WO2016109892A1 - Ultrasound triggered delivery of growth factors from liposomes for tissue regeneration - Google Patents

Abstract

A composition for use in promoting bone repair compromising liposomes encapsulating one or more bone morphogenetic proteins, BMPs. The liposomes are adapted to release the BMPs upon exposure to ultrasound energy. A use of the liposome composition for the controlled or triggered release of BMPs from liposomes is also provided. Bioimplants comprising the liposome composition are also provided.

Description

ULTRASOUND TRIGGERED DELIVERY OF GROWTH FACTORS FROM LIPOSOMES

FOR TISSUE REGENERATION

CROSS REFERENCE TO PRIOR APPLICATIONS

[0001] This application claims priority under the Paris Convention from US Application 62/100,032, filed on January 5, 2015. The entire contents of such prior application is incorporated herein by reference.

FIELD OF THE DESCRIPTION

[0002] This present description relates to a composition and method of delivering growth factors, in particular bone morphogenetic proteins, BMPs. In particular, the description relates to liposomes that encapsulate the growth factors and which are adapted to release the growth factors at a treatment site upon exposure to ultrasound energy. The description provides a composition and method for tissue, in particular bone regeneration.

BACKGROUND

[0003] Tissue healing involves a complex series of events that ideally leads to restoration of the mechanical and structural stability of the tissue. The interdependent cascades of events involve timed recruitment and differentiation of appropriate cell populations (including neutrophils, macrophages, endothelial cells, mesenchymal stem cells and for bone, osteoprogenitors and osteoblasts), which are dependent on the expression of various signaling proteins, growth factors and cytokines at appropriate times, places and concentration. Several external factors including age and health status can influence the natural process to slow, interrupt or completely stop the healing process (Hayda et al.

Clinical Orthopedics Related Research. 1998; 355(Supplement):S31-40)

[0004] When tissue fails to heal, surgical intervention is often required. This may include the use of autogenous tissue grafts to provide a scaffold, and cells and biological signals to promote healing. However, the use of autogenous grafts is often associated with various drawbacks. For example, the harvesting of such tissue grafts requires added operative and post-operative time, as well as donor site pain and morbidity. Further, such procedures can have unpredictable performance and suffer from limited tissue supply, particularly in the pediatric population (Rogers et al. Journal of Craniofacial Surgery. 2012; 23:323-7).

Alternatives to autogenous tissue grafts are allografts (tissue from others of the same species) or xenografts (tissue from a different species) and synthetic grafts. However, these procedures have limited efficacy. A promising alternative solution for promoting tissue repair is the use of growth factor containing implants, such as Bone Morphogenetic Protein (BMP) containing bioimplants for bone repair and Platelet Derived Growth Factor (PDGF) containing bioimplants for skin and other connective tissue repair.

[0005] Bone Morphogenetic Proteins

[0006] BMPs belong to the transforming growth factor beta (TGFp) superfamily of secreted growth and differentiation factors with over 30 members in mammals alone. The proteins are dimeric and are characterized by a distinct conserved seven cysteine knot based structure. They have been implicated in diverse roles where they have been shown to regulate the proliferation, differentiation and migration of many cell types, and have important roles in morphogenesis, organogenesis, tissue maintenance and wound healing. The TGFp superfamily of growth factors can be subdivided into several subfamilies including the transforming growth factor beta subfamily, the bone morphogenetic protein and growth and differentiation factor (GDF) family (also called the BMP subfamily), and the inhibin and activin subfamily.

[0007] At least twenty proteins form the BMP subfamily including BMP-2, BMP-3 (also known as osteogenin), BMP-3b (also known as growth and differentiation factor 10, GDF- 10), BMP-4, BMP-5, BMP-6, BMP-7 (also known as osteogenic protein-1 , OP-1), BMP-8 (also known as osteogenic protein-2, OP-2), BMP-9, BMP-10, BMP-1 1 (also known as growth and differentiation factor 8, GDF-8, or myostatin), BMP-12 (also known as growth and differentiation factor 7, GDF-7), BMP-13 (also known as growth and differentiation factor 6, GDF-6), BMP-14 (also known as growth and differentiation factor 5, GDF-5), and BMP-15 (Bragdon et al. Cellular Signalling. 201 1 ;23(4):609-20).

[0008] Bone Morphogenetic Protein-2 (BMP-2) is a protein that plays a critical role in bone formation and repair. It is involved in promoting mesenchymal stem cell (MSC) chemotaxis (migration), proliferation and differentiation into osteoblasts (bone forming cells) (Tsuji et al. Nature Genetics. 2006; 38: 1424-29. Scheufler et al. Journal of Molecular Biology. 1999; 287: 103-15). Bioimplants containing recombinant human BMP-2 (rhBMP-2) have been approved for use clinically to promote bone repair in spinal fusions, long bone non-unions and bone defect healing (Wozney JM. Spine. 2002; 27(Supplement 1):S2-8). Garrison et al., 2007 reported the use of rhBMP-2 was associated with reduced operating time, improvement in clinical outcomes and shorter hospital stays. In addition the proportion of secondary interventions were lower in patients treated with rhBMP-2 (Garrison et al. Health Technology Assessment. 2007; 1 1 (30)).

[0009] Protein therapeutics including growth factors are susceptible to neutralization, degradation and rapid clearances from the implant site in vivo and can cause toxicity and non-desirable clinical outcomes. To circumvent these problems when using BMPs to promote bone repair, the BMP is typically combined with a carrier, which acts to retain the BMP at the implant site protecting it from degradation and releasing it into the healing tissue at the required time and concentration. Often the carrier also acts as a scaffold to maintain space and provide a structure for cell ingrowth (Haidar et al. Biotechnol Lett. 2009; 31 : 1825- 35). The current clinically approved bioimplant uses a bovine derived absorbable acellular collagen sponge (ACS) as the carrier. This is soaked in an rhBMP-2 solution immediately prior to placement into the implant site. Upon placement of the BMP soaked ACS into the implant site, the rhBMP-2 is released quickly from the sponge, with an estimated 80 to 90% of the BMP released over the first 24 hours and sequentially less released each day (this is typically referred to as burst release). In order to ensure that sufficient amounts of BMP remains when MSCs migrate into the wound site (usually day 4 and later),

supraphysiological doses of rhBMP-2 (1.5 mg/ml) are initially applied to the ACS.

[0010] While rhBMP-2 bioimplants have been shown to be as effective as autogenous bone grafts (ABG), their use has also been associated with reports of bone overgrowth and formation outside of the implant site, inflammation, edema, neurologic events and cancer (Sasso et al. Journal of Spinal Disorders & Techniques. 2005; 18:S77-S81). The incidence of these adverse events appears to be related to the total amount of BMP applied, suggesting that a bioimplant that delivers less BMP more efficiently may be as effective as current bioimplants, but be significantly safer.

[0011] Therefore there is a need for a BMP bioimplant that provides controlled release of rhBMP-2 when the appropriate responsive cells are present. An effective and safe bioimplant should combine a controllable rate of BMP release and high degree of spatial localization control to ensure the bioimplant is both effective and safe.

[0012] Further, healing environments differ due to factors including site, degree of trauma, age and underlying health of the patient, which would be expected to alter the timing and potentially the optimal concentration of rhBMP-2 required to promote healing. Therefore there is also a desire that the BMP implant have a modifiable BMP release profile so the appropriate amount of BMP can be released at different times and concentrations depending on the situation.

[0013] Carriers for protein delivery tend to work in one or a combination of two ways (Schliephake H. Oral Maxillofac Surg. 2010; 14(1): 17-22). The first approach involves the binding of the protein to the carrier. In these carriers the release profile is controlled by the strength of bonding. When the bonding is weak, the release profile will follow a rapid exponential decline and a rapid burst release. The current clinical rhBMP-2 ACS carrier acts in this manner.

[0014] The second approach is to encapsulate the protein within the carrier so that, as the carrier degrades over time, it releases the protein. The release rate is then dependent on the amount loaded and the degradation rate of the carrier.

[0015] An alternative approach is to use a carrier where release of the protein is stimulated by some external event. In this case the timing and amount of protein released is dependent on exposure to the external trigger.

[0016] Liposomes

[0017] Liposomes are nanocarriers that are composed of cholesterol and phospholipids like phosphatidycholines (PC), phosphatidylethanolamines (PE) and phosphatidylserines (PS) that form spontaneously when placed in an aqueous environment. Due to their structure and lipid composition, liposomes are biocompatible, biodegradable and relatively non-toxic. Liposomes offer several advantages, such as ease of preparation, less or no immunological response and stability, which make ideal vectors.

[0018] As drug carriers, liposomes display several advantages as their physical characteristics (size, shape, charge) can be easily modified to optimize entrapment of proteins or drugs and targeted delivery.

[0019] Liposomes are prepared by a variety of techniques and are classified into three types based on size and structure. These are multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs) and small unilamellar vesicles (SUVs). MLVs range from 500 to 5,000 nm and consist of several concentric bilayers of lipids. LUVs and SUVs are created by downsizing MLVs via extrusion. LUVs are commonly used for clinical applications. Large unilamellar vesicles range from 200 to 800 nm and small unilamellar vesicles are 100 nm and smaller formed with a single lipid bilayer (Lim et al. Journal of Controlled Release.

2012;163(1):34-45).

[0020] Currently liposomes have only been approved for delivery of small molecule drugs including doxorubicin (Doxil®, Caelyn® and Adriamycin®), Daunoxome

(Daunorubicin), Ambisome (Amphotericin B), Visudyne (Verteporfin), Depocyt (Cytarabine), Marqibo (Vincristine) and the vaccine Epaxal (Hepatitis A vaccine) (Swaminathan J, Ehrhardt C. Liposomal delivery of proteins and peptides. Expert opinion on drug delivery. 2012; 9(12):1489-503).

[0021] Animal studies have investigated the potential of using liposomes to deliver growth factors including TGF-βΙ , rhBMP-4 (Ferreira et al Arch. Oral Biol. 2013; 58:646-56) and rhBMP-2 (Matsuo et al. J Biomed Mater Res A. 2003; 66:747-54). However in these studies no external trigger was applied to release the growth factor. Further in these studies the growth factor is released uncontrollably as the liposome degrades. The reported stability of liposomes in vivo is typically a matter of hours (i.e. 24 hours, Kanaoka et al. JPP 2001 , 53: 295-302). A recent review noted that significant obstacles still needed to be overcome to develop practical liposomal delivery of proteins including formulation and stability of the proteins, leakage of proteins from the liposomes, and stability of the liposomes

(Swaminathan & Ehrhardt Expert Opin. Drug Deliv. 2012; 9: 1489-1503).

[0022] Several different triggers can be utilized to release drugs encapsulated in liposomes. Ideally these should be external triggers than can be localized to the site of action to such as change in temperature or exposure to light or ultrasound. For example, US Application Publication No. US 2007/0184085 teaches the use of ultrasound stimulation to release drugs from vesicles, such as liposomes. In the current state of the art, these triggers are applied shortly after administration (as soon as visualized at the site of action, Liang et al. Arterioscler Thromb Vase Biol. 201 1 ; 31 :1357-1359) or within a few hours after administration (6 hours after administration Ta et al. Journal of Controlled Release 2014; 194:71-81). However these times were much shorter than is desired for application of bone morphogenetic proteins such as rhBMP-2 or rhBMP-7 where the actions of these proteins during bone healing occur over several weeks. Further it has been reported that the exposure to ultrasound can modulate the effect of therapeutics (Shaw et al. Thrombosis Research 2009; 124: 306-310) and that the ultrasound induced effects that induce release of the payload from liposomes may cause tissue damage and so the ultrasound parameters must be carefully controlled (Pitt et al Expert Opin Drug Deliv. 2004; 1 : 37-56). Finally a recent review noted that while numerous in vitro studies have shown ultrasound can release bioactive payloads from liposomes in vitro, it is much more challenging for effective release in vivo (Husseini et al. Colloids and Surfaces B: Biointerfaces 2014; 123: 364-386).

[0023] It was not previously known nor could it be predicted that BMP could be encapsulated in a liposome composition in such a way as to prevent its release and/or activity until its release is triggered by exposure to ultrasound - in fact the only known liposome compositions used for BMPs or other growth factors would not do so. It was also not known or predicted if exposure to ultrasound would negatively affect the activity of BMP or if exposure to ultrasound would release sufficient amounts of bioactive BMP (and if so under what ultrasound parameters and duration of exposure) to induce bone formation when implanted into the body. Further it was not known whether, even if such liposomes existed, they would remain stable and still be able to release BMP upon exposure to ultrasound when stored prior to implantation for extended periods of time (months). For these same reasons, it was also not known or predicted what ultrasound parameters would be needed to trigger the release of BMP from liposomes at a concentration sufficient to result in bone formation.

SUMMARY OF THE DESCRIPTION

[0024] In one aspect, there is provided a composition for use in promoting bone repair, the composition compromising liposomes encapsulating one or more bone morphogenetic proteins, BMPs. In one aspect, the liposomes are formed from a phospholipid material, wherein the phospholipid material is a phosphatidylcholine and/or a phosphoethanolamine, and cholesterol.

[0025] In one aspect, the liposomes are adapted to release the one or more BMPs upon exposure to ultrasound stimulation. In one aspect, the peak negative pressure of the ultrasound exposure is from about 200 kPa to about 6 MPa. In another aspect, the ultrasound frequency is from about 1 MHz to about 20 MHz. In another aspect, the ultrasound mechanical index is from about 0.5 to about 1.5. In another aspect the ultrasound exposure is from about 5 seconds to about 60 minutes.

[0026] In one aspect, the BMPs released by the liposomes are one or more of BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-1 1 , BMP- 12, BMP-13, BMP-14, BMP-15, or combinations thereof. In one aspect, the BMPs are mammalian BMPs, or preferably human BMPs. [0027] In one aspect, there is provided a use of the liposome composition for the controlled or targeted release of BMPs at a target site, specifically the site of implantation of the composition.

[0028] In another aspect, there is provided a bioimplant having thereon or therein the liposome composition described above.

[0029] In another aspect, there is provided a method of releasing BMPs at a target site comprising: a) providing the composition described herein; b) implanting the composition at an implantation site within an animal; and, c) subjecting the target site to ultrasound exposure sufficient to rupture the liposomes and release the one or more BMPs.

BRIEF DESCRIPTION OF THE FIGURES

[0030] The features of certain embodiments will become more apparent in the following detailed description in which reference is made to the appended figures wherein:

[0031] Figure 1 shows TEM images of DSPC (Panel A) and DSPC-DSPE-PEG (Panel B) formulations that were obtained post size exclusion chromatography. The TEM images confirm the size of particles obtained through dynamic light scattering technique.

[0032] Figure 2 shows the effect of ultrasound on a selected liposome composition.

[0033] Figure 3 shows the release of rhBMP-2 from liposomes of selected formulations upon ultrasound exposure.

[0034] Figure 4 shows that the amount of rhBMP-2 released can be varied by varying the ultrasound pressure and duration of exposure.

[0035] Figure 5 shows the bioactivity of various preparations of liposomes with and without ultrasound exposure.

[0036] Figure 6 shows that an implanted select liposome composition containing rhBMP- 2 induces bone formation following exposure to ultrasound in vivo, and does not do so in the absence of ultrasound exposure.

[0037] Figure 7 shows that the timing of ultrasound exposure affects the amount of bone formed by a select liposome formulation containing rhBMP-2. [0038] Figure 8 shows the effect of the number of exposures and timing of those exposures on bone formation by a select liposome formulation containing rhBMP-2.

[0039] Figure 9 shows the effect of varying the duration of ultrasound exposure on the amount of BMP-2 from liposomes implanted within a tissue phantom.

DETAILED DESCRIPTION

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0041] As used herein the term "bioimplant" refers to a material which is suitable for implantation. A bioimplant may contain, be provided with, or have disposed thereon, an exogenous growth or biologically active factor. For example, the growth or biologically active factor may be applied on a surface of the bioimplant. In the present description, a bioimplant associated in any way with a growth or biologically active factor may be described, for convenience, as "containing" the factor.

[0042] As used herein the term "growth factor", or "GF", refers to peptides and proteins that stimulate growth and/or differentiation of cells via the interaction of the GFs with specific cell surface receptors. Examples of growth factors include the bone morphogenetic proteins (BMPs), transforming growth factor beta (TGFp), the insulin-like growth factors (IGF), the fibroblast growth factors (FGFs), platelet derived growth factor (PDGF) and vascular endothelial growth factor. In preferred embodiments the growth factors are BMPs.

[0043] By "recombinant" is meant a protein produced by a transiently transfected, stably transfected, or transgenic host cell or animal as directed by an expression construct containing the cDNA for that protein. The term "recombinant" also encompasses pharmaceutically acceptable salts of such a polypeptide

[0044] As used herein, the term "polypeptide" or "protein" refers to a polymer of amino acid monomers that are alpha amino acids joined together through amide bonds.

Polypeptides are therefore at least two amino acid residues in length, and are usually longer. Generally, the term "peptide" refers to a polypeptide that is only a few amino acid residues in length. A polypeptide, in contrast with a peptide, may comprise any number of amino acid residues. Hence, the term polypeptide included peptides as well as longer sequences of amino acids. [0045] As used herein, the terms "bone morphogenetic protein" or "bone morphogenic protein" or "BMP" are used interchangeably and refer to any member of the bone morphogenetic protein (BMP) subfamily of the transforming growth factor beta (TGFp) superfamily of growth and differentiation factors, including BMP-2, BMP-3 (also known as osteogenin), BMP-3b (also known as growth and differentiation factor 10, GDF-10), BMP-4, BMP-5, BMP-6, BMP-7 (also known as osteogenic protein-1 , OP-1), BMP-8 (also known as osteogenic protein-2, OP-2), BMP-9, BMP-10, BMP-1 1 (also known as growth and differentiation factor 8, GDF-8, or myostatin), BMP-12 (also known as growth and differentiation factor 7, GDF-7), BMP-13 (also known as growth and differentiation factor 6, GDF-6), BMP-14 (also known as growth and differentiation factor 5, GDF-5), and BMP-15.

[0046] The terms "bone morphogenetic protein" and "BMP" also encompass allelic variants of BMPs, function conservative variants of BMPs, and mutant BMPs that retain BMP activity. The BMP activity of such variants and mutants may be confirmed by any of the methods well known in the art (see the section Assays to measure BMP activity, below).

[0047] In preferred embodiments, the BMP is BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 or BMP-9. In particularly preferred embodiments the BMP is BMP-2, BMP-4 or BMP- 7.

[0048] In preferred embodiments the BMP is a mammalian BMP (e.g., mammalian BMP- 2 or mammalian BMP-7). In particularly preferred embodiments, the BMP is a human BMP (hBMP) (e.g. hBMP-2 or hBMP-7).

[0049] It will also be understood that references to "BMPs" herein is intended to include recombinant BMPs, mammalian BMPs and human BMPs unless otherwise indicated.

[0050] As used herein the term "scaffold" refers to a material whose purpose is to provide a structure which supports cell adhesion, migration and ingrowth into a tissue repair site.

[0051] As used herein the term "triggered release" refers to the release of growth factor upon exposure to an external stimulus (e.g. ultrasound).

[0052] As used herein, and as known in the art, the term ultrasound "mechanical index" (Ml) is defined as the quotient of the peak negative pressure (in Mega Pascals, MPa) divided by the square root of the center frequency of the ultrasound beam (in MHz). [0053] The present description provides nanoparticles composed of liposomes with one or more growth factors encapsulated within the aqueous core. Following implantation of such liposomes, ultrasound can be applied on one or more occasions to release the encapsulated material in a controlled manner to vary the amount and timing of release of the growth factor.

[0054] The compositions, uses and methods described herein can be used for a variety of therapeutic and clinical applications, including, but not limited to, fracture repair; spine fusion and regeneration of bone defects.

[0055] As described above, the use of liposomes for delivering or releasing proteins, such as BMPs, at a given site of action has proven challenging. The present inventors, however, have developed vesicles, such as liposomes, that are stable (e.g. do not collapse for at least 24 hours at 37 °C) and that can contain desired proteins, in particular BMPs, for delivery at an implantation site. Furthermore, the inventors have developed methodologies for the controlled delivery of such proteins from the liposomes at the site of action using ultrasound stimulation.

[0056] It will be understood from the present description that the ultrasound stimulation to release the proteins, such as BMPs, from the liposomes can be applied either internally or externally with respect to the animal within which the liposomes are implanted.

[0057] EXAMPLES

[0058] Aspects of the subject matter described above will now be illustrated by means of the following examples. It will be understood that the examples only provided to illustrate the described subject matter are not intended to limit the scope of the description in any way.

[0059] EXAMPLE 1 : Identifying potentially suitable liposome formulations

[0060] The present example shows how to screen liposome formulations and identify those which may be suitable for use in the current invention.

[0061] Material and Methods:

[0062] Formulation of the liposomes:

[0063] Various mixtures of cholesterol and lipids were combined with chloroform in a round bottom flask at the ratios given in Table 1. The formulation was roto-evaporated at 40°C until all the chloroform was removed. The flask was then frozen and lyophilized overnight to remove any residual organic solvent. The formulation was rehydrated with 6 mL of buffered solution (buffer composition used are listed in Table 2) at 45°C for 30 minutes with rhBMP-2 at various concentrations up to 1.5 mg/mL and sonicated 3 times at 30 seconds each to encapsulate rhBMP-2 and remove any residual lipid film on the flask. After application of 13 freeze thaw cycles, the formulation is extruded ten times with a 200 nm polycarbonate filter to convert multilamellar vesicles to unilamellar vesicles.

[0064] Table 1 : Liposome Formulations investigated

[0065] DPPC: 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine

[0066] DSPC: 1 ,2-distearoyl-sn-glycero-3-phosphocholine

[0067] CH: Cholesterol

[0068] DSPE-PEG: 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000]

[0069] POPE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

[0070] POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

[0071] Table 2: Buffers used for Liposome Preparation

[0072] IFB buffer

5. Polysorbate 80 0.1 mg/mL

pH of 4.3±0.2

[0073] AGN buffer

[0074] Purification of liposomes and estimation of encapsulation efficiency:

[0075] The extruded unilamellar vesicles were further purified using size exclusion chromatography (SEC) to remove unencapsulated rhBMP-2. The solution containing liposomes were applied on a GE Healthcare Life Sciences Hi Load 16/600 Superdex column. GE Healthcare Life Science AKTAprime was used to collect fractions. The flow rate was maintained at 1 ml/min using AGN buffered solution. Fractions collected in 5 ml volumes were further analyzed using an ELISA to measure unencapsulated rhBMP- 2.

[0076] The amount of encapsulated protein was estimated utilizing the following formula:

_ . . . (Total protein loaded)-( protein recovered after SEC )x 100

[0077] Encapsulation efficiency (%) = Total protein

[0078] Estimation of liposome size and zeta potential:

[0079] Purified liposome formulations obtained from fraction collector post size exclusion chromatography were analyzed for size and zeta potential using dynamic light scattering principles.

[0080] Estimation of liposome stability at 37°C for 24 hours by TEM and ELISA:

[0081] Further qualitative confirmation of particle size, structure and uniformity was evaluated by imaging the formulations. The formulations were maintained at 37°C and then applied onto a formvar grid and stained with uranyl acetate (negative staining) and imaged using Transmission electron Microscopy as described by Chetanachan et al. (Advanced Materials Research 2008; 55-57: 709-71 1). [0082] Samples were collected from the exposure chamber and BMP-2 concentration was analyzed by ELISA (Quantikine ELISA, R&D Systems Minneapolis, MN) following the manufacturer's instructions to assess BMP release.

[0083] Experimental liposome disruption by ultrasound:

[0084] The ability of ultrasound to release BMP from formulations 1 , 2, 3, 4 and 5 was assessed using an 18 mm diameter immersion type V314-SU videoscan ultrasound transducer (Olympus, Quebec City, Canada) in a water bath. This single-element, untuned, focused transducer was operated at 1 .0 MHz with a focal length of 36 mm. The transducer was driven by a programmable arbitrary waveform generator (AWG520; Tektronix,

Beaverton, OR) connected to an RF power amplifier (RPR-400; Ritec, Warwick, Rl).

Acoustic pressures emitted by the transducer in water were quantified using a wideband PVDF polymer membrane hydrophone probe (Sonic 804; Sonora Medical Systems, Longmont, CO). Ultrasound exposure at 1000kPa for the various test formulations (1 ml) were done in a stirred cuvette, containing liposomes containing 10 μg equivalent of rhBMP-2 in 1 ml of AGN, maintained at 37°C in the water bath. The solution was then collected and the amount of BMP-2 released measured by ELISA as described above.

[0085] Evaluation of BMP solution formulation:

[0086] Studies also compared the stability of formulations 2 and 4 (Listed in Table 1) when using BMP-2 1 mg/mL in IFB buffer or AGN buffer. Transmission Electron Microscopy and an ELISA assay carried out to evaluate stability of formulations in IFB and AGN buffer.

[0087] Results:

[0088] The results are summarized in Table 3. TEM images revealed that formulations 1 , 3, 5, 6 (all of which contained DPPC) broke apart when kept at 37°C for 24 hours.

[0089] Encapsulation efficiency estimates for formulations 7, 8 and 9 which included those containing POPE (1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) and POPC (1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) were less than 70%.

[0090] Two formulations (2, 4) containing DSPC (1 ,2-distearoyl-sn-glycero-3- phosphocholine) and cholesterol with or without DSPE-PEG (1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N[amino(polyethylene glycol)-2000] ) were stable at 37°C (Figure 1) and had an encapsulation efficiency of over 70%. [0091] Formulations 1 and 3 released only limited amounts of BMP upon exposure to ultrasound, while formulations 2, 4 and 5 released greater than 60% of the estimated amount of BMP present.

[0092] Studies showed that liposome formulations 2 and 4 when rehydrated with BMP in AGN buffer were not stable, while those rehydrated using BMP in IFB buffer were.

[0093] Table 3: Summary of Characterization studies carried out on Liposome formulations

[0094] DPPC: 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine

[0095] DSPC: 1 ,2-distearoyl-sn-glycero-3-phosphocholine

[0096] CH: Cholesterol

[0097] DSPE-PEG: 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000]

[0098] POPE: 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine

[0099] POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

[00100] nd: not detected [00101] EXAMPLE 2: Release of rhBMP2 from liposomes upon ultrasound exposure

[00102] The present example demonstrates how to determine whether ultrasound can trigger the release of a growth factor such as rhBMP-2 from selected liposome formulations as determined from Example 1.

[00103] Materials and Methods:

[00104] Liposomes of formulations 2, and 4 were prepared as described in Example 1 . The liposome solution were placed in an exposure chamber and gently stirred to provide uniform ultrasound exposure to the sample. Ultrasound (US) exposure comprised 80 cycles ultrasound pulses at 1 MHz and 0.8% duty cycle with peak negative pressures ranging from 200 kPa to 1000 kPa and total ultrasound exposure times ranging from 30 seconds to 60 seconds. Samples were collected from the exposure chamber and BMP-2 concentration was analyzed by ELISA (Quantikine ELISA, R&D Systems Minneapolis, MN) following the manufacturer's instructions or were processed for examination by TEM as described in example 1.

[00105] Results:

[00106] Upon US exposure, the liposome formulations disintegrated as demonstrated by TEM (Figure 2). The DSPC:CH formulation released approximately 72% (7.560 ± 0.991 μg) of the encapsulated rhBMP-2 after a 60 second US exposure at 1000kPa. The DSPC:DSPE- PEG:CH formulation released approximately 77% (8.150 ± 0.532 μg) of rhBMP-2 after a 60 second exposure of US at 1000kPa (Figure 3). In the absence of US no rhBMP-2 was measured indicating that there is no leakage of BMP prior to US exposure.

[00107] The amount of BMP released could be varied by varying the time of exposure and the ultrasound pressure used (Figure 4). The amount of rhBMP-2 released was approximately 1.5 times more for 60 second exposure compared to 30 second exposure at 1000kPa. In addition, of prime importance pressures ranging from 200 up to 700 kPa had a limited amount of protein released.

[00108] EXAMPLE 3: The bioactivity of rhBMP2 released from the liposomes. [00109] Methods:

[00110] The bioactivity of rhBMP-2 protein was measured by using a C2C12 cell based assay. Liposomes containing rhBMP-2 were exposed to ultrasound as described in Example 2. The amount of BMP released into buffer was then measured by ELISA and then known amounts of recovered BMP were incubated with C2C12 cells. After 48 to 72 hours the cells were lysed and the alkaline phosphatase activity was measured using a colorimetric assay as described by Peel et al. J Craniofacial Surg. 2003; 14:284-291.

[00111] Results:

[00112] The results are summarized in Figure 5. No significant differences were observed in the ALP activity stimulated by equivalent amounts of rhBMP-2 released from both formulations when exposed to 30 or 60 second exposure and was comparable to the amount of ALP stimulated by an equivalent amount of rhBMP-2 that had not been formulated into the liposomes (N=3, n=3. 3 individual trials and triplicates within each trial).

[00113] Further we also validated the effect of US on rhBMP-2 alone. No significance difference was observed up to 30 minute exposure and no loss of rhBMP-2 activity was observed up to 15 minute exposure. (N=3, n=3. 3 individual trials and triplicates within each trial)

[00114] EXAMPLE 4: Stability of liposome formulations with extended storage

[00115] The stability of liposome formulations post preparation is critical in the process of developing a useful liposome preparation for delivery of growth factors. In order to identify the stability of liposomes encapsulating rhBMP-2, stability studies were performed.

[00116] Methods

[00117] For both DSPC:CH and DSPC:DSPE-PEG:CH formulations long term stability was assessed by storing formulations at 2-8°C and -80°C for periods of 1 week to 6 months. To assess physically stability, the formulations were imaged post negative staining via TEM at day 1 , 3 months and 6 months post storage (as described in example 1). To further verify if the liposome formulations could still be triggered to release rhBMP-2, the samples were exposed to US and the recovered rhBMP-2 measured by ELISA assay (as described in example 2).

[00118] Results:

[00119] TEM images confirmed samples stored at 2-8°C and -80°C were stable up to 3 and 6 months respectively. The structural integrity at 3 months for samples stored at 2-8°C and 6 months for samples store at -80 °C was directly comparable to sample imaged on day 1. At 3 months, samples stored at 2-8 °C and -80 °C exposed to ultrasound released BMP-2 as measured by ELISA.

[00120] EXAMPLE 5: Bone formation in vivo by a selected liposome formulation containing rhBMP-2.

[00121] To determine whether the selected liposome formulations remain stable when implanted in vivo and release sufficient amounts of BMP to stimulate de novo bone formation only when exposed to ultrasound the following study was performed.

[00122] Methods:

[00123] DSPC:DSPE-PEG:CH formulations with and without rhBMP-2 were prepared as described in Example 1.

[00124] Preliminary studies were conducted to assess the appropriate exposure time needed to release the BMP from the liposomes using a tissue phantom made of agarose. Two percent agarose was poured into a plastic cup and allowed to set. A pocket was prepared where liposome preparations were then placed. This was then covered with a further slab of agarose. Ultrasound gel was then applied to the agarose and the preparations exposed to ultrasound for various times. The agarose was then cut open and shaken at room temperature overnight and the solution collected and the amount of BMP-2 present in solution was determined by ELISA as described in Example 1.

[00125] For in vivo studies liposome formulations with and without rhBMP-2 were applied to a piece of absorbable collagen sponge (INFUSE kit, Medtronic Canada) to form a bioimplant. The ACS was then placed in a gelatin capsule. Each gelatin capsule was estimated to contain approximately 35-40 μg of rhBMP-2 protein encapsulated within the liposome formulation.

[00126] The capsule was then implanted into a biceps femoris of a mouse as described by Barr et al. (Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and

Endodontics. 2010; 109:531 -540).

[00127] Certain bioimplants were exposed to US for 5 minutes at 1000KPa following implantation. The experimental groups for this study are described Table 4. After 28 days the mice were euthanized and the hind limbs removed, fixed and analyzed by microCT and histology. [00128] Agarose phantom tissues and mice were exposed to ultrasound using a preclinical Vevo 2100 scanner with a MS-250 probe (Fujifilm Visualsonics, Toronto, Canada). In both cases, the probe was used at approximately 1 cm distance from the implant for a duration of 5 min at a frequency of 16 MHz (100% power in B-mode, frame rate 30 fps). For treatment of mice, hindlegs were shaved to allow probe contact with skin.

[00129] Table 4: Experimental Design

[00130] N= 8 mice per group

[00131] Results:

[00132] Results from the tissue phantom experiment showed that ultrasound exposure durations of several minutes were required to ensure significant BMP-2 release (Figure 9). It was noted that it took significantly longer to release most of the BMP-2 from the liposomes when embedded in the tissue phantom than when the liposomes were in solution (compare Figure 9 with Figure 4). Based on the results of these studies a time of 5 minutes was selected for the duration of ultrasound in the in vivo studies.

[00133] Results show that the bioimplants comprising liposome encapsulated BMP did not induce detectable amounts of bone unless they were exposed to ultrasound. The amount of bone formed when the liposome bioimplant was exposed to a single ultrasound exposure immediately following implantation was equivalent to the amount produced when similar amount of BMP was applied directly to the ACS and implanted (Figure 6).

[00134] Histological evaluation confirmed that the bone formed was similar in both the positive control and ultrasound exposed liposome bioimplant. No bone or cartilage formation was observed in liposome bioimplants that had not been exposed to ultrasound. [00135] These results show that the liposome formulations used to form bioimplants do not form any bone following implantation for up to 28 days in the absence of exposure to ultrasound, while exposure to ultrasound releases bioactive BMP that is capable of inducing bone formation.

[00136] EXAMPLE 6: Determination of the effect of timing of ultrasound exposure on the amount of bone formed in vivo

[00137] To determine the effect of varying the time when ultrasound was applied on the amount of bone formed the following study was performed.

[00138] Methods:

[00139] DSPC:DSPE-PEG:CH formulation were prepared as described in Example 1. These were then applied to a piece of absorbable collagen sponge (taken from an INFUSE kit, Medtronic Canada) to form a bioimplant. This was then placed in a gelatin capsule. Each gelatin capsule was estimated to contain approximately 35-40 μg of rhBMP-2 protein encapsulated within the liposome formulation. The capsule was then implanted into a biceps femoris of a mouse as described by Barr et al. (Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics. 2010; 109:531-540).

[00140] Bioimplants were exposed to one or two ultrasound exposures of 5 minutes at 1 MPa following implantation at various times after implantation as described Table 05. A Vevo 2100 scanner with a MS-250 probe (Fujifilm Visualsonics, Toronto, Canada) was used. The probe was used at approximately 1 cm distance from the implant for a duration of 5 min at a frequency of 16 MHz (100% power in B-mode, frame rate 30 fps). Before treatment of mice, hindlegs were shaved to allow probe contact with skin.

[00141] After 28, 35 or 42 days the mice were euthanized and the hind limbs removed, fixed and analyzed by microCT and histology.

[00142] Table 5: Experimental Design

4 LIP+ACS X X H H

5 LIP+ACS X X H

6 LIP+ACS X X H H

7 ACS H H

[00143] Legend: ACS - acellular collagen sponge; LIP-ACS - liposome containing rhBMP-2 on ACS; Ultrasound treatment (X); Harvest (H)

[00144] Results:

[00145] Results show that the timing of a single exposure of ultrasound to the Liposome- ACS bioimplant affects the amount of bone formed with significantly less bone formed after 42 days by bioimplants exposed to ultrasound on day 14 than bioimplants exposed to ultrasound on day 0 (day of implantation) or day 7 (Figure 7).

[00146] Results also show that multiple exposures to ultrasound increased the amount of bone formed, with most bone being formed when the first exposure was on the day 0 and the second exposure was either on day 0 or day 7 (Figure 8).

[00147] EXAMPLE 7: Determination of the ability of preparing a P407-liposome gel for delivery of rhBMP-2 by ultrasound.

[00148] Methods:

[00149] Poloxamer 407 was weighed and mixed slowly into chilled liposome formulations 2 or 4 (prepared as described in Example 1) at a ratio of 3.3g P407 in 10mL liposome solution. The mixture was left mixing overnight at 2-8 °C.

[00150] 100μΐ_ of the each poloxamer-liposome solution was mixed in 900μΐ_ of phosphate buffered saline (PBS). Samples were then exposed to ultrasound and the amount of BMP which was released from each preparation was measured by ELISA as described in Example 2.

[00151] Results:

[00152] Pegylated liposomes (formulation 4) were found to be leaky when mixed with P407, releasing large amounts of BMP into the solution in the absence of ultrasound. In contrast, the non-pegylated liposomes (formulation 2) only released BMP when exposed to ultrasound.

[00153] Although the above description includes reference to certain specific

embodiments, various modifications thereof will be apparent to those skilled in the art. Any examples provided herein are included solely for the purpose of illustration and are not intended to be limiting in any way. Any drawings provided herein are solely for the purpose of illustrating various aspects of the description and are not intended to be drawn to scale or to be limiting in any way. The scope of the claims appended hereto should not be limited by the preferred embodiments set forth in the above description, but should be given the broadest interpretation consistent with the present specification as a whole. The disclosures of all prior art recited herein are incorporated herein by reference in their entirety.

Claims

CLAIMS:

1 . A composition for use in promoting bone repair, the composition compromising liposomes encapsulating one or more bone morphogenetic proteins, BMPs.

2. The composition of claim 1 , wherein the liposomes are formed from a phospholipid material, wherein the phospholipid material is a phosphatidylcholine and/or a

phosphoethanolamine, and cholesterol.

3. The composition of claim 1 or 2, wherein the phospholipid material includes a polyethylene glycol moiety.

4. The composition of any one of claims 1 to 3, wherein the liposomes are formed from 1 ,2-distearoyl-sn-glycero-3-phosphocholine, DSPC, and cholesterol.

5. The composition of claim 4, wherein the ratio of DSPC:cholesterol is about 7:3 (w/w).

6. The composition of any one of claims 1 to 3, wherein the liposomes are formed from: 1 ,2-distearoyl-sn-glycero-3-phosphocholine, DSPC; 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000], DSPE-PEG; and cholesterol.

7. The composition of claim 6, wherein the ratio of DSPC: DSPE-PEG: cholesterol is about 45:45: 10 (w/w).

8. The composition of any one of claims 1 to 7, wherein the liposomes are stable for at least 24 hours at 37 °C.

9. The composition of any one of claims 1 to 7, wherein the protein encapsulation efficiency of the liposomes is at least 70%.

10. The composition of any one of claims 1 to 9, wherein the one or more BMPs is/are BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-

1 1 . BMP-12, BMP-13, BMP-14, BMP-15, or a combination thereof.

1 1. The composition of claim 10, wherein the one or more BMPs is/are mammalian BMPs.

12. The composition of claim 10, wherein the one or more BMPs is/are human BMPs.

13. The composition of claim 12, wherein the one or more BMPs is/are hBMP-2 and/or hBMP-7.

14. The composition of any one of claims 1 to 13, wherein the liposomes are adapted to release the one or more BMPs upon exposure to ultrasound stimulation.

15. The composition of claim 14, wherein the peak negative pressure of the ultrasound exposure is from about 200 kPa to about 6 MPa.

16. The composition of claim 14 or 15, wherein the ultrasound frequency is from about 1 MHz to about 20 MHz.

17. The composition of claim 16, wherein the ultrasound mechanical index is from about 0.5 to about 1.5.

18. The composition of any one of claims 14 to 17, wherein the ultrasound exposure is from about 5 seconds to about 60 minutes.

19. The composition of any one of claims 1 to 18, wherein the composition further comprises a polymer in the form of a liquid, gel or putty.

20. The composition of claim 19, wherein the polymer is a poloxamer.

21. Use of the composition of any one of claims 1 to 20 in the delivery of the BMPs to a target site.

22. The use of claim 21 , wherein the delivery of the BMPs is triggered by exposing the liposomes to ultrasound stimulation.

23. The use of claim 22, wherein the peak negative pressure of the ultrasound exposure is from about 200 kPa to about 6 MPa.

24. The use of claim 22 or 23, wherein the ultrasound frequency is from about 1 MHz to about 20 MHz.

25. The use of claim 24, wherein the ultrasound mechanical index is from about 0.5 to about 1.5.

26. The use of any one of claims 22 to 25, wherein the ultrasound exposure is from about 5 seconds to about 60 minutes.

27. The use of any one of claims 22 to 26, wherein the liposomes are exposed to a first ultrasound stimulation within the first seven days after implantation of the liposomes at an implantation site.

28. The use of claim 27, wherein the liposomes are subjected to one or more further ultrasound exposures.

29. The use of claim 28, wherein the one or more further ultrasound exposures occur within the first seven days after implantation and/or later.

30. The use of claim 29, wherein the first and one or more further ultrasound exposures comprise ultrasound exposures of different frequencies, peak negative pressures, mechanical indices, and/or exposure times.

31 . A bioimplant having thereon or therein, a composition according to any one of claims 1 to 20.

32. The bioimplant of claim 31 , wherein the bioimplant comprises a scaffold material.

33. The bioimplant of claim 32, wherein the scaffold material is a collagen sponge.

34. A method of delivering one or more bone morphogenetic proteins, BMPs, at a target site, the method comprising: a) providing the composition of any one of claims 1 to 20;

b) implanting the composition at an implantation site within an animal; and, c) subjecting the target site to ultrasound exposure sufficient to rupture the liposomes and release the one or more BMPs.

35. The method of claim 34, wherein step (a) comprises applying the composition to a region within the implantation site.

36. The method of claim 34, wherein step (a) comprises combining the composition with a bioimplant, and wherein step (b) comprises implanting the bioimplant at the implantation site.

37. The method of any one of claims 34 to 36, wherein the peak negative pressure of the ultrasound exposure is from about 200 kPa to about 6 MPa.

38. The method of any one of claims 34 to 37, wherein the ultrasound frequency is from about 1 MHz to about 20 MHz.

39. The method of claim 38, wherein the ultrasound mechanical index is from about 0.5 to about 1.5.

40. The method of any one of claims 34 to 39, wherein the ultrasound exposure is from about 5 seconds to about 60 minutes.

41. The method of any one of claims 34 to 40, wherein the liposomes are exposed to a first ultrasound stimulation within the first seven days after implantation of the liposomes at the implantation site.

42. The method of claim 41 , wherein the liposomes are subjected to one or more further ultrasound exposures.

43. The method of claim 42, wherein the one or more further ultrasound exposures occur within the first seven days after implantation and/or later.

44. The method of claim 43, wherein the first and one or more further ultrasound exposures comprise ultrasound exposures of different frequencies, peak negative pressures, mechanical indices, and/or exposure times.