t undergo any surgical procedures. All the rats in the defect group and the ICG / BP group were anesthetized using isoflurane via inhalation. The fur on the right leg was shaved and skin was sterilized using iodine solution. A small incision was made to expose the tibia in right leg. A cylindrical bone defect (4 mm diameter x 2 mm thickness) was created in the proximal portion of the tibia using a surgical drill while irrigating with cold 0.9 % sterile saline solution. The defect was either not treated (defect group) or fully filled with lyophilized ICG / BPs (ICG / BP group, FIG. 8A). Muscle over the defect was sutured with 6-0 sutures, and skin was closed over the wound with 4-0 sutures. Animals were monitored over the course of 8 weeks for any leg abnormalities and possible compound fractures, in which case animals were euthanized. All animal protocols were approved by the Institutional Animal Care and Use Committees (IACUC) at the Medical College of Wisconsin.

[0113] Evaluation of Bone Grow th using Computed Tomography (CT): IVIS Spectrum CT was used to analyze the bone healing of tibia defects with imaging parameters of 90 kV, 8 mA, and 0.3-mm voxel sizes. The total area of remining defect detected from CT images was measured with the ImageJ® software package. 3D renderings of the tibia and defect areawere created using the Living Image® software package included with the IVIS Spectrum CT. Total percentage of the bone healing was calculated using following equation (4):Bone regeneration (%) = [1- (total area of the remining defect (mm3)) / (total area of initial defect(mm3))] * 100%.

[0114] In situ monitoring of ICG / BP degradation with SWIR: Fluorescent intensity' was used to evaluate in vivo degradation of ICG / BPs after their local implantation in tibia, using the SWIR imaging system and methodology’ previously described in section 2.6. After ICG / BPs implantation, the tibia was imaged immediately after surgery and 1 and 3 days and every yveek post-surgery up to 8 weeks. SWIR imaging data yvas analyzed, converted, and filtered into a raw image using algorithms developed for MATLABr. A region-of-interest (ROI) was drawn around the defect area, and signal intensity values were calculated using mean filtering.

[0115] Histology: Eight yveeks after the tibia surgery, all the tibias in right legs yvere isolated and fixed with 10% neutral buffered formalin for 24 hours followed yvith washing three times with PBS. For tibias used for the Goldner's Masson trichrome staining, samples yvere dehydrated with increasing concentrations of ethanol and embedded in methacrylate. Longitudinal sections of the tibia (200-300 pm) yvere prepared with a diamond-precision parallel saw (EXAKT300CP, Norderstedt Germany). The defect region was then reduced to a thickness of 10-15 pm by micro grinding and polishing. The slides were stained with the Goldner's Masson trichrome staining kit (Sigma, St. Louis, Missouri) following manufacturer's instructions. For tibias used for Hematoxylin and Eosin (H&E) staining, tibias yvere decalcified using OSTEOMALL solution (SigmaAldrich, St. Louis, Missouri) following manufacturer’s instructions. Samples were then dehydrated with increasing concentrations of ethanol, washed with xylene solution, and placed into paraffin for embedding. Cross sections measuring 8 um thickness were used placed on glass slides for H&E staining (Sigma, St. Louis, Missouri) and observed using a brightfield microscope.

[0116] In vivo Evaluation of ICG / BPs for systemic administration: SD rats (8-10-week-old, males and females) were used for systematic administration of ICG / BPs and compared with control groups including 1) No injection, 2) Saline injection, and 3) ICG dye injection. ICG was dissolved in distilled water at 400 pM and ICG / BPs were dissolved in PBS solution at 5 mg / ml based on the dry weight of ICG / BPs (-440 uM ICG). Rats under isoflurane anesthesia were placed on a heating pad underneath the NIR imaging system and 0.5 ml of ICG, ICG / BPs, or saline solution was intravenously (IV) injected via tail vein at a rate of 0. 1 ml / s using a syringe pump.

[0117] In situ monitoring of ICG / BPs using dynamic SWIR imaging: Immediately after IV injection, each rat was in situ, real-time imaged to capture the in vivo delivery and biodistribution of ICG or ICG / BPs up to 7 minutes post-administration. Dynamic imaging was collected using the SWIR imaging system and methodology previously described in section 2.6. The free ICG dose for dynamic contrast enhanced imaging was approximately 0.25mg / kg. while the ICG / BPs dose was 4 mg / kg. Both free ICG and ICG / BPs were administered IV via tail vein with a 24-gauge catheter and syringe pump set to 0.2mL / s. Dynamic imaging was performed for 6 minutes to assess biodistribution. Data was analyzed, filtered, and converted into video using algorithms developed for MATLAB®. Principal component analysis (PCA) was performed in accordance with previously published methods [Ref. 76], At the completion of the experiment, rats were kept on a heating pad and monitored for any signs of complications.

[0118] Biodistribution and clearance: To investigate the subsequent accumulation and clearance of ICG / BPs in major organs after systematic administration, rats were euthanized at different timepoints (0.5, 1, 2, 4. 8, 12, 24, 48. and 72 hours, n=3 at each timepoint) after IV injection of the ICG / BPs and the hearts, lungs, livers, and kidneys were harvested for ex vivo analysis. The fluorescent intensity of each organ was measured by the IVIS spectrum CT, with preset settings for ICG in the NIR window (Excitation = 745nm / Emission = 840nm).

[0119] Toxicity: Both liver and kidney function were evaluated for rats that received an IV injection of saline, ICG and ICG / BPs (n=3). Whole blood was collected from all the rats after 5- and 10-days post administration. Blood plasma was separated from the whole blood via centrifugation and tested with Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), and Alkaline phosphatase (ALP) assay kits for liver function, and Creatinine and Urea for kidney function, respectively. All assays were performed according to the manufacturer's instructions (Sigma Aldrich, MO, USA) to characterize the potential toxicity of the ICG / BPs using markers indicative for abnormal liver and kidney function.

[0120] Histology: To evaluate any potential damage to the major organs after ICG / BPs administration, all rats from different groups were euthanized 15 days post-administration and major organs including the lungs, livers, kidneys, and spleens were harvested. Samples were washed with PBS solution, fixed with 10% paraformaldehyde for 24 hours, dehydrated with increasing concentrations of ethanol, washed with xylene solution, and placed into paraffin for embedding. Ground sections measuring 8 urn were used placed on glass slides for H&E staining (Sigma, St. Louis. Missouri) and observed using a brightfield microscope.

[0121] Statistical Analysis: Data was analyzed using Excel spreadsheet or GraphPad Prism® and expressed as mean ± standard error. Comparisons were determined by ANOVA (SAS 9.0). The Holm-Sidak Test, which can be used for pairwise comparisons and comparisons versus a control group, was used for post-hoc comparisons. Data correlations were determined from mean values using a Pearson-product moment correlation (parametric) or Spearman Rank Order Correlation (nonparametric). Results are considered significantly different at p < 0.05.2, Results

[0122] Morphology and characterization of ICG / BPs

[0123] TEM images of the ICG / BPs showed a small, spherical morphology with an average size of 79.5 ± 25.4nm (FIG. 5A). It was not uncommon to see the particles agglomerate into small clusters. The distribution of particle diameters based on TEM images showed a large percentage within 75-100 nm, but there were small counts of larger or smaller particles also found (FIG. 5B). After re-suspension of the ly ophilized ICG / BPs into distilled water, DLS measurements found the average size of ICG / BPs to be 171 ± 11.41nm (FIG. 5C). The discrepancy in particle size differences between TEM and DLS measurements may be attributed to observations of the particles in a dry or wet state, or the size ranging from a single NP up to particle assemblies [Ref. 96, 97], The surface charge, also measured as the zeta potential (£,), falls in the range of -6.3 ± 1.9mV when resuspended in water (FIG. 5D). NPs with a zeta potential between -10 and +10 mV are considered neutral, and a zeta potential between -30 mV to +30 mV is considered to have sufficient repulsive forces in order to attain better physical colloidal stability. These parameters are critical in understanding the in vitro and in vivo behavior of the ICG / BPs with cells and in the bloodstream, respectively. Protein characterization of ICG / BPs by mass spectrometry (FIG. 5E) showed that several major ECM proteins were preserved and this result was consistence with previous findings [Ref. 68], A total of 48 proteins were found and identified by two or more unique peptides, including collagens, such as collagens I, II, V, VI, XI, XII, XIV, and XXII, non-collagenous proteins, such as osteomodulin (OMD), fibronectin, aggrecan, biglycan. decorin, chondroadherin, fibromodulin, calcium-binding proteins (CBPs), and albumin, and grow th factors, such as transforming growth factor-beta (TGF-|3), beta-enolase (ENO3), phosphoglycerate kinase (PGK), peptidyl-prolyl cis-trans isomerase (PPIA), pigment epithelium-derived factor (PEDF). Thermal characterization of ICG / BPs using thermogravimetric analysis (TGA) showed the mass loss of ICG / BPs was consistent with protein-only degradation.

[0124] Loading efficiency and optical characterization of ICG / BPs

[0125] Loading efficiency: After synthesis, the ICG / BPs appeared whitish in color with a slight green appearance, indicating entrapped ICG, which was confirmed using fluorescence imaging in the near-infrared spectrum (FIG. 6 A). The loading efficiency of ICG in the ICG / BPs was determined to be -34% in ICG / BPs with 5% ICG starting loading concentration, and the loading efficiency decreased with higher amounts of ICG pre-mixed with the stock prior to encapsulation (FIG. 6B). It was examined whether the decreased loading efficiency had any impact on the resulting fluorescent intensity. When starting loading with 5% ICG (w / w) and increasing to 10%, there is nearly double the resulting fluorescent output, however any increase in ICG after 10% (up to 20%) shows a quenching effect. That is the reason that all the ICG / BPs used herein are fabricated with 10% ICG starting loading concentration.

[0126] Optical characterization: The optical characterization of free ICG and ICG / BPs was done using absorption and emission spectroscopy. It was observed that a significant enhancement in both the absorption spectrum and the emission curve of ICG / BPs. In comparison to free ICG in water, a noticeable bathochromic shift of approximately 10-20 nm in the absorption spectrum was identified. This shift slightly altered the position of the second peak, moving it approximately 15-30 nm from around 780 to 810 nm (FIG. 6C). The red shift towards longer wavelengths observed in the ICG / BPs is indicative of changes in the physiochemical environment, likely attributed to polaric-n* transitions resulting from electrostatic interactions between ICG molecules and the nanoparticles [Ref. 77], The florescent intensity of ICG / BPs based on total concentration was determined using the NIR-I window (840 nm). Using a starting sample of 5mg of ICG / BPs resuspended into Milli-Q water. lOOuL aliquots that would equate to 40, 20, 15, 10, and 5 pM ICG (Assuming a 100% loading efficiency) were measured out. It was found that each aliquot of ICG / BPs was equivalent to ~ 12.5, 4.85, 3.52, 2.75, and 1.52 pM of encapsulated ICG, respectively (FIG. 6D). The average percent of expected signal from each concentration is consistent with the average loading efficiency previously determined (-30%). The characterization demonstrated a linear relationship in the intensity relative to increasing ICG / BP concentration. However, the resulting intensity of the ICG / BPs when compared to their ICG counterpart (pM) is not equivalent, more than likely due to their average loading efficiency. This would mean that when measuring out ICG / BPs that would equate to 40pM of ICG, the resulting fluorescent output more closely resembles that of ~15pM ICG.

[0127] Pho testability: The long-term photostability of ICG / BPs was examined in both light and dark conditions (FIG. 6E), using the SWIR window (950 nm). The ICG / BPs kept in darkconditions retained up to 80% of their original fluorescent value, whereas the ICG / BPs exposed to constant light retained ~ 47% of their original fluorescent value after 12- weeks. There were observed instances of samples retaining more than 50% of the original fluorescence up to 1 year after synthesis and lyophilization under dark conditions. This increased retention of fluorescent performance allows for extended storage and use of the ICG / BPs, providing ease- of-use advantages for clinicians.

[0128] Material stability of ICG / BPs

[0129] Material Degradation Rate: To assess the in vitro degradation rate of ICG / BPs, the cumulative protein loss by mass (mg) was quantified. Over a 42-day period, the ICG / BPs gradually degraded, exhibiting an approximately 12% total mass loss over 7 weeks without external perturbation (FIG. 6F).

[0130] ICG Release Rate: To measure the release profile of the ICG / BPs, pre-weighed amounts of particles were studied in rat plasma at a temperature of 37°C to best simulate physiological conditions at different timepoints over the course of 5 days (FIG. 6G). It was found that the amount of ICG released from ICG / BPs was initially (0.5 - 4 hours) very high, and then slowly declined to stabilize over 5 days. There was an initial large decrease over the first 4 hours, indicating -40% of ICG by mass had been released. This initial loss was followed by a slow linear decay over the next 5 days, during which an additional 11% of ICG was released. Cumulatively, -51% of the ICG had been released over 5 days of incubation.

[0131] Intracellular uptake, cytotoxicity, and osteogenic differentiation of BMSCs after ICG / BP co-culture

[0132] Intracellular uptake: After 24 hours of co-culturing BMSCs with ICG / BPs, the cellular uptake of particles was analyzed using a confocal laser scanning microscope (FIG. 7A) and TEM (FIG. 7B). Both results demonstrated the intracellular localization of particles within the cellular environment. The percentage of cellular uptake was determined by flow cytometry after co-culturing BMSCs with ICG / BPs for 24 and 48 hours (FIG. 7C), which showed that the cellular uptake rate was about 44.1 ± 4.6 % after 24 hours and 64.5 ± 4.3 % after 48 hours of co-culturing.

[0133] Cytotoxicity and osteogenic differentiation: The cytotoxicity of the ICG / BPs was determined by Live / Dead staining. After 4-week cell culture, most of the cells in different groups retained green staining (FIG. 7D, Row 1), which demonstrated the co-culturing of ICG / S s with the BMSCs did not affect the viability and survival of cells. The osteogenic differentiation BMSCs in different groups after 4-week cell culture was evaluated with RUNX- related transcription factor 2 (RUNX2) and osteocalcin (OCN) staining, and calcium depositionwas verified by the alizarin red staining. Enhanced RUNX2 and OCN fluorescence (FIG. 7D, Rows 2 & 3, respectively) and mineralized nodules (FIG. 7D, Row 4) were found in the BMSCs that were co-cultured with the ICG / BPs, suggesting that the ICG / BPs could additionally enhance the osteogenic differentiation of stem cells.

[0134] Bone regeneration and in situ monitoring of ICG / BPs in tibial defects

[0135] In vivo bone regeneration: For in vivo evaluation of any bone-regenerating stimulation effects. ICG / BPs were implanted into a critical sized tibial defect (FIG. 8A). New bone formation was assessed using CT after 8 weeks of implantation. Comparing each group, CT analysis and 3D-CT reconstruction indicated that the ICG / BPs stimulate bone growth (FIG. 8B, Columns 1 & 2). The calculated bone regeneration using CT imaging indicated a highest bone regeneration (89 ± 5.1%) after 8 weeks, compared to the incomplete bone healing (17 ± 3.8%) in that of the defect group (FIG. 8C). Histological analysis with H&E showed a large amount of fibrous tissue invasion in the defect group, whereas there is significantly less present in the ICG / BP group (FIG. 8B, Column 3). Goldner’s Trichrome stain on un-decalcified samples indicated that the defect area that is treated with ICG / BPs showed comparable bone healing and morphology to the tibia in the control group (FIG. 8B, Column 4).

[0136] In situ monitoring of ICG / BP degradation: The SWIR window was used to monitor and quantify the degradation of the ICG / BPs after their implantation in tibia defect (FIG. 8D, Row71). By distinguishing the ROI using MATLAB®, the NIR signal was able to be quantified using the mean intensity value of the measured area. The ROI was created using known anatomical distances measured on the leg and re-creating the measured defect size (4x4mm). Immediately following implantation, ICG / BPs exhibited intense fluorescent signal within the defect site. The fluorescence signal emanating from the implanted ICG / BPs exhibited an abrupt decline during the initial week post-implantation. Following the first week, a discernible, consistent reduction in fluorescence signal was observed, and after 8 weeks of ICG / BPs implantation, only limited fluorescent signal remained. Quantitative analysis using the mean values calculated from the ROI revealed a continual decrease in fluorescence signal over time, indicating the degradation of the particle graft with approximately 5-10% of the signal remaining after 8 weeks (FIG. 8E). Additionally, bone samples were measured ex vivo via CT to verify the bone growth corresponding to fluorescence decay (FIG. 8D, Rows 2 & 3). At the end of 8 weeks, there is roughly 10 ± 3.8% fluorescence remaining, which is consistent with the near complete bone regeneration shown in FIG. 8D.

[0137] In vivo evaluation of ICG / BPs for systemic administration

[0138] In situ monitoring using Dynamic SWIR imaging: SWIR dynamic imaging demonstrated the in-situ monitoring capability of the ICG / BPs after IV injection (FIG. 9A, Columns 1-3). Using the previously established methods for PCA decomposition, the liver was segmented and the biodistribution was quantified of the ICG / BPs and free ICG, demonstrating the difference in fluorescent intensity and the pharmacokinetics of each substance over time (FIG. 9A, Column 4). The data extracted from each ROI were used partially to determine that intensity was true ICG / BP signal and not just free ICG dye, and it was determined that the lower intensity and difference in kinetics through the liver verifies the ICG is still bound within the BPs (FIG. 9C).

[0139] Biodistribution and clearance of ICG / BPs: Visual comparisons of free ICG and ICG / BP clearance over 6 minutes showed the initial pass through the plural cavity and eventual passage into the liver and intestines. The PCA analysis further showed the initial increase as the ICG / BPs were filtered into the liver and the gradual decrease after roughly 4 minutes postadministration. To determine the full clearance method of ICG / BPs, the biodistribution was further analyzed ex vivo using fluorescent analysis of each organ in the NIR window (FIG. 9B). It was found that there was minimal ICG / BP presence in the heart after 1 hour (FIG. 9D), and a strong decrease in the lung during the first 4 hours (FIG. 9E). The large majority of signal was present in the liver (FIG. 9F) and the kidney (FIG. 9G). While the liver contains the highest signal for that of any tissue, the kidney is the organ that retains most of its original signal over the first 12 hours after injection. Excluding the heart, each organ showed a gradual decrease in fluorescence, with negligible signal in any organ after 72 hours. The only exception to this trend was the spleen; The ICG / BPs were cleared from the spleen after 72 hours, but vary ing amounts of the ICG / BPs were found at each time point (SI. 7).

[0140] Systemic biocompatibility of ICG / BPs: The first observations came from the animals themselves immediately after IV administration; Upon returning from the state of anesthesia, animals were alert and mobile with no typical indication of stress (rough hair, hunched posture, lethargy ), indicating the initial biocompatibility7of the IC G / BPs. H&E histological analysis was also performed on major organs including lung, liver, kidney, and spleen (FIG. 10). After 15 days, anatomical markers relevant for each organ were used to evaluate potential damage, including necrosis, scarring, or any other abnormalities, and it was determined that there was no noticeable damage.3, Discussion

[0141] Bone ECM-based scaffolds derived from decellularized bone have been widely utilized as a regenerative therapy for bone repair because of their close-to-native microstructures,mechanical properties, and osteoinductivity / osteoconductivity features [Ref. 56, 79-82], To better meet clinical needs, ECM-based biomaterials can be made or incorporated into a variety of forms that can include patches, scaffolds, powders, bio-inks, hydrogels, and nanomaterials. This study is the first instance of transforming the entire ECM of bone tissue into NPs using the combinative methodology7.

[0142] Protein-based NPs can be fabricated with many methods, including desolvation, coacervation, self-assembly, thermal gelation, spray-drying, emulsification, and chemical crosslinking [Ref. 32, 43, 83, 84], During particle formation, proteins undergo conformational changes depending on the composition and concentration of protein. Other environmental factors also play an important role in NP formation, such as solution pH, temperature, stirring rate, ionic strength, ratios of chemical agents, and crosslinking method, all of which influences the size, electrostatic interactions, surface charge, and stability of the final particles [Ref. 85], During design, the NPs can be generated to encapsulate various types of molecules, drugs, or dyes as an in vivo guidance and nano-delivery system, capable of targeting and binding to specific moieties and then releasing cargo with controlled and sustained release mechanisms. Herein, ICG were encapsulated within these novel BPs, designed for fluorescent-guided imaging practices in bone treatments and therapy, and further demonstrated their potential use in localized or systemic applications in rats.

[0143] The size, shape, and surface properties of NPs are critical parameters on particle performance in vivo. These parameters will ultimately influence the therapeutic and diagnostic applications for the NPs, as they impact many different physiological functions including cellular uptake, blood circulation half-life, organ accumulation, and subsequent clearance [Ref. 86-90], Herein, a large discrepancy is reported in the size of the ICG / BPs using TEM (79.5 ± 25.4nm) and DLS (171 ± 11.41nm) measurements. Classifying the "true" size of NPs is difficult, as there is no metrological technique that can accurately characterize the morphological features of a NP. Electron microscope techniques, including SEM and TEM, are still considered the gold standard, as their high spatial resolution (< 5 nm) allows for visualizing the morphology7of particles with excellent quality [Ref. 91-95], Overall, both TEM, DLS. and zetasizer measurements provide us with a good estimate of the “true” size of the ICG / BPs and their corresponding surface charge. In general, NPs with an overall size range of 10 nm to 200 nm are considered optimal for intravenous injection without embolization concerns. NPs that are smaller than 10 nm will be rapidly cleared by the kidney, whereas NPs that are larger than 200 nm will be mostly cleared by the mononuclear phagocytic system [Ref. 89, 90, 98-100],

[0144] Recent attempts, especially in cancer related research [Ref. 115-117]. have been made to encapsulate ICG in nanocarriers to overcome limitations of ICG that include concentrationdependent aggregation, non-specific binding, short half-life (1.5-3 minutes in rats [Ref. 76, 111] and 2-4 minutes in humans [Ref. 112-114]), and poor photostability. Successful encapsulation of ICG within the BPs was demonstrated and the effect this encapsulation has on its newly enabled optical capabilities based on these experimental findings.

[0145] First, the impact of various starting concentrations of ICG on the loading efficiency of BPs was investigated. The objective was to determine an optimal ICG-to-ECM ratio that maximizes loading efficiency without wasting excess ICG and without compromising the final fluorescent output. During encapsulation, it was observed that an increase in the initial amount of ICG had an adverse effect on loading efficiency, establishing an inverse relationship between the initial ICG quantity and subsequent loading efficiency. However, it was found that fluorescence saturation with values above 10%, leading us to conclude that 10% is optimal for the experiments.

[0146] Second, the in vitro degradation of the ICG / BPs was examined and it was found that the ICG / BPs showed a slow degradation rate, with -12% total protein loss over the course of 7 weeks without perturbation. The degradation rate of a biomaterial is an important characteristic for bone-related applications. An ideal biomaterial should have a degradation rate that is commensurate with the new bone growth-ratio to better maintain the growth of new bone. Moreover, the degradation rate of protein-based NPs is also closely related to the release rate of any encapsulated cargos [Ref. 118-123],

[0147] Third, the release rate of ICG from ICG / BPs during in vitro incubation with blood plasma was determined. It was found that the amount of ICG released from ICG / BPs was initially very high, suggesting that the interaction of the ICG / BPs in the bloodstream disrupts the bonds of proteins that are relatively weak, resulting a quick release of ICG that is most likely near the surface of the particles. It is noted that other synthesis variables such as pH, temperature, desolvating and crosslinking agent concentrations, have an effect on the properties of ICG / BPs. These properties, in turn, would likely influence parameters such as particle size, zeta potential, degradation, and the loading efficiency and release rate of ICG.

[0148] After particle characterization, basic optical properties of the ICG / BPs using absorbance and fluorescence spectra were explored. It was found that there is a broad enhancement of the absorption spectrum and the ICG emission curve. The much broader peak in absorbance within the ICG / BPs is most likely due to the complex diffraction patterns of the many proteins within the ICG / BP solution itself, causing uneven aggregation. It has beenpreviously reported that encapsulation of ICG may have each spectrum due to stokes’ shifting and changes in the chemical structure of ICG [Ref. 124. 125],

[0149] While traditional NIR windows serve well in various clinical applications, the use of shortwave IR (SWIR) extends the observation depth to over 1 cm within living animals. This feature proved invaluable for the study on the in-situ degradation and release of ICG / BPs in a tibial defect model, as SWIR enables robust fluorescence monitoring through skin, periosteum, muscle, and bone tissue. With the promising results in the NIR window, their performance in the SWIR window (950nm) was also evaluated. In this window, ICG / BPs exhibited a high fluorescent profile when compared to increasing concentrations of free ICG in water. The encapsulation of ICG within the BPs altered the fluorescent profile of free ICG, a change attributed to the exposure to proteins during the synthesis process. During this step, free ICG binds to various proteins in the stock solution, though the specific nature of this binding and the proteins involved remain unexplored. Importantly, the encapsulation of ICG within the BPs resulted in increased photostability, likely stemming from the interaction between ICG and ECM proteins during synthesis, subsequently modifying the initial fluorescence.

[0150] The cellular fate of NPs is intricately governed by their physiochemical attributes, encompassing composition, size, shape, surface charge, and hydrophobicity / hydrophilicity [Ref. 126, 127], To test the cellular uptake and cellular compatibility7, ICG / BPs were cocultured with BMSCs and the success of entry into cells and intracellular localization of ICG / BPs were confirmed by confocal microscopy and TEM imaging after 24 hours of culture time, with no apparent toxic effects on BMSCs. ICG / BPs not only demonstrated biocompatibility but also augmented osteogenic differentiation of stem cells, evidenced by upregulated expression of OCN and RUNX2 transcription factors, along with increased matrix mineralization as observed through alizarin red staining. These findings underscore the potential of ICG / BPs to not only be efficiently taken up intracellularly but also to induce osteogenic differentiation in stem cells, indicating promising applications in bone regeneration.

[0151] Building upon the encouraging outcomes of cellular outcomes, the translational potential of ICG / BPs for localized and systemic applications in orthopedic treatments was investigated. Initial experiments focused on ICG / BPs as a bone grafting substance for repairing tibial defects in rats. Eight weeks after locally implanting the ICG / BPs in a surgically created tibial defect, both CT and histological evaluations demonstrated that the ICG / BPs could efficiently promote new bone regeneration and formation compared to a defect without any graft filling. Furthermore, the locally implanted ICG / BPs were capable of in-situ monitoring in the SWIR window over the course of 8 weeks. The observed phenomena of rapid degradationat the onset of the experiment may be due to the clearance of loosely associated material by the lymphatic system, resulting in the confinement of the ICG / BP signal to the defect site. Additionally, a plausible contributor to this initial steep decline is the prompt release of ICG due to the disruption of weak protein bonds within the ICG / BPs upon contact with blood or alterations in temperature and pH. It was found that the ICG / BPs aid in the reconstruction and regeneration of local bone defects while simultaneously offering continuous qualitative assessment of bone healing and regeneration using NIR-II fluorescence imaging. The outcomes align with prior studies utilizing NIR optical imaging techniques for monitoring the degradation of bone scaffolds [Ref. 131, 132],

[0152] Moving to the systemic level, the second objective involved in-situ monitoring of ICG / BPs after intravenous administration. NIR-II dynamic imaging enabled quantitative assessment of in vivo biodistribution, organ accumulation, and systemic clearance. Following intravenous administration, all the animals exhibited normal behavior without acute or chronic reactions. No observable signs of physical abnormalities or weight loss were noted. The in vivo biodistribution of ICG / BPs was systematically tracked using NIR imaging over 7 minutes, and Principal Component Analysis (PCA) decomposition of epifluorescence images enabled anatomical segmentation of the liver, revealing a delayed arrival of ICG / BPs compared to free ICG that is indicative of an extended circulation time in the bloodstream. The results underscore the in-situ monitoring capabilities, in vivo stability, and initial biocompatibility of ICG / BPs. Ex vivo analysis confirmed preferential accumulation in the liver and kidneys, suggesting efficient clearance through hepatic and renal pathways to mitigate potential toxicity. Histological examinations revealed no notable changes in organs, identified by observing anatomical landmarks, affirming the biocompatibility and safety of ICG / BPs.4, Conclusions

[0153] This Example introduced a new type of NPs, denoted ICG / BPs, formed by encapsulating ICG dye within a bone-based NP crafted from the complete decellularized bone ECM. The ICG / BPs demonstrated a nano-sized structure, incorporating organic matrix proteins reminiscent of those naturally occurring in native bone. This composition not only promotes biocompatibility but also augments osteoblast differentiation during in vitro coculturing with BMSCs. In vivo assessments demonstrated the capacity of ICG / BPs to promote bone regeneration when utilized for locally repairing large-size tibial defects in animals. Additionally, the degradation of ICG / BPs is able to be monitored using SWIR imaging in vivo and aligns temporally with bone regeneration and healing.

[0154] To explore the systemic potential of the ICG / BPs, in vivo evaluations were conducted on rats, focusing on biodistribution, systemic clearance, and overall safety following intravenous administration of ICG / BPs. The use of SWIR imaging revealed the biodistribution pattern, highlighting significant accumulation primarily in the liver and kidneys. This indicated effective elimination through hepatic and renal pathways, mitigating the risk of accumulation-related toxicity. The observed well-being and normal behavior of treated animals, coupled with the absence of acute and chronic toxicities in vital organs during histological examination, affirmed the favorable safety profile of ICG / BPs. Notably, the real-time, in-situ monitoring capabilities of ICG / BPs in both NIR I and SWIR windows provided valuable insights into particle dynamics, enhancing safety assessment, determining applicability, and identifying potential downstream effects.

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J Biomech, 2006. 39(12): p. 2264- 73.The citation of any document or reference is not to be construed as an admission that it is prior art with respect to the present invention.Example 2

[0156] The above BP formation processes may be used to encapsulate one or more bioactive agents. FIG. 11 provides a cranial defect methodology where the defects are filled with ICG / BMP2 / BPs (middle right). As shown in FIGS. 12A-12B, the bioactive agent BMP2 affects ICG / BP performance after implantation in vivo. FIG. 13 further illustrates bone regeneration of both ICG / BPs and ICG / BMP2 / BPs in a cranial defect model. After 8 weeks, bone regeneration of the untreated defect was 17.64 %, while ICG / BPs and ICG / BMP2 / BPs showed 70.67% and 85.99% bone regeneration, respectively.Example 3

[0157] In a non-limiting example, the degree of crosslinking may affect the NP formation. For example, uncrosslinked (UnX) BPs may by formulated as described previously inExample 1. but skipping the addition of glutaraldehyde to the solution as used to form the crosslinked (X) BPs. and instead letting the solution stir for 30 minutes after desolvation. FIGS. 14A-14B illustrate that the UnX BPs are more irregularly shaped with a more diverse size range compared to the X BPs. Bioactive agents may be added as previously described in Example 1 above to either UnX or X BPs.

[0158] Referring to FIG. 15. the performance of X and UnX BPs was evaluated in an alveolar cleft palate defect model. Repair of the cleft palate defect using X ICG / BPs (FIG. 16A) and UnX ICG / BPs (FIG. 16B) was monitored by NIR imaging. FIGS. 16A-16B illustrate that crosslinking affects the localization and intensity over time. The plot of NIR fluorescence over time in FIG. 17 further suggests that two distinct trends between X ICG / BPs and UnX ICG / BPs, where the latter has a less stable degradation rate.

[0159] Referring to FIGS. 18A-18B, the X ICG / BPs stimulate more bone regeneration over 8 weeks compared to the UnX ICG / BPs, shown by gross observation, CT scanning, and 3D CT reconstruction.

[0160] Thus, the present disclosure provides nanoparticles for orthopedic regeneration and imaging.

[0161] In light of the principles and example embodiments described and illustrated herein, it will be recognized that the example embodiments can be modified in arrangement and detail without departing from such principles. Also, the foregoing discussion has focused on particular embodiments, but other configurations are also contemplated. In particular, even though expressions such as "in a non-limiting example embodiment", or the like are used herein, these phrases are meant to generally reference embodiment possibilities, and are not intended to limit the invention to particular embodiment configurations. As used herein, these terms may reference the same or different embodiments that are combinable into other embodiments. As a rule, any embodiment referenced herein is freely combinable with any one or more of the other embodiments referenced herein, and any number of features of different embodiments are combinable with one another, unless indicated otherwise.

[0162] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise.

[0163] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term w hich are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately”will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0164] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0165] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0166] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together. A and C together, B and C together, and / or A. B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0167] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0168] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how tomake or use an aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb '‘may’’ has the same meaning and connotation as the auxiliary verb “can.”

[0169] Although the invention has been described in considerable detail with reference to certain embodiments, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which have been presented for purposes of illustration and not of limitation. Therefore, the scope of the appended claims should not be limited to the description of the embodiments contained herein. Various features and advantages of the invention are set forth in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising: bone extracellular matrix particles, wherein the particles have an average particle size in a range of 1 nanometer to 500 nanometers.

2. The composition of claim 1, wherein the particles have an average particle size in a range of 100 nanometer to 400 nanometers.

3. The composition of claim 1 wherein, the particles have an average particle size in a range of 10 nanometer to 250 nanometers.

4. The composition of claim 1 wherein, the particles have an average particle size in a range of 125 nanometer to 250 nanometers.

5. The composition of claim 1 further comprising: one or more bioactive agents associated with each particle of at least a portion of the particles.

6. The composition of claim 5, wherein the one or more bioactive agents is selected from the group consisting of enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, allograft bone, gene constructs, therapeutics, and mixtures thereof.

7. The composition of any one of claims 1-6 further comprising: a fluorescent dye associated with each particle of at least a portion of the particles.

8. The composition of claim 7, wherein: an amount of the dye is encapsulated by each particle of the portion of the particles.

9. The composition of claim 8, wherein a ratio of the dye to the particles has an encapsulation efficiency in the range of 13-15 % weight / weight (w / w) based on total weight of the nanoparticles.

10. The composition of claim 7, wherein the composition has a photostability of 45% intensity or greater over a 12-week period.

11. The composition of claim 7, wherein the fluorescent dye includes indocyanine green (ICG).

12. The composition of claim 1, wherein: the bone extracellular matrix comprises demineralized and decellularized bone.

13. The composition of claim 12, wherein: the bone extracellular matrix comprises an entire extracellular matrix of mammalian bone.

14. The composition of claim 13, wherein: the bone extracellular matrix comprises an entire extracellular matrix of acellular porcine bone.

15. The composition of claim 1, wherein: the particles comprise at least two of collagens I, II, V, VI, XI, XII, XIV, and XXII, osteomodulin (OMD), fibronectin, aggrecan, biglycan, decorin, chondroadherin, fibromodulin, calcium-binding proteins (CBPs), and albumin, and growth factors, such as transforming grow th factor-beta (TGF-P), beta-enolase (ENO3), phosphoglycerate kinase (PGK), peptidyl-prolyl cis-trans isomerase (PPIA), and pigment epithelium-derived factor (PEDF).

16. A composition comprising: bone extracellular matrix particles, wherein the particles have an average particle size in a range of 1 nanometer to 500 nanometers; and a fluorescent dye associated with each particle of at least a portion of the particles.

17. The composition of claim 16, further comprising: one or more bioactive agents associated with each particle of at least a portion of the particles.

18. The composition of claim 16, wherein the one or more bioactive agents is selected from the group consisting of enzymes, organic catalysts, ribozymes, organometallics, proteins, glycoproteins, peptides, polyamino acids, antibodies, nucleic acids, steroidal molecules, antibiotics, antimycotics, cytokines, growth factors, carbohydrates, oleophobics, lipids, pharmaceuticals, allograft bone, gene constructs, therapeutics, and mixtures thereof.

19. The composition of claim 16, wherein the one or more bioactive agents includes bone morphogenic protein 2 (BMP2).

20. A method of making particles, the method comprising:(a) providing a solution including particles comprising bone extracellular matrix, wherein the particles have an average particle size in a range of 1 nanometer to 500 nanometers; and(b) adding a fluorescent dye to the solution to associate an amount of the dye with each particle of at least a portion of the particles.

21. A method of making particles, the method comprising:(a) providing a solution including particles comprising bone extracellular matrix, wherein the particles have an average particle size in a range of 1 nanometer to 500 nanometers; and(b) adding one or more bioactive agents to the solution to associate an amount of the bioactive agent with each particle of at least a portion of the particles.

22. The method of claim 21 wherein step (b) further comprises: adding a fluorescent dye to the solution to associate an amount of the dye with each particle of at least a portion of the particles.

23. The method of claim 20 or claim 21 wherein step (a) comprises: demineralizing a bone sample with one or more acids;decellularizing the bone sample with at least one of a detergent and a surfactant; lyophilizing the demineralized and decellularized bone sample; and milling the lyophilized bone sample into a powder of the particles.

24. The method of claim 20 or claim 21 wherein step (a) further comprises: reconstituting the powder in one or more acids; digesting the reconstituted bone sample with one or more proteolytic enzymes; and desalting the digestion to produce the solution.

25. The method of claim 20 or claim 21, wherein the fluorescent dye includes indocyanine green (ICG).

26. The method of claim 20 or claim 21, wherein the amount of the dye is encapsulated by each particle of the portion of the particles.

27. The method of claim 20 or claim 21. wherein step (b) comprises adding the fluorescent dye to the solution in the range of 5-20% weight / weight (w / w) based on total weight of the solution.

28. The method of claim 20 or claim 21. wherein the particles comprise at least two of collagens I, II, V, VI, XI, XII, XIV, and XXII, osteomodulin (OMD), fibronectin, aggrecan, biglycan, decorin, chondroadherin, fibromodulin, calcium-binding proteins (CBPs), and albumin, and growth factors, such as transforming growth factor-beta (TGF-P), betaenolase (ENO3), phosphoglycerate kinase (PGK). peptidyl-prolyl cis-trans isomerase (PPIA), and pigment epithelium-derived factor (PEDF).

29. The method of claim 20 or claim 21, further comprising (c) crosslinking the solution using a crosslinking agent.

30. The method of claim 29, wherein the crosslinking agent includes glutaraldehyde.

31. A method of treating an orthopedic defect, the method comprising:(a) filling the orthopedic defect with the composition of claim 1 or claim 16.

32. The method of claim 31 wherein step (a) comprises: lyophilizing the composition of claim 1; filling the orthopedic defect with the lyophilized composition; and monitoring the orthopedic defect over a duration using a medical imaging technique.

33. The method of claim 32, wherein the medical imaging technique includes at least one of fluorescence imaging and fluoroscopic imaging.

34. The method of claim 31, wherein the orthopedic defect is in bone.

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