Liposomal drug delivery system for bone cements

Abstract

The invention relates to a novel antibiotic delivery vehicle for impregnating bone cement wherein said vehicle is an antibiotic encapsulated liposome having a block co-polymer on its surface; a method for the manufacture of a bone cement impregnated with antibiotic or a mixture of antibiotics using said vehicle; and also a novel bone cement made therewith and/or thereby.

Claims

1. A bone cement having dispersed therein a vehicle for delivering and dispersing at least one antibiotic in said bone cement, wherein said vehicle comprises a liposome containing said at least one antibiotic, further wherein said liposome also comprises a block co-polymer adsorbed or absorbed onto the liposome, the block co-polymer having an average molecular weight less than 2000 and a higher proportion of polypropylene oxide to polyethylene oxide.

2. The bone cement according to claim 1 wherein said liposome is selected from the group comprising: a liposome less than 600 nm in diameter when measured using laser diffraction; a liposome less than 150 nm in diameter when measured using Transmission Electron Microscopy (TEM); and a liposome about 100 nm in diameter using TEM.

3. The bone cement according to claim 1 wherein said liposome is made from a phospholipid selected from the group consisting of: cationic phospholipids, neutral phospholipids, anionic phospholipids and one or more combinations thereof.

4. The bone cement according to claim 1 wherein said liposome is made from at least one phospholipid selected from the following groups: phospatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidic acid, phospatidylglycerol, phospatidylserine and phospatidylinositol.

5. The bone cement according to claim 1 wherein said liposome is made from at least one cationic lipid selected from the following groups: 1,2 dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammonium chloride (DODAc), 1,2-dimyristoyloxypropyl-3-dimethyl-hydroxyethyl ammonium (DMRIE), 2,3-dioleoyloxy-N-(2(sperminecarboxamide)ethyl)-N,N-dimethyl-1 propananninium (DOSPA), 1,2-dimethyl-dioctadecylammoniumbromide (DDAB), 2-dioleyl-3-N,N,N-trimethylaminopropanechloride (DOTMA), 1,2-dimyristoyl-3-trimethylammoniumpropane DMTAP, 1,2-distearoyl-3-trimethylammoniumpropane (DSTAP), 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ) and dioctadecylamidoglycylspermine (DOGS).

6. The bone cement according to claim 1 wherein said liposome is selected from the group consisting of: dimyristoyl-phosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylglycerol-cholesterol (DSPG), cholesteryl sulfate, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 1.2-dipalmitoyl-sn-3-glycero-[phosphorrac-(1-glycerol)] (DPPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), egg yolk phosphatidylcoline (EPC), Dioleoylphosphatidylethanolamine, egg phosphatidylglycerol (ePG), Polyethylene glycol (PEG)-dendron phospholipids, Dipalmitoylphosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG200 DSPE), monostearoylphosphatidylcholine (MSPC), 1-stearoyl-L--phosphatidyl (SHPC), Soybean phospholipids (SPC), egg sphingomyelin, stearylamine (SA), and 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyl ethyl ammonium bromide (DMRIE).

7. The bone cement according to claim 1 wherein said block co-polymer is selected from the group consisting of: Pluronic L31, Pluronic L43 and Pluronic L61.

8. The bone cement according to claim 1 wherein said antibiotic is selected from the list consisting of: gentamicin, vancomycin, tobramycin, ampicillin, benzylpenicillin, erythromycin, kanamycin, methicillin, neomycin, streptomycin, tetracycline, co-trimoxazole, cloxacillin, chloramphenicol, cephaloridine, cephazolin, oxacillin, ciprofloxacin, aztreonam and imipenem.

9. The bone cement according to claim 1 wherein said antibiotic comprises two or more antibiotics.

10. The bone cement according to claim 1 wherein said cement is selected from the group consisting of: poly(methyl methacrylate) (PMMA), methacrylate-cements and acrylic resins.

11. The bone cement according to claim 1 wherein said cement comprises a plurality of said liposomes.

12. The bone cement according to claim 11 wherein said plurality of liposomes is selected from one or more of the groups consisting of: liposomes made from the same lipid; liposomes made from the same two or more different lipids; different liposomes made from a different lipid; different liposomes made from two or more different lipids; liposomes made from the same one or more lipid(s) but containing different antibiotics; and liposomes made from different one or more lipids but containing different antibiotics.

13. The bone cement according to claim 1 wherein said vehicle or said cement also comprises an agent selected from the group consisting of: other antimicrobial agents, drugs to stimulate bone formation, therapeutic agents, strontium, bisphosphonates and bone morphogenetic proteins.

14. A method for the manufacture of bone cement comprising mixing together a polymer suitable for making bone cement, or a precursor thereof, with a vehicle for delivering and dispersing at least one antibiotic within said bone cement; wherein said vehicle comprises a liposome containing said antibiotic; and further wherein said liposome comprises a block co-polymer adsorbed or absorbed onto the liposome, the block co-polymer having an average molecular weight less than 2000 and a higher proportion of polypropylene oxide to polyethylene oxide.

15. The method according to claim 14 wherein said precursor is a monomer.

16. The method according to claim 14 wherein a suspension of said vehicle is mixed into a liquid component of the bone cement prior to mixing this liquid component with the polymer or precursor thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Shows the structure of a liposome;

(2) FIG. 2. Shows a diagrammatic arrangement of Pluronics on the surface of a liposome;

(3) FIG. 3. Shows fluorescence microscopy images of liposomes in (a-b) water and (c-d) Palacos R, with L61 Pluronic;

(4) FIG. 4. Shows particle sizes of the most effective Pluronic-coated liposomes in methyl methacrylate obtained by laser diffraction;

(5) FIG. 5. Shows TEM images of (a) uncoated liposomes and (b) liposomes coated with L61 in water and (c) uncoated liposomes and (d) liposomes coated with L61 in methyl methacrylate (bar=1 m);

(6) FIG. 6. Shows TEM image of liposomes coated with L61 in methyl methacrylate (bar=2 m);

(7) FIG. 7. Shows sedimentation rates of different Pluronic-coated liposomes in methyl methacrylate over time obtained by absorption measurements at 420 nm;

(8) FIG. 8. Shows cumulative percentage gentamicin release after 72 hours from Cemex Genta, CMW smartest, Palacos R+G and Palacos R cement containing liposomes coated with Pluronics L31, L43 and L61 at the amount specified;

(9) FIG. 9. Shows cumulative percentage gentamicin release after 1440 hours from Cemex Genta, CMW smartest, Palacos R+G and Palacos R cement containing liposomes coated with L31, L43 and L61 at the amount specified;

(10) FIG. 10. Shows zones of S. aureus growth inhibition on agar plates for Palacos R and Palacos R+G bone cement and Palacos R cement containing gentamicin-loaded liposomes coated with Pluronic L61;

(11) FIG. 11a. Shows SEM images of CMW smartest, Cemex, Cemex Genta, Palacos R before and after 1440 hours in Ringer's solution;

(12) FIG. 11b. Shows SEM images of Palacos R+G and Palacos R containing liposomes coated with Pluronics L31, L43 and L61 cement samples before and after 1440 hours in Ringer's solution;

(13) FIG. 12. Shows compressive strength of commercial Palacos R and Palacos R+G cements, with and without antibiotic respectively, when compared with Palacos R containing liposomes coated with Pluronics L31, L43 and L61;

(14) FIG. 13. Shows bending strength of commercial Palacos R and Palacos R+G cements, with and without antibiotic respectively, when compared with Palacos R containing liposomes coated with Pluronics L31, L43 and L61;

(15) FIG. 14. Shows bending modulus of commercial Palacos R and Palacos R+G cements, with and without antibiotic respectively, when compared with Palacos R containing liposomes coated with Pluronics L31, L43 and L61;

(16) FIG. 15. Shows fracture toughness of commercial Palacos R and Palacos R+G cements, with and without antibiotic respectively, when compared with with Palacos R containing liposomes coated with Pluronics L31, L43 and L61;

(17) FIG. 16. Shows the structure of a typical phospholipid. This consists of two hydrophobic fatty acid chains connected by a glycerol molecule and a phosphate molecule to an hydrophilic head group. The presence of both hydrophobic and hydrophilic parts result in an amphiphatic molecule. Phospholipids form the bilayer of liposomes, with a layer of lipids orientated with the hydrophilic head groups facing outwards and a layer of the lipids with the hydrophilic head groups orientated inwards (FIG. 2). The different head groups of naturally-occurring phospholipids are also shown. Phospatidylcholine and phosphatidylethanolamine are neutral as the positive charge of the head group and the negative charge of the phosphate molecule neutralise one another. These lipids are also referred to as zwitterionic, i.e. they comprise both positive and negative charged groups. Sphingomyelin containing liposomes are also considered neutral. Phospholipids with a neutral head group are negatively charged due to the negative charge of the phosphate molecule. Negatively charged liposomes are those which contain phosphatidic acid, glycerol, serine and inositol head groups. Certain lipids exist which do not have phosphatidyl moieties. The charge on these lipids is governed mainly by the charge on the hydrophilic head group. This has allowed for the manufacture of synthetic cationic (positively charged) liposomes. Examples of cationic liposomes include DOTAP, DODAc, DMRIE, DOSPA, DMTAP, DSTAP, DODAP, DOBAQ, DDAB and DOGS.

(18) Table 1. Particle sizes of Pluronic-coated liposomes in methyl methacrylate measured by laser diffraction;

(19) Table 2. Pluronics and their properties (tested Pluronics underlined);

(20) Table 3. Shows the liposomal-based therapeutics that have been clinically approved; and

(21) Table 4. Shows the liposome-based therapeutics currently undergoing clinical trials. Both tables show the liposome composition for each product; the overall charge of the liposomal system; the therapeutic agent encapsulated and the approved indication.

LIST OF ABBREVIATIONS

(22) DDAB, 1,2-dimethyl-ioctadecylammoniumbromide

(23) DMPC, 1--dimyristoylphosphatidylcholine

(24) DMPG, l--dimyristoylphosphatidylglycerol

(25) DMRIE, (1,2-dimyristoyloxypropyl-3-dimethyl-hydroxyethyl ammonium)

(26) DMTAP, 1,2-dimyristoyl-3-trimethylammoniumpropane

(27) DOBAQ, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium

(28) DOGS, (dioctadecylamidoglycylspermine).

(29) DOPC, 1,2-Dioleoyl-sn-glycero-3-phosphocholine

(30) DODAc, (dioctadecyldimethylammonium chloride)

(31) DOTAP, 1,2 dioleoyl-3-trimethylammoniumpropane

(32) DODAP, 1,2-Dioleoyl-3-dimethylammonium-propane

(33) DOTMA, 2-dioleyl-3-N,N,N-trimethylaminopropanechloride

(34) DOPC, dioleoylphosphatidylcholine

(35) DOPE, dioleoylphosphatidylethanolamine

(36) DOSPA, (2,3-dioleoyloxy-N-(2(sperminecarboxamide)ethyl)-N,N-dimethyl-1 propananninium)

(37) DPPC, dipalmitoylphosphatidylcholine

(38) DPPG, dipalmitoylphosphatidylglycerol

(39) DSPC, distearoylphosphatidylcholine

(40) DSPE, distearoylphosphatidylethanolamine

(41) DSPG, distearoylphosphatidylglycerol

(42) DSTAP, 1,2-distearoyl-3-trimethylammoniumpropane

(43) EPC, egg phosphatidylcholine

(44) EPG, egg phosphatidylglycerol

(45) HSPG, hydrogenated soy phosphatidylcholine

(46) mPEG 2000-DSPE, methoxy-polyethylene glycol-distearoyl phosphatidylethanolamine

(47) MSPC, monostearoylphosphatidylcholine

(48) PEG 2000-DSPE, polyethylene glycol 2000-distearoylphosphatidylethanolamine

(49) SPC, soy phosphatidylcholine

DETAILED DESCRIPTION

Materials and Methods

Preparation of Pluronic-Coated Liposomes

(50) Phosphatidyl choline (PC) from egg yolk (99.0%), cholesterol (C, 99.0%), uranyl acetate (98.0%), gentamicin sulphate (590 ug gentamicin base per mg), o-phthaldialdehyde (97%, HPLC), ethanol (99.8%, HPLC), 2-mercaptoethanol (99.0%), sodium borate (99.0%), methyl methacrylate (MMA, 99%) and Pluronics L31, L61, F68 and F127 were purchased from Sigma Aldrich (Gillingham, UK). Chloroform (HPLC grade) was purchased from Fisher Scientific (Fisher Scientific UK Ltd, Loughborough, UK). Pluronics L43, L44, L62, L64, P65, P84, P104, P123 were provided by BASF (BASF Corporation, Connecticut, USA). TopFluor cholesterol was purchased from INstruchemie (INstruchemie BV, Delfzijl, Netherlands). Cemex and Cemex Genta were obtained from Tecres (Sommacampagna, Italy), Palacos R and Palacos R+G were provided by Heraeus (Newbury, UK) and CMW Smartset GHV was provided by Depuy (Depuy CMW, Blackpool, UK).

(51) Phosphatidylcholine (PC) and cholesterol (C) were weighed and combined in a w/w ratio of 7:1 respectively and added to a 50 mL round-bottom flask. 5 mL of chloroform was added to the flask and the suspension was vortex mixed until the lipids dissolved. The flask was attached to a rotary evaporator with a water bath set at 60 C. (above the phase transition temperature of PC) with a vacuum pump and rotation set to 1 revolution per second. Once all the chloroform had evaporated and a thin film of lipids had formed, deionised water heated to 60 C. (above the phase transition temperature of the lipid) was added and the flask vortex mixed to create a suspension of liposome vesicles at a concentration of 5 mg/mL. The lipid suspension was held at 60 C. for 30 minutes for the liposomes to form. The suspension was extruded 10 times under nitrogen pressure (8 bars maximum) using a Lipex extruder (Northern Lipids Inc., British Columbia, Canada) through a 400 nm polycarbonate membrane (Whatman, UK) followed by further extrusion 10 times using a 100 nm polycarbonate membrane.

(52) The average liposome diameter was measured by laser diffraction using a Beckman Coulter N4 PLUS particle size analyzer (Beckman Coulter Ltd, High Wycombe, UK) to ensure an average 100 nm diameter liposome suspension was obtained. 2% w/w of Pluronic (L31, L43, L44, L61, L62, L64, P65, F68, P84, P104, P123 or F127) was added to the liposome suspension after extrusion. The liposome-Pluronic suspension was centrifuged at 100,000 g (25,000 RPM) for 1 hour at 4 C. using a Beckman Optima LE-80K centrifuge (Beckman Coulter Ltd., High Wycombe, UK) with a SW28 rotor to create a pellet in order to minimize the amount of water in the final cement mixture.

Laser Diffraction

(53) 10 mg PC:C liposome pellets with and without the Pluronics stated above were prepared as described and resuspended in 10 mL of methyl methacrylate (MMA) by the process of titruration (progressive aliquot mixing in a mortar and pestle i.e. a small amount of MMA added to the liposome pellet to create a paste, gradually adding more MMA to produce a homogeneous mixture) using a glass mortar and pestle, followed by vortex mixing. The liposome diameters in MMA were measured by laser diffraction using a Beckman Coulter N4 PLUS particle size analyzer (Beckman Coulter Ltd, High Wycombe, UK).

Sedimentation Rate

(54) The sedimentation rate was established by measuring the absorption of incident light at 420 nm by the liposome-Pluronic suspension in MMA over time using a Hitachi U-1900 spectrophotometer (Hitachi High-Technologies Europe GmbH, Mannheim, Germany) against an MMA blank. The suspensions were agitated initially and placed in the spectrophotometer, where the suspension was allowed to stand undisturbed over a period of one hour whilst absorption readings were taken.

Transmission Electron Microscopy

(55) Transmission electron microscopy (TEM) was used to assess the dispersion of liposomes in MMA and water. PC:C liposomes and PC:C liposomes with L61 Pluronic were prepared as described and mixed with 4% w/v aqueous uranyl acetate in a ratio of 1:1 and left for 60 minutes. Ratio relates to the ratio of uranyl acetate added to the liposome suspension (e.g. 1 mL of liposome suspension and 1 mL of 4% w/v uranyl acetate).

(56) The liposomes were then pelleted as described. The pellets were resuspended in water or MMA. A 10 uL droplet of each suspension was added to a Formvar carbon film on a 400 mesh Nickel grid (EM Systems Support Ltd., Macclesfield, UK) and allowed to dry in air. MMA alone was also dried on a grid as a control. The grids were observed using a Philips CM12 TEM (Philips Research, Eindhoven, Netherlands) operating at 80 kV. Images were recorded using an SIS MegaView III digital camera (Olympus Soft Imaging Solutions GmbH, Munster, Germany).

Fluorescence Microscopy

(57) To assess the dispersion of the liposomes in a commercial cement, 100 mg of liposomal material was suspended in water at a concentration of 5 mg/mL, as described. TopFluor Cholesterol (FC, 23-(dipyrrometheneboron difluoride)-24-norcholesterol, INstruchemie BV, Delfzijl, Netherlands) was used to substitute for a portion of the cholesterol component to give a ratio of 7:0.9:0.1 of PC:C:FC. The fluorescent liposome suspension was sized using laser diffraction to ensure 100 nm liposomes were formed. 1 mL of the suspension was diluted in 4 mL of distilled deionised water to obtain a concentration of 1 mg/mL and fluorescent images were taken for observation. The remaining 5 mg/mL liposomal suspension was divided into four aliquots of 4 mL. 2% w/w of Pluronic L31, L43 or L61 was added to three of the aliquots and the suspension was pelleted as previously described. Similarly, the remaining 4 mL aliquot of 5 mg/mL liposomal suspension alone was pelleted. The four pellets were individually resuspended in 2 mL of the liquid component of Palacos R (MMA) which contains N,N-dimethyl-p-toluidine (DMPT, the initiator for the polymerisation reaction) and colorant E141 for better visibility, however it mainly consists of the same MMA as above.

(58) This was mixed with 4 g of the Palacos R powder according to the manufacturer's instructions. The cement was compressed between two glass slides to create a thin sample capable of transmitting light. All cement samples were inspected under a light microscope for pores and transparency and stored in the dark until observed using an Olympus IX50 fluorescent microscope. A green filter (495-570 nm) was used to excite the fluorescent lipids and images of the emitted red fluorescence were taken.

Liposome-Pluronic Preparation with Antibiotic

(59) A similar method for liposome preparation was undertaken as previously outlined. 175 mg of PC and 25 mg C were weighed and added to a 50 mL round-bottom flask. 5 mL of chloroform was added to the flask and the suspension was vortex mixed until the lipids dissolved.

(60) The flask was attached to a rotary evaporator with a water bath set at 60 C. with a vacuum pump and rotation set to 1 revolution per second. Once the chloroform had evaporated and a thin film of lipids had formed, 40 mL of 5 mg/mL gentamicin sulphate solution, heated to 60 C., was added and the flask vortex mixed to create a suspension of liposome vesicles at a concentration of 5 mg/mL. The liposome suspension was held at 60 C. for 30 minutes for the liposomes to form. The suspension was extruded 10 times under nitrogen pressure (8 bars maximum) using a Lipex extruder (Northern Lipids Inc., British Columbia, Canada) through a 400 nm polycarbonate membrane (Whatman, UK) followed by further extrusion 10 times using a 100 nm polycarbonate membrane. A Beckman Coulter N4 PLUS particle size analyzer was used to ensure an average 100 nm liposome diameter was obtained. 2% w/w of L31 Pluronics was added and the solution was centrifuged at 100,000 g (25,000 RPM) for 1 hour at 4 C. using a Beckman Optima LE-80K centrifuge with a SW28 rotor to create a pellet. The 200 mg pellet was resuspended in 20 mL of the liquid component of Palacos R (MMA) by the process of titruration, using a glass mortar and pestle, followed by vortex mixing. The method was repeated for L43 and L61 Pluronics.

Antibiotic Release

(61) Commercially available antibiotic-loaded cements (Cemex Genta, Palacos R+G and CMW Smartset GHV) were prepared according to the manufacturer's instructions. Standard Cemex and Palacos R bone cement samples were prepared and tested to ensure leaching components of the cement did not affect measurement of antibiotic release. A 200 mg liposomal gentamicin sulphate pellet (with Pluronics L31, L43 or L61) was prepared as described and mixed with 20 mL of the MAA-based liquid component of Palacos R Cement. This was mixed with 40 g of Palacos R cement according to the manufacturer's instructions.

(62) A high-density PTFE mould was manufactured to produce 10 mm diameter by 2 mm thick cylindrical samples. All samples were finished with a 250 grit silicon carbide sandpaper to the stated dimensions with a tolerance of 0.2 mm. Each sample weighed 0.400.01 g and five samples for each test group were examined. Each sample was stored in 5 mL of Ringer's solution (8.6 mg/mL NaCl, 0.3 mg/mL KCl and 0.33 mg/mL CaCl.sub.2, buffered to a pH of 7.4 with NaHCO.sub.3 [1]) at 37 C. After 6 hours, 1, 2, 3, 7, 15, 30 and 60 days the Ringer's solution was removed and stored in the dark at 20 C. before assaying; 5 mL of fresh Ringer's was added as replacement until the next time point.

(63) The solutions were thawed overnight at room temperature in the dark and the concentration of gentamicin was determined using an o-phthaldialdehyde (PHT) method developed by Sampath et al. (1990) [2] and Zhang et al. (1994) [3], whereby a PHT reagent reacts with the amino groups of gentamicin sulphate to yield measurable fluorogenic products. The reagent was prepared by adding 2.5 g of o-phthaldialdehyde, 62.5 mL of ethanol and 3 mL of 2-mercaptoethanol to 560 mL of 0.04M sodium borate solution in distilled water. The PHT reagent was stored in an amber glass bottle in the dark for 24 hours prior to use.

(64) Twelve calibration solutions with gentamicin concentrations from 0 g/mL to 100 g/mL in Ringer's solution were prepared for the calibration curve. 1 mL of the calibration solution was added to 1 mL PHT reagent and 1 mL isopropanol and left for 40 minutes to react. The absorbance was then measured at 340 nm using a Hitachi U-1900 spectrophotometer (Hitachi High-Technologies Europe GmbH, Mannheim, Germany) and a linear relationship between concentration and absorbance was produced. 1 mL of the sample eluate was mixed with 1 mL PHT reagent and 1 mL isopropanol and left for 40 minutes to react and its absorbance compared against the calibration graph, in order to determine the concentration of gentamicin sulphate released by the samples at each time point. Average gentamicin concentrations for each time point were calculated from the 5 samples and the cumulative gentamicin release was calculated as a percentage of the theoretical maximum amount of gentamicin sulphate in each sample over 60 days.

Scanning Electron Microscopy

(65) Scanning electron microscopy (SEM) images of the surfaces following release of the antibiotic into Ringer's solution for 60 days were compared with fresh samples. Prior to imaging with an EBT1 Scanning Electron Microscope (SEM Tech Ltd, Southampton, UK) at 15 KeV, the samples were gold coated using an E65x sputter coater (Emitech, Kent, UK).

Microbial Growth and Zones of Inhibition

(66) 10 mm diameter by 2 mm thick cylindrical bone cement samples were prepared as previously described for Palacos R, Palacos R+G and Palacos R with 200 mg of L61 liposomal gentamicin sulphate. Tryptone soya agar (TSA) was prepared by dissolving 40 g of tryptone soy agar medium in one liter of distilled water. The solution was boiled for one minute then sterilized in an autoclave at 121 C. for 15 minutes. The solution was allowed to cool to 45-50 C. before being dispensed into petri dishes (9 cm diameter). The petri dishes were cooled to room temperature then stored at 8-15 C. until use. Staphylococcus aureus (S. aureus) was cultured in tryptic soy broth (TSB) for 18-24 hours at 37 C. A sterile cotton swab was used to spread the inoculum across the TSA petri dish. The petri dish was turned by 60 and the process was repeated to ensure complete surface coverage. A 10 ug gentamicin standard disc was placed on the petri dish as a control and pressure was applied to the top of the disc to ensure full surface contact. The dish was divided into segments and the bone cement samples were placed well separated on the agar, in the same manner. The petri dish was then incubated at 37 C. for 18-24 hours, after which, the zones of inhibition around the samples and gentamicin disc were measured. Images of the zones of inhibition were taken and measured using ImageJ software (National Institutes of Health, Maryland, USA). The zones of inhibition were measured as the radius of the zone minus the radius of the sample. Two measurements for each zone of inhibition were taken, perpendicular to one another. The experiment was repeated in triplicate (n=3). Observations on the appearance of the bacterial cultures and the zones of inhibition were also made.

Compressive Strength

(67) Compressive strength was determined as specified in the ISO 5833:2002 standard.[4] Cylindrical bone cement samples of Palacos R, Palacos R+G and Palacos R with 200 mg of liposomal gentamicin (with L31, L43 or L61 Pluronic) were prepared with 120.1 mm length and 60.1 mm diameter. Prior to loading, the dimensions of the samples were recorded to an accuracy of 0.01 mm. Each individual sample was then loaded incrementally in compression using a Zwick Roell ProLine table-top Z050/Z100 materials testing machine (Zwick Testing Machines Ltd., Herefordshire, UK) at a constant cross-head speed of 20 mm/min. Load and displacement was recorded and loading was stopped when failure occurred or the upper yield point had been passed. Five samples per group were tested and the compressive strength was calculated according to the ISO 5833 standard.

Bending Strength and Modulus

(68) Bending modulus and strength was determined as specified in the ISO 5833:2002 standard.[4] Rectangular bone cement samples were prepared for Palacos R, Palacos R+G and Palacos R with 200 mg of liposomal gentamicin (with L31, L43 or L61 Pluronic) with a length of 750.1 mm, width of 100.1 mm and thickness of 3.30.1 mm. Prior to loading, the width and thickness of the samples were recorded to an accuracy of 0.01 mm. A four-point bending test rig was used with a distance between the outer loading points of 601 mm and a distance between the inner loading points of 201 mm. Each individual sample was carefully placed in the centre of the four-point bending rig and loaded incrementally using a Zwick Roell ProLine table-top Z050/Z100 materials testing machine at a constant cross-head speed of 5 mm/min. Displacement as a function of applied force was recorded. Loading was stopped when failure of the specimen occurred. Five samples per group were tested and the average bending strength and average bending modulus were calculated as described in the ISO 5833 standard.

Fracture Toughness

(69) The ISO 13586:2000 standard was used to determine the fracture toughness of Palacos R, Palacos R+G and Palacos R with 200 mg of liposomal gentamicin (with L31, L43 or L61 Pluronic) samples.[5] This was similar to the bending tests but with a sharp chevron notch (roughly 4.5-5.5 mm) through the centre of the sample, created using a sharp razor blade. Prior to loading in three-point bending at 10 mm/min using a Zwick Roell ProLine table-top Z050/Z100 materials testing machine, a travelling (multi-axis) microscope was used to measure the length of the crack and the width and length of each sample was measured using a vernier caliper. The results from five samples for each group were recorded to obtain an average and the critical stress intensity was calculated as specified by the ISO 13586 standard.

RESULTS

Pluronic-Coated Liposomes Exhibit Different Particle Size in Methyl Methacrylate Using Laser Diffraction

(70) Table 1 shows the particle sizes of the liposomes in methyl methacrylate measured by laser diffraction. L31, L43 and L61 Pluronic gave significantly smaller particle sizes when compared to the other Pluronics. FIG. 4 shows nascent liposomes suspended in methyl methacrylate and the Pluronic coated liposomes that gave particle sizes below 600 nm using laser diffraction.

(71) Although laser diffraction shows the effective diameter of Pluronic coated liposomes in methyl methacrylate to be roughly 400 nm, TEM images show the liposomes to in fact be closer to 100-120 nm in diameter and relatively well dispersed in methyl methacrylate when using Pluronics such as L61 (FIG. 5 and FIG. 6).

(72) Table 3 shows examples of clinically approved liposomal-based therapeutics, with details of the liposome composition and the encapsulated drug. This table demonstrates the potential variety of stable lipid-drug combinations. Similarly, Table 4 shows the liposomal-based therapeutics currently undergoing clinical trials.

Formation of Stable Pluronic-Coated Liposome-Polymer Suspensions

(73) Absorbance values at 420 nm over time of the Pluronic coated liposomes in methyl methacrylate shows the three Pluronic-liposome combinations that achieved the smallest particle sizes (L31, L43 and L61) stay suspended in methyl methacrylate more effectively than all other liposome-Pluronic combinations tested (FIG. 7). It was clear when resuspending the liposome pellets that L31, L43 and L61 created stable suspensions in methyl methacrylate while the other Pluronic systems agglomerated and sedimented rapidly. L31, L43 and L61 coated liposomes could also be readily resuspended by agitation after extended periods of storage.

(74) From this data it is apparent that L31, L43 and L61 Pluronics on the surface of 100 nm diameter liposomes create the most stable and well dispersed suspension of liposomes in methyl methacrylate. Analysis of the structure of these three Pluronics has found that all three Pluronics have an average molecular weight less than 2000 and a higher polypropylene oxide to polyethylene oxide ratio (see Table 2). Therefore Pluronics with an average M.sub.w2000 and a higher polypropylene oxide to polyethylene oxide ratio form the most stable polymer suspensions.

Polymer-Coated Liposomal Cement Exhibits Superior Antibiotic Release

(75) FIG. 8 shows the cumulative percentage release of the samples after 72 hours and FIG. 9 shows the cumulative percentage release of the samples over 1440 hours. The cumulative percentage release was calculated as the total amount of gentamicin detected by the assay at each time point as a percentage of the theoretical total amount of gentamicin in each sample. From the results it is clear that the liposomal system is much more efficient at releasing gentamicin than the current powdered antibiotic systems. The release is also sustained over the course of 1440 hours, whilst the powdered antibiotic bone cements experience a dumping effect which plateaus after 72 hours.

(76) To demonstrate the antimicrobial efficacy of the liposomal system, coupons of Palacos R, Palacos R+G and the liposomal cement system with L61 Pluronic were placed on agar plates streaked with the bacteria S. aureus. The plates were incubated for 24 hours at 37 C. to allow the bacteria to grow. FIG. 10 shows the zones of inhibition obtained. The 10 ug gentamicin sulphate control created a zone of inhibition against S. aureus demonstrating the bacteria is susceptible to gentamicin. There was no antibacterial activity for Palacos R. Sample 1 of Palacos R+G gave the largest zone of inhibition; however sample 2 and 3 demonstrates how inconsistent the zones were due to poor antibiotic dispersion on the surface of the sample, regardless of the high levels of gentamicin sulphate present. Palacos R L61 created consistent zones of inhibition, highlighting that not only is the gentamicin sulphate well dispersed but also that the encapsulated gentamicin sulphate is readily accessible to inhibit bacterial growth.

Pluronic-Coated Liposomal Cement Exhibits Reduced Porosity

(77) FIG. 11 shows SEM images taken of the samples prior to storage in Ringer's solution and after storage in Ringer's solution for 1440 hours. All commercial cements which used powdered antibiotics resulted in large pores appearing after the antibiotic was released whilst cements without antibiotics and the liposomal bone cements demonstrated no substantive porosity after 1440 hours in Ringer's solution. Closer inspection of the surface of liposomal bone cements after 1440 hours shows barely visible sub-micron sized pores where the liposomes were released.

Pluronic-Coated Liposomal Cement Shows Acceptable Compressive and Improved Bending Strength and Fracture Toughness

(78) FIG. 12 shows the compressive strength of Palacos R, Palacos R+G and the liposomal antibiotic bone cement using L31, L43 or L61 Pluronics. Although a reduction was observed for the liposomal system, the compressive strength of the cement remained above the ISO5833 mininum requirement (70 MPa).

(79) FIG. 13 shows the bending strength of Palacos R, Palacos R+G and the liposomal antibiotic bone cement using L31, L43 and L61 Pluronics. Overall the liposomal system had significantly better (ANOVA, P0.05) bending strength than the commercially available powdered gentamicin system (Palacos R+G), but not significantly different when compared to Palacos R. Palacos R was found to have significantly higher bending modulus than Palacos R+G (FIG. 14). There were no significant differences when comparing the liposomal antibiotic cements with Palacos R and Palacos R+G.

(80) FIG. 15 shows the fracture toughness (critical stress intensity factor) of Palacos R, Palacos R+G and the liposomal antibiotic bone cement using L31, L43 or L61 Pluronics. All liposomal cements had significantly better fracture toughness over Palacos R and Palacos R+G. The increased fracture toughness may be attributed to the well dispersed 100 nm liposomes, inducing toughening mechanisms in the cement perhaps similar to rubber particles.

SUMMARY

(81) We show that the encapsulation of antibiotics within Pluronic-coated liposomal vesicles results in a well dispersed suspension. This system is stable throughout a range of temperatures (4-80 C.) and has been shown to survive the exothermic process of setting bone cement. When this liposomal suspension is incorporated into the liquid component of the bone cement and thus the polymer matrix, a better overall dispersion of the antibiotic is observed compared to conventional techniques which tend to agglomerate antibiotic powder particles, thereby preventing uniform and controlled release of the antibiotic from the bulk of the cement. Therefore this invention improves the dispersion of the antibiotic within the bone cement matrix and, as a result of this, improves the release characteristics. Moreover, advantageously it has been found that by adsorbing amphiphilic block copolymers (Pluronics) on the surface of the liposomes and incorporating these into cements, then those cements including the same exhibit enhanced structural and mechanical properties.

REFERENCES

(82) 1. Davis E J R, International A. Handbook of Materials for Medical Devices: ASM International; 2003. 2. Sampath S S, Robinson D H. Comparison of new and existing spectrophotometric methods for the analysis of tobramycin and other aminoglycosides. J Pharm Sci. 1990; 79(5):428-431. 3. Zhang X, Wyss U P, Pichora D, Goosen M F A. Biodegradable Controlled Antibiotic Release Devices for Osteomyelitis: Optimization of Release Properties. J Pharm Pharmacol, 1994; 46(9):718-724. 4. Institution BS. ISO5833:2002 Implants for surgery: Acrylic resin cements. BSI, London. 2002. 5. Institution BS. ISO13586:2000 Plastics: Determination of fracture toughness (Gic and Kic). Linear elastic fracture mechanics (LEFM) approach. BSI, London. 2000.

(83) TABLE-US-00001 TABLE 1 Particle diameters of Pluronic-coated PC:C liposomes in methyl methacrylate measured by laser diffraction Size/nm PC:C 870 400 L31 380 180 L43 390 150 L44 1580 120 L61 450 200 L62 2020 860 L64 1080 520 F68 1100 490 P84 840 370 P104 920 440 P123 750 250 F127 6160 2860

(84) TABLE-US-00002 TABLE 2 Pluronics and their properties (tested Pluronics underlined): % Mw No of No of Calcu- Pluronic Cloud Pour Mw of PEO of Average PPO PEO HLB HLB lated BASF State Number point point PPO Content PEO Mw Chains Chains HLB Group Description HLB HLB L 10 32 5 300 0 0 3200 5 0 14 13to15 Detergents- 6 12to18 O/W emulsifier L 31 37 32 900 10 110 1100 15 2 4.5 3to6 W/O 5 1to7 emulsifier L 35 73 7 900 50 950 1900 15 21 18.5 N/A N/A N/A 18to23 F 38 >100 48 900 80 3760 4700 15 85 >24 N/A N/A N/A >24 L 43 42 1 1200 30 555 1850 21 12 12 8to12 O/W 7 7to12 emulsifier L 44 65 16 1200 40 880 2200 21 20 16 15to18 Solubilizing N/A 12to18 agent L 61 17 30 1800 10 200 2000 31 4 3 3to6 W/O 4 1to7 emulsifier L 62 35 1 1800 20 472 2360 31 10 7 7to9 Wetting agent 5 1to7 L 64 58 16 1800 40 1160 2900 31 26 15 13to15 Detergents- N/A 12to18 O/W emulsifier P 65 82 27 1800 50 1700 3400 31 38 17 15to18 Solubilizing N/A 12to18 agent F 68 >100 52 1800 80 6720 8400 31 152 29 N/A N/A N/A >24 F 77 >100 48 2100 70 4620 6600 36 104 >24 N/A N/A N/A >24 L 81 20 37 2400 10 275 2750 41 6 2 N/A N/A 3 1to7 P 84 74 34 3200 40 1680 4200 55 38 14 13to15 Detergents- N/A 12to18 O/W emulsifier P 85 85 34 3200 50 2300 4600 55 52 24 N/A N/A N/A 12to18 F 87 >100 49 3200 70 5390 7700 55 122 24 N/A N/A N/A >24 F 88 >100 54 3200 80 9120 11400 55 207 28 N/A N/A N/A >24 L 92 26 7 2700 20 730 3650 46 16 5.5 3to6 W/O 4 1to7 emulsifier F 98 >100 58 2700 80 10400 13000 46 236 >24 N/A N/A N/A >24 L 101 15 23 3000 10 380 3800 52 8 1 N/A N/A 2 1to7 P 103 86 30 3000 30 1485 4950 52 33 7to12 8to12 O/W N/A 7to12 emulsifier P 104 81 32 3000 40 2360 5900 52 53 12to18 15to18 Solubilizing N/A 12to18 agent P 105 91 35 3000 50 3250 6500 52 73 12to18 15to18 Solubilizing N/A 12to18 agent F 108 >100 57 3000 80 11680 14600 52 265 >24 N/A N/A N/A >24 L 121 14 5 3600 10 440 4400 62 10 0.5 N/A N/A 1 1to7 P 123 90 31 3600 30 1725 5750 62 39 7to12 8to12 O/W N/A 7to12 emulsifier F 127 >100 56 3600 70 8820 12600 62 200 22 N/A N/A N/A 18to23

(85) TABLE-US-00003 TABLE 3 Clinically approved liposomal-based therapeutics Liposome Liposome Form/storage Trade name Company composition charge Drug Drug type time Indications Abelcet Enzon, Cephalon DMPC and DMPG Negative Amphoterecin B Polyene Suspension/24 Fungal infections (7:3 molar ratio) antimycotics months AmBisome Gilead Sciences, HSPC, DSPG Negative Amphoterecin B Polyene Powder/36 Fungal and NeXstar and cholesterol antimycotics months protozoal infections (2:0.8:1 molar ratio) Amphotec Sequus Cholesteryl sulfate Negative Amphotericin B Polyene Powder/24 Fungal infections antimycotics months DepoCyt SkyePharma, DOPC, DPPG, Negative Cytarabine Antineoplastics Suspension/18 Malignant Napp Cholesterol and months lymphomatous Triolein (7:1:11:1 meningitis molar ratio) DaunoXome Gilead Sciences, DSPC and cholesterol Neutral Daunorubicin Antineoplastics Emulsion/12 HIV-related NeXstar, Galen (2:1 molar ratio) citrate months Kaposi's sarcoma Myocet Zeneus, Cephalon EPC and cholesterol Neutral Doxorubicin Antineoplastics Powder/18 Combination (55:45 molar ratio) hydrochloride months therapy with cyclophosphamide in metastatic breast cancer Epaxal Berna Biotech, DOPC and DOPE Neutral Inactivated Vaccine Suspension/36 Hepatitis A Janssen-Cilag hepatitis A virus months (haemagglutinin) Inflexal V Berna Biotech, DOPC and DOPE Neutral Influenza virus Vaccine Suspension/12 Influenza Janssen-Cilag surface antigens months (haemagglutinin and neuraminidase) DepoDur SkyePharma, DOPC, DPPG, Negative Morphine Analgesic Suspension/24 Postsurgical Endo cholesterol, Triolein sulphate months analgesia (7:1:11:1 molar ratio) pentahydrate Visudyne QLT, Novartis EPG and DMPC Neutral Verteporfin Photosensitizing Powder/48 Age-related (3:5 molar ratio) agent months macular degeneration, pathologic myopia, ocular histoplasmis Doxil/Caelyx Ortho Biotech, HSPC, cholesterol Neutral Doxorubicin Antineoplastics Suspension/20 HIV-related Schering-Plough, and PEG 200-DSPE hydrochloride months Kaposi's sarcoma, Seqqus, (56:39:5 molar ratio) metastatic breast Janssen-Cilag cancer, metastatic ovarian cancer and prostate cancer Estrasorb Novavax HSPC Neutral Estradiol Hormone Emulsion/36 Menopausal hemihydrate months therapy

(86) TABLE-US-00004 TABLE 4 Liposome-based therapeutics in clinical trials Liposome Trade name Company Liposome composition charge Drug Drug type Indication LEP-ETU NeoPharm DOPC, cholesterol and Negative Paclitaxel Mitotic inhibitor Ovarian, breast, cariolipin (90:5:5 molar lung cancer ratio) LEM-ETU NeoPharrn DOPC, cholesterol and Negative Mitoxantrone Antineoplastics Leukemia, breast, cariolipin (90:5:5 molar stomach, liver, ratio) ovarian cancers EndoTAG-1 Medigene DOTAP, DOPC and Positive Paclitaxel Mitotic inhibitor Anti-angiogenic paclitaxel (50:43:3 molar properties, breast ratio) cancer, pancreatic cancer Arikace Insmed DPPC and cholesterol Neutral Amikacin Aminoglycoside Lung infection antibiotic Marqibo Talon therapeutics Egg sphingomyelin and Negative Vincristine Mitotic inhibitor Metastatic cholesterol (55:45 molar malignant uveal ratio) melanoma ThermoDox Celsion DPPC, MSPC and PEG Neutral Doxorubicin Antineoplastics Non-resectable 200-DSPE (90:10:4 molar hydrochloride hepatocellular ratio) carcinoma Atragen Aronex DMPC and soybean oil Neutral Tretinoin Antineoplastics Acute promyelocytic leukemia hormone-refractory prostate cancer T4N5 liposome AGI Dermatics Unknown Bacteriphage T4 Bacteri- Xeroderma lotion endonuclease 5 phage/Enzyme pigmentosum Liposomal Bio-Path Unknown Grb2 antisense, Synthetic DNA Acute myeloid Grb-2 oligodeoxynucelotide leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia Nyotran Aronex DMPC, DMPG and Negative Nystatin Polyene Systemic fungal cholesterol antimycotics infections LE-SN38 NeoPharm, lInsys DOPC, cholesterol and Negative SN-38 Metabolite Metastatic Therapeutics cardiolipin colorectal cancer Aroplatin Antigenics DMPC and DMPG Negative Cisplatin Antineoplastics Metastatic colorectal cancer Liprostin Endovasc Unknown Prostaglandin E1 Antiulcerative Peripheral vascular disease Stimuvax Merck KGaA Monophosphoryl lipid A, Negative BLP25 lipopeptide Vaccine Cancer vaccine for cholesterol, DMPG and multiple myeloma DPPC developed encephalitis SPI-077 Sequus SHPC, cholesterol, Neutral Cisplatin Antineoplastics Head and neck DSPE-PEG cancer, lung cancer Lipoplatin Regulon SPC, DPPG, cholesterol Negative Cisplatin Antineoplastics Pancreatic cancer, and mPEG 2000-DSPE head and neck cancer, mesothelioma, breast and gastric cancer, non- squamous non- small-cell lung cancer S-CKD602 Alza DPSC and DSPE-PEG Neutral Camptothecin Drug intermediate Recurrent and (95:5 molar ratio) progressive carcinoma of the uterine cervix OSI-211 OSI HSPC, cholerstol (2:1 Neutral Lurtotecan Anti-histamine Ovarian cancer, Pharmaceuticals molar ratio) head and neck cancer INX-0125 Inex Egg sphingomyelin and Neutral Vinorelbine Mitotic inhibitor Advanced solid cholesterol (55:45 molar tumors ratio) INX-0076 Inex Egg sphingmyelin and Neutral Topoitecan Antineoplastics Advanced solid cholesterol (55:45 molar tumors ratio) Liposome- Callisto DSPC and DSPG Negative Annamycin Anthracycline Acute lymphocytic Annamycin antibiotic leukemia SLIT Cisplatin Transave DPPC and cholesterol Neutral Cisplatin Antineoplastics Cancer treatments AeroLEF Delex Therapeutics EPC/SPC, cholesterol Neutral Fentanyl Analgesic Pain treatment Onco TCS Inex, Enzon DSPC, cholesterol Neutral Vincristine sulfate Antineoplastic Cancer treatments Agents Allovectin-7 Vical DMRIE and DOPE Positive HLA-B7 plasmid Gene Gene therapy of metastatic cancers Annamycin Aronex DMPC, DPPC, DMPG, Negative Annamycin Anthracycline Breast cancer Sterylamine (SA), antibiotic cholesterol