Liposomes, methods for their production and use

Abstract

Acoustically activatable liposomes, which can be used as drug delivery vehicles, methods for production and methods using said liposomes.

Description

[0001] LIPOSOMES, METHODS FOR THEIR PRODUCTION AND USE FIELD

[0002] The present invention generally relates to liposomes, particularly but not exclusively acoustically activatable liposomes, which can be used as drug delivery vehicles, methods for their production and methods using said liposomes.

[0003] BACKGROUND

[0004] Liposomes have been used as drug delivery vehicles for encapsulating various therapeutic agents. Liposomes can provide a non-toxic and biodegradable delivery vehicle that can act as a drug carrier, and enable the modification of pharmacokinetics, system toxicity and degradation or inactivation of an encapsulated agent.

[0005] Difficulties may arise in controlling the amount and size of drug particles present within liposomes and this may impact on dose control when administered for therapeutic application. There has also been ongoing difficulty in developing liposome-based systems and compositions that are capable of an effective localised and controlled release of an encapsulated agent.

[0006] Further, there are significant difficulties in safely and effectively delivering drugs to the brain, for example to treat neurological disorders. Administration methods can be ineffective, invasive and / or come with the risk of damaging tissue.

[0007] Bibliographic details of the publications referred to herein, if any, are collated at the end of the description.

[0008] OBJECT

[0009] It is an object of the present invention to provide novel liposomes, acoustically activatable liposomes, methods for their production and / or methods using said liposomes, which overcome or ameliorate at least one of the disadvantages of known liposomes and methods, or at least to provide the public with a useful choice.

[0010] STATEMENT OF INVENTION

[0011] Method of producing liposomes

[0012] In a first aspect, the invention provides a method for the production of acoustically activatable liposomes comprising an amphipathic agent nanoparticle, the method comprising at least the steps of: a) Providing a suspension of liposomes, wherein and the concentration of ammonium ions inside the liposomes is the equivalent of from approximately 200 to approximately lOOOmM of an ammonium salt and is greater than the concentration of ammonium ions outside the liposomes, b) Adding one or more amphipathic agent to the suspension of liposomes, wherein on adding, the one or more amphipathic agent is taken into the liposomes to a final concentration within the liposomes which is greater than outside the liposomes and forms a nanoparticle inside the liposome.

[0013] In one preferred embodiment of the first aspect, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of from approximately 300mM to approximately 500mM of an ammonium salt. In one particular embodiment, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of approximately 300mM of an ammonium salt. In one particular embodiment, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of approximately 500mM of an ammonium salt.

[0014] In one preferred embodiment of the first aspect, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of from approximately 300mM to approximately 500mM of ammonium sulfate. In one particular embodiment, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of approximately 300mM of ammonium sulfate. In one particular embodiment, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of approximately 500mM of ammonium sulfate.

[0015] In one embodiment of the first aspect, the liposomes provided in step a) are from approximately 80 nm to approximately 400 nm in diameter. In one embodiment, the liposomes provided in step a) are from approximately 120 nm to approximately 300 nm in diameter. In a preferred embodiment, the liposomes provided in step a) are approximately 200 nm in diameter.

[0016] In one embodiment of the first aspect, the ammonium salt is ammonium sulfate. In other embodiments, the ammonium salt is chosen from the group comprising ammonium oxalate, ammonium citrate, ammonium trimesate and / or carboxyfluorescein ammonium salt.

[0017] In one embodiment of the first aspect of the invention, the liposomes comprise a single amphipathic agent nanoparticle.

[0018] In one embodiment of the first aspect, the amphipathic agent nanoparticle is approximately 10% to approximately 60% of the size of a liposome once loaded.

[0019] In one embodiment of the first aspect, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of approximately 300mM of an ammonium salt and the amphipathic agent nanoparticle is from approximately 20% to approximately 30% of the size of a liposome once loaded. In another embodiment, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of approximately 500mM of an ammonium salt and the amphipathic agent nanoparticle is approximately 40% to approximately 50% of the size of a liposome once loaded. In one embodiment, the ammonium salt is ammonium sulfate.

[0020] In a preferred embodiment of the first aspect, in step a) the ratio of the concentration of ammonium ions inside the liposomes compared to outside the liposomes is approximately 200-300 mM and the amphipathic agent nanoparticle is from approximately 20% to approximately 30% of the size of a liposome once loaded. In another embodiment, the ratio of the concentration of ammonium ions inside the liposomes compared to outside the liposomes is approximately 500 mM and the amphipathic agent nanoparticle is approximately 40% to approximately 50% of the size of a liposome once loaded.

[0021] In one embodiment of the first aspect, the amphipathic agent is added to the suspension of liposomes in a loading medium having an osmotic concentration substantially equivalent to that inside the liposomes. In one embodiment, the loading medium comprises sodium chloride, sucrose and / or phosphate buffered saline.

[0022] In one embodiment of the first aspect, the one or more amphipathic agent is a drug or therapeutic agent. In certain embodiments an amphipathic agent comprises an amine group. In another aspect, the amphipathic agent comprises a positively ionisable functional group, for example but not limited to a primary or secondary amine, an imine, an isoxazole, a nitrogen-based heterocycle such as a pyridine, a pyrrole, or a quinoline, etc. In one embodiment, the amphipathic agent is a neuromodulatory agent. In one embodiment, the neuromodulatory agent(s) is chosen from the dopamine group comprising but not limited to: apomorphine, bromocriptine, carbergoline, ciladopa, dihydrexidine, disnapsoline, doxanthrine, epicriptine, fenoldopam, lisuride, pergolide, piribedil, pramipexole, propylnorapomorphine, quinagolide, ropinirole, rotigotine, roxindole, sumanirole, and / or SKF38393. In one embodiment, the neuromodulatory agent(s) is chosen from the dopamine antagonist group comprising but not limited to: amisulpride, aripiprazole, azaperone, benperidol, bromopride, chlorpromazine, clopenthixol, clozapine, domperidone, droperidol, eticlopride, fluphenazine, flupentixol, fluspirilene, haloperidol, quetiapine, lurasidone, metoclopramide, olanzapine, paliperidone, penfluridol, perazine, perphenazine, pimozide, prochlorperazine, promazine, raclopride, risperidone, remoxipride, SCH23390, sertindole, sulpiride, suitopride, thiethylperazine, thiothixene, thioridazine, tiapride, trifluperidol, triflupromazine, trifluoperazine, ziprasidone. In one embodiment, the neuromodulatory agent(s) is selected from the group of anti-epileptic agents comprising but not limited to: muscimol, perampanel, levetiracetam, gabapentin, pregabalin, progabide, vigabatrin. In one embodiment, the neuromodulatory agent(s) is chosen from the group of serotonin modulators comprising but not limited to: alosetron, dolasetron, granisetron, ondansetron, palonosetron, topisetron, almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan, zolmitriptan, buspirone, citalopram, clozapine, desipramine, fluoxetine, m-chlorophenylbiguanide mCPG, m-chlorophenylguanidine mCPP, m- chlorophenylpiperazine, imipramine, olanzapine, propranolol, quetiapine, risperidone, tranylcypromine, trazodone, ziprasidone, zotepine. In one embodiment, the neuromodulatory agent(s) is selected from the group of cholinergic system modulators comprising but not limited to: acetylcholine, methacholine, carbachol, bethanechol, tacrine, muscarine, pilocarpine, cevimeline, nicotine, varenicline tartrate, galantamine hydrobromide, suxamethonium chloride, epibatidine, oxotremorine, atropine, benztropine mesylate, clidinium, cyclopentolate, darifenacin, dicylomine, fesoterodine, flavoxate, glycopyrrolate, homatropine hydrobromide, hyoscyamine, orphenadrine, oxybutynin, propantheline, scopolamine, methscopolamine, solifenacin, tiotropium, tolterodine, trihexyphenidyl, trospium. In preferred embodiments, the neuromodulatory agent(s) is chosen from apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride .

[0023] Liposomes made by method of first aspect

[0024] In a second aspect, the invention provides acoustically activatable liposomes comprising an amphipathic agent nanoparticle made by a method of the first aspect.

[0025] In a third aspect, the invention provides a composition comprising liposomes of the second aspect.

[0026] Liposome generic - not linked to method of first aspect

[0027] In a fourth aspect, the invention provides acoustically activatable liposomes comprising an amphipathic agent nanoparticle, wherein the amphipathic agent nanoparticle is positioned proximal to an area of an internal face of the liposomal membrane.

[0028] In one embodiment of the fourth aspect, the nanoparticle is positioned proximal to an area of an internal face of the liposomal membrane such that a region of the liposomal membrane partially wraps the nanoparticle and is distended relative to the curve of adjacent regions of the membrane.

[0029] In one embodiment of the fourth aspect, the liposome is from approximately 80nm to approximately 400nm in diameter. In one embodiment, the liposome is from approximately 200nm in diameter to approximately 300nm in diameter.

[0030] In certain embodiments of the fourth aspect, the amphipathic agent nanoparticle(s) comprise approximately from 10% to approximately 60% of the volume of the liposome. In one embodiment of the fourth aspect, the amphipathic agent nanoparticle comprises at least one amphipathic agent. In one embodiment, the at least one amphipathic agent is a drug or therapeutic agent. In certain embodiments, an amphipathic agent comprises an amine group. In a preferred embodiment, the at least one amphipathic agent is a neuromodulatory agent. In preferred embodiments, the neuromodulatory agent is chosen from apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride.

[0031] In a fifth aspect, the invention provides a composition comprising liposomes of the fourth aspect.

[0032] In one embodiment of the second or fourth aspects, the liposomes are activatable on application of an effective acoustic signal. In a preferred embodiment, the effective acoustic signal is ultrasound. It will be appreciated that the ultrasound signal is provided with parameters effective for releasing or unloading the agent from the liposomes. The liposomes may be provided in a sample or administered to a subject. The parameters may therefore vary depending on the medium, attenuation or remoteness of the ultrasound signal from the liposomes.

[0033] A continuous or pulsed irradiation mode involving the ultrasound signal may be used. In one embodiment, a pulsed signal is used to provide a controlled step-wise release of an amount of the agent from the liposomes to the liposomes' surrounding. It will be appreciated that the amount of agent released will depend on various factors including the initial amount of agent loaded in the liposome, the permeability characteristics of the agent and liposome membrane composition, characteristics of surrounding medium, and ultrasound signal parameters.

[0034] Methods of use of liposomes

[0035] In a sixth aspect, the invention provides a method for delivering one or more amphipathic agent to a subject, the method comprising: a) Administering liposomes or a composition comprising liposomes of any one of the second to fifth aspects to the subject.

[0036] In a preferred embodiment of the sixth aspect, the method comprises the steps of: a) Administering liposomes or a composition comprising liposomes of any one of the second to fifth aspects to the subject and, b) Applying an effective acoustic signal to the subject to release the one or more amphipathic agent from the liposomes. In one embodiment of the sixth aspect, the invention provides a method for the delivery of one or more amphipathic agent to the brain of a subject, the method comprising at least the steps of: a) Administering liposomes or a composition comprising liposomes of any one of the second to fifth aspects to the subject; and, b) Applying an effective acoustic signal to an area of the head of the subject to release the one or more amphipathic agent from the liposomes and deliver it to the subject's brain.

[0037] In one embodiment of the sixth aspect, the at least one amphipathic agent is a drug or therapeutic agent. In certain embodiments, an amphipathic agent comprises an amine group. In a preferred embodiment, the at least one amphipathic agent is a neuromodulatory agent. In preferred embodiments, the neuromodulatory agent is chosen from apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride .

[0038] In a seventh aspect, the invention provides a method for the treatment of a neurological disorder in a subject, the method comprising the steps of the sixth aspect, wherein the one or more amphipathic agent is a neuromodulatory agent. In one embodiment, the neuromodulatory agent is chosen from apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride . In one embodiment, the neurological disorder is Parkinson’s disease.

[0039] In one embodiment of the sixth or seventh aspects, the liposomes or composition comprising liposomes of any one of the second to fifth aspects is administered systemically to the subject. In another embodiment, the liposomes or composition comprising liposomes of any one of the second to fifth aspects is administered intranasally to the subject.

[0040] In a preferred embodiment of the sixth or seventh aspects, the acoustic signal is ultrasound. Preferably, the ultrasound signal has a frequency between about 200 kHz and 1 MHz, and the ultrasound signal's intensity is between about 0.01 and 10 W cm'2.

[0041] Swiss style claims equivalent to method of use

[0042] In an eighth aspect, the invention provides the use of liposomes or a composition comprising liposomes of any one of the second to fifth aspects in the manufacture of a composition for the delivery of one or more amphipathic agent to a subject.

[0043] In one embodiment of the eighth aspect, the composition is formulated for delivery to a subject followed by the application of an effective acoustic signal to an area of the body of the subject to release the one or more amphipathic agent from the liposomes.

[0044] In one embodiment of the eighth aspect, the invention provides the use of liposomes or a composition comprising liposomes of any one of the second to fifth aspects in the manufacture of a composition for the delivery of one or more amphipathic agent to the brain of a subject. In one embodiment of the eighth aspect, the one or more amphipathic agent is a drug or therapeutic agent. In certain embodiments, an amphipathic agent comprises an amine group. In a preferred embodiment, the at least one amphipathic agent is a neuromodulatory agent. In preferred embodiments, the neuromodulatory agent is chosen from apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride .

[0045] In a ninth aspect, the invention provides the use of liposomes or a composition comprising liposomes of any one of the second to fifth aspects in the manufacture of a composition for the treatment of a neurological disorder in a subject, wherein the composition is formulated for delivery to the subject, followed by the application of an effective acoustic signal to an area of the head of the subject, wherein the one or more amphipathic agent is a neuromodulatory agent. In one embodiment of the nineth aspect, the one or more neuromodulatory agent is chosen from apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride . In one embodiment, the neurological disorder is Parkinson’s disease.

[0046] In one embodiment of the eighth or nineth aspects, the composition is formulated for systemic delivery to a subject. In another embodiment, the composition is formulated for intranasal or intrathecal administration to the subject.

[0047] In a preferred embodiment of the eighth or nineth aspects, the effective acoustic signal is ultrasound. Preferably, the ultrasound signal has a frequency between about 200 kHz and 1 MHz, and the ultrasound signal's intensity is between about 0.01 and 10 W cm'2.

[0048] European use

[0049] In a tenth aspect, the invention provides liposomes or a composition comprising liposomes of any one of the second to fifth aspects for use in the delivery of one or more amphipathic agent to a subject.

[0050] In one embodiment of the tenth aspect, the composition is formulated for delivery to a subject followed by the application of an effective acoustic signal to an area of the body of the subject to release the one or more amphipathic agent from the liposomes.

[0051] In one embodiment of the tenth aspect, use is for delivery of one or more amphipathic agent to the brain of a subject.

[0052] In one embodiment of the tenth aspect, the one or more amphipathic agent is a drug or therapeutic agent. In certain embodiments, an amphipathic agent comprises an amine group. In a preferred embodiment, the at least one amphipathic agent is a neuromodulatory agent. In preferred embodiments, the neuromodulatory agent is chosen from apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride . In an eleventh aspect, the invention provides liposomes or a composition comprising liposomes of any one of the second to fifth aspects for use in the treatment of a neurological disorder in a subject, wherein the composition is formulated for delivery to the subject, followed by the application of an effective acoustic signal to an area of the head of the subject, wherein the one or more amphipathic agent is a neuromodulatory agent. In one embodiment of the nineth aspect, the one or more neuromodulatory agent is chosen from apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride . In one embodiment, the neurological disorder is Parkinson’s disease.

[0053] In one embodiment of the tenth or eleventh aspects, the composition is formulated for systemic delivery to a subject. In another embodiment, the composition is formulated for intranasal administration to the subject.

[0054] In a preferred embodiment of the eighth or nineth aspects, the effective acoustic signal is ultrasound. Preferably, the ultrasound signal has a frequency between about 200 kHz and 1 MHz, and the ultrasound signal's intensity is between about 0.01 and 10 W cm'2.

[0055] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any and all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0056] FIGURES

[0057] These and other aspects of the invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures:

[0058] Figure 1: (A) Fluorescence intensity of apomorphine in a concentration range from 10 to 1000 pM. (B) Apomorphine standard curves taking into consideration the effect of liposomes and MTAB. Note: excitation and emission wavelengths of 320 and 435 nm respectively.

[0059] Figure 2: Schematic illustration depicting the active loading of apomorphine against an ammonium salt gradient.

[0060] Figure 3: Raw fluorescence intensity apomorphine liposomes before and after lysis with MTAB containing increasing initial intraliposomal an ammonium salt concentrations.

[0061] Figure 4: Active loading of apomorphine into liposomes containing 2 M an ammonium salt with and without the inclusion of an equal concentration of sodium chloride, monitored by fluorescence intensity. Figure 5: (A) Reaction scheme for the pH dependent equilibrium between the ionised and unionised state of apomorphine. (B) Active loading of apomorphine in response to liposomes containing 300 mM an ammonium salt in various pH environments monitored by fluorescence intensity over time. (C) Control of apomorphine under equivalent pH conditions with the absence of an ammonium salt liposomes over an equivalent period of time.

[0062] Figure 6: (A) Phosphotidylcholine standard curve; equation for the line: y = 10.866x + 0.0033, R2= 0.9987 at 485 nm. (B) Total phospholipid concentration, (C) total apomorphine concentration, and (D) apomorphine to phospholipid ratio for apomorphine liposomes prepared against increasing initial ammonium salt gradients.

[0063] Figure 7: Morphology of apomorphine liposomes prepared via active loading against a 300 mM an ammonium salt internal concentration (pH ~5) with an external pH of (A) 5.5, (B) 6.0, (C) 6.5, and (C) 7.0.

[0064] Figure 8: TEM images of apomorphine containing liposomes prepared against (A) 50 mM, (B) 100 mM, (C) 300 mM, (D) 500 mM, (E) 1000 mM, (F) 1500 mM, and (G) 2000 mM internal an ammonium salt concentration gradient. (H) Apomorphine liposomes prepared using a 2000 mM an ammonium salt gradient at twice the apomorphinediposome ratio.

[0065] Figure 9: (A) Cryo-TEM of apomorphine liposomes prepared from 300 mM an ammonium salt liposomes. (B) DLS of 300 mM an ammonium salt liposomes (solid line) and the resulting apomorphine liposomes (dashed line).

[0066] Figure 10: Illustrated mechanism of apomorphine sulfate nanoparticle growth in response to an ammonium salt gradient with increasing initial internal concentration.

[0067] Figure 11: (A) Absolute and (B) percentage apomorphine released from liposomes prepared using an ammonium salt liposomes with an increasing initial concentration in response to ultrasound applied at an intensity of 2.4 W cm'2.

[0068] Figure 12: (A) Percentage of apomorphine released from Aposomes upon application of ultrasound for 60 seconds at an intensity of 1.4 W cm'2at frequencies of 1 MHz (blue) and 3 MHz (orange). (B) Percentage of apomorphine released after the application of ultrasound at a frequency of 1 MHz at a duty cycle of 100% for 60 seconds (blue) or 50% for 120 seconds (orange). (C) Percentage of apomorphine released passively over 12 hours at 37 °C. Equation of the line: y = 0.5977x - 0.1779; R2= 0.9767.

[0069] Figure 13: (A) Percentage of apomorphine released in response to 60 seconds of ultrasound exposure at intensities between 0 and 2.4 W cm'2. (B) First derivative of the percentage of apomorphine released with respect to ultrasound intensity demonstrating the greatest rate of change occurring at an intensity of 2.4 W cm'2. Figure 14: (A) Cryo-TEM of an Aposome suspension prior to the application of ultrasound at 2.4 W cm'2for a cumulative exposure time of 360 seconds. Note presence of a rod-shaped nanocrystal indicated by the black arrow. (B) Cryo-TEM of the Aposome suspension after ultrasound exposure. Note the presence of empty, "wrinkled" liposomes (blue arrow), small lipid discs (green arrows), and small black nanoparticles, possibly the remnants of undissolved apomorphine sulfate (red arrows).

[0070] Figure 15: Representative examples of liposomal nanostructues (A) prior to, and (B) postapplication of ultrasound, demonstrating a change in the degree of membrane wrapping of the internal apomorphine sulfate nanoparticle. Note: hexagonal ice crystal in (A) far right panel.

[0071] Figure 16: Proposed mechanism of ultrasound triggered drug release: acoustically accelerated budding of the intraliposomal apomorphine sulfate nanoparticle, which subsequently dissolves upon release from the liposome body.

[0072] Figure 17: (A) Negative-stain TEM of HSPC liposomes containing apomorphine. (B) Release of apomorphine from HSPC Aposomes in response to ultrasound applied with an intensity of 2.4 W cm' 2

[0073] Figure 18: Chemical structures of citrate, carboxyfluorescein, oxalate and trimesate anions used to form transmembrane ammonium gradients (A). Negative stain TEM images of apomorphine liposomes loaded using 300 mM ammonium citrate (B), 200 mM carboxyfluorescein ammonium salt (C), 250 mM ammonium oxalate (D) and 300 mM ammonium trimesate (E).

[0074] Figure 19: Structures of Dl-selective agonist dihydrexideine and D2-selective agonist ropinirole (A). Negative-stain TEM of dihydrexidine actively loaded inside a liposome in response to a 300 mM an ammonium salt gradient (B). Actively loaded ropinirole in response to a 250 mM ammonium oxalate gradient at pH 6.0 (C) and pH 7.5 (D).

[0075] Figure 20: In vitro dihydrexidine and apomorphine release measured using fast-scan cyclic voltammetry. A) mean (+ / - SEM) dihydrexidine liposome release (n=ll) evoked by 60 seconds ultrasound (top). (*) and (+) indicate 1.5 and 59 seconds post ultrasound onset, respectively; points for which representative cyclic voltammagrams (CVs) are shown in the bottom plots. B) mean dihydrexidine release evoked by 2 second ultrasound application (n=9). (#) indicates 1.5 second post ultrasound for which a representative CV is plotted bottom left. Bottom right plot shows a typical CV generated by 1 pM free dihydrexidine. C) mean apomorphine release (n=5) produced by 30 seconds of ultrasound. (*) and (+) indicate 1.5 and 29 seconds post ultrasound onset, for which typical CVs are shown (bottom plots). D) mean apomorphine release (top, n=3), typical CV at 1.5 seconds post ultrasound onset (bottom, left) and representative CV generated by 1 pM free apomorphine (bottom right). E) mean current profile (left, n=8) produced by exposing buffer filled liposomes to 60 seconds ultrasound, and typical CVs observed 1.5 (middle) and 59 seconds (right) after ultrasound onset. All plots generated after subtracting ultrasound-only control data. In some CV plots residual ultrasound artifacts have been truncated to show the electrochemical profile of each drug in detail. Release measured between +0.5 to 0.6 V for all experiments.

[0076] Figure 21: In vivo apomorphine release. A) step (top) and cylinder test (bottom) measures of impairment right forepaw function by the 6-hydroxydopamine lesioned animals used in the in vivo experiment. B) maximal dopamine release detected in the right (intact) and left (lesioned) striatum after electrical stimulation (100 pulses at 100 Hz) of the ipsilateral substantia nigra pars compacta. (*) and (+) indicate the points at which cyclic voltammograms (CV) data were measured, plotted bottom left and right, respectively. C) apomorphine release in the lesioned striatum produced by different durations of ultrasound (132 V intensity). (#) and (x) indicate points (»1.5 seconds post ultrasound onset) at which CV data were examined, plotted bottom left and center, respectively. Bottom right plot shows the calibration CV generated by 1 iM free apomorphine. CV ultrasound artifacts in C have been truncated to show the electrochemical profile in detail. Release measured between +0.5-0.6 V for all experiments. NB reduced peak release in 7 seconds experiment is likely due to adsorption.

[0077] Figure 22: Results of behavioural analysis of rats treated with liposomes of the invention in combination with ultrasound applied non-invasively through the skull.

[0078] DETAILED DESCRIPTION

[0079] The following is a description of the invention, including preferred embodiments thereof, given in general terms. The invention is further elucidated from the disclosure given under the heading “Examples" herein below, which provides experimental data supporting the invention, specific examples of various aspects of the invention, and means of performing the invention.

[0080] The present invention generally relates to methods for preparing liposomes comprising an internal nanoparticle of one or more chemical agent(s), liposomes made by the methods, and methods for non-invasive delivery and / or controlled release of agents to a subject, particularly neuromodulatory agents to the brain using acoustic activation. The internal nanoparticle can surprisingly be created at certain sizes, position and / or nanoparticle volume to liposome volume ratios which impart membrane imperfections and deformations which create inverse curvature within the liposome membrane and confers a degree of sensitivity to acoustic energy. While the inventors note that this surprising phenomenon was most evident when liposomal nanostructures were manufactured from liposomes containing an ammonium salt at an initial concentration of about 300 mM to about 500 mM, they contemplate that it will occur using concentrations from approximately 200 and 600mM and with the use of alternative ammonium salts. The liposomes made by the methods of the invention may provide one or more of a number of potential advantages, particularly in the case of delivery of agents to the brain: for example, dose control of agents delivered; targeting delivery to a specific brain area or areas; the ability to deliver a higher concentration of agents; improved release of agents with acoustic activation (eg ultrasound); reduced toxicity; release of agents using acoustic activation applied directly through the skull of a subject; and / or the ability to control the timing of the release of agents delivered, including repeated step-wise release of the agent contained within the liposomes. When the agent-containing liposomes are inside the blood vessels of the brain and acoustically activated at a specific defined region of the brain, they release their agents at that specific defined region where the agents are then able to cross the blood brain barrier. Then following such acoustic activation, and after an increment of time, the blood flow in the blood vessels causes the specific region of the brain to be replenished with intact agent-containing liposomes, which can then be acoustically activated. It is through repeated cycles of acoustic activation and replenishment that pulsed release of the agent can be caused in a specific defined region of the brain. In addition, the liposomes of the invention can be activated using a level of one or more parameters of acoustic activation which may lower the risk of damaging the blood brain barrier and / or other tissue in the subject.

[0081] Definitions

[0082] Throughout this specification and any claims which follow, unless the context requires otherwise, the word “comprise", “comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of “including, but not limited to".

[0083] Singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes mixtures of two or more such agents, and the like. In addition, reference to plural forms include singular references unless the context clearly dictates otherwise. For example, reference to "liposomes" should be taken to include reference to "a liposome".

[0084] “Nanoparticle" and like terms, such as “drug particle" and / or “agent nanoparticle" should be taken broadly to mean an aggregated form of a number of molecules of one or more amphipathic agent. The particle will comprise a defined region having a high density of molecules of one or more agent. It should not be taken to imply that the particle is in a particular form or structure such as a precipitate, a solid, or a crystal. In the Examples section herein, words such as “solid, “nanocrystal", “nanoprecipitate", “nanoprecipitation", “precipitate", “crystallisation", “precipitation" and like words may be used in reference to an agent nanoparticle. Such wording has been used for ease of description but should not be considered limiting.

[0085] An “amphipathic agent" as used herein is an agent possessing both hydrophilic and lipophilic properties. For example, "amphipathic agent" may be used herein to describe an agent with aqueous solubility possessing a degree of hydrophobicity and an ionisable group(s), for example an organic compound with a nitrogen-containing moiety, e.g. an amine.

[0086] A “therapeutic agent" is any agent that may assist in the treatment of a condition or disorder in a subject, whether a disease or a cosmetic condition or disorder. Reference to such agents herein should be taken to include reference to pharmaceutically acceptable salts thereof.

[0087] A “neuromodulatory" agent or drug is one which is adapted to treat one or more condition or disorder of the central nervous system. In a preferred embodiment, neuromodulatory agent is one which is adapted to treat one or more condition or disorder of or in the brain.

[0088] Reference to “treatment" should be taken broadly to include controlling, inhibiting, preventing or slowing the progression of a disorder and / or ameliorating one or more symptoms associated with a disorder. It should not be taken to mean that a subject is treated until total recovery, although that may be preferred. Treatment may be therapeutic or cosmetic.

[0089] A “subject" should be taken to include reference to any animal. In preferred embodiments, the subject is a mammal, more preferably a human.

[0090] A “neurological condition" or like terms should be taken broadly to mean any condition that affects the central nervous system, in a particularly preferred embodiment the brain, of a subject. It is intended to include cancers and other tumours, neurodegenerative disorders, infectious diseases, metabolic diseases, endocrinological disorders, inflammatory disorders, congenital disorders, autoimmune disorders, psychological disorders, haematological disorders, genetic / inherited disorders. In preferred embodiments, the condition or disorder is a neurological or psychological disorder. In certain embodiments, the neurological condition is Parkinson’s disease.

[0091] The phrase "pharmaceutically acceptable carrier, diluent and / or excipient" or the like, refers to any useful carriers, excipients, and diluents which are nontoxic to a cell or animal to which a composition is administered at the dosages and concentrations used. "Carriers, diluents and / or excipients" include but are not limited to fillers, colouring agents, flavouring agents, preservatives, stabilising agents, bulking agents, agents which help control release of active agents, agents which enhance delivery, binders, solvents, emulsifiers, suspending agents, lubricants, agents which alter viscosity of the composition, and moisturisers. n the context of the invention an "effective amount" of an agent to be administered is an amount necessary to at least partly attain a desired response.

[0092] References to “iso-osmotic" or “substantially equivalent osmotic concentration" and like terms, should be taken to mean that the osmotic concentration of one composition compared to another (for example a medium in which liposomes are suspending or a loading medium compared to the concentration of ammonium salts / ions inside a liposome) is sufficient to substantially retain the integrity or prevent the disruption of a desired proportion of the liposomes in a population. The term should not be taken to mean the osmotic concentration of two compositions are exactly the same, although that may be preferred. Preferably, it is sufficient to retain the integrity of at least approximately 80% of the liposomes, at least approximately 90%, at least approximately 95% of the liposomes, or at least approximately 99% of the liposomes in a population.

[0093] “Acoustically activatable" means the liposomes can be activated to release a drug or agent contained therein by application of an acoustic signal.

[0094] "Effective acoustic signal", and like terms, including reference to "effective ultrasound signal" and like terms, generally refers to providing acoustic parameters effective to release at least a portion of an amount of one or more amphipathic agent present within the inner volume of a population of liposomes to an area outside of the liposomes. The term should not be taken to mean that 100% of the one or more amphipathic agent contained in the inner volume of liposome or population of liposomes is released, although this may be preferred in some instances. In certain embodiments, at least about 0.001%, at least about 0.01%, at least about 0.1%, at least about 1%, at least about 1.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.5%, at least about 7%, at least about 7.5%, at least about 8%, at least about 8.5%, at least about 9%, at least about 9.5% of the amphipathic agent is release. In certain embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of the amphipathic agent is released. As noted elsewhere herein, a signal may be applied in a pulsed manner such that there is a staged release of one or more amphipathic agent with each pulse and over time. It will be appreciated that the amount of the one or more amphipathic agent released will depend on various factors including the amount of agent loaded into or contained within a liposome or population of liposomes, the characteristics of the agent(s) and liposome membrane composition, and acoustic signal parameters applied.

[0095] Methods of the invention described herein may be described in terms of an acoustic signal being applied to a subject on or at an area of the body that is in “effective proximity" to a target site of delivery or treatment. “Effective proximity" means a distance which is sufficient to allow an effective acoustic signal to reach the liposomes of the invention or compositions comprising them.

[0096] A “target site" for delivery of one or more agent or for treatment is a site at which it is desirable to release or deliver the one more agents contained within a liposome or liposomes. For example, it may be desirable to release the one or more agents at or near a site within a subject where a particular group of cells, tissue or organ affected by a condition or disorder resides. By way of example, in the case of Parkinson’s disease, the target site may be the caudate nucleus, the putamen, the substantia nigra, the globus pallidus, any other nucleus of the basal ganglia, the thalamus, and / or associated areas of the cerebral cortex.

[0097] A “liposomal membrane" is the outer lipid bilayer membrane of a liposome. The “internal face" of a liposomal membrane is the side of the membrane facing the interior volume of the liposome (ie inside the liposome). The “inner volume" or “interior volume" of a liposome is the area sequestered inside the liposomal membrane.

[0098] Liposomes may be referred to herein as having a particular size or diameter. While liposomes generally have a spherical shape, reference to diameter should not be taken to imply that individual liposomes are completely spherical. In addition, references to liposomes having a particular size should not be taken to mean that every liposome within a population of liposomes is of that size, although that may be preferred, and includes reference to the average size of liposomes contained within a population of liposomes.

[0099] In certain aspects of the invention, liposomes are described as including an amphipathic agent nanoparticle which is positioned within the liposome proximal to an area of an internal face of the liposomal membrane. In certain embodiments, a region of the liposomal membrane is distended. “Distended" is intended to refer to an outward distortion of the membrane where it partially wraps around a nanoparticle, relative to the curve of adjacent regions of the liposomal membrane. “Proximal" should not be taken to mean that the nanoparticle is necessarily touching, attached to or integral with the liposomal membrane, although this may be the case. In the Examples section which follows, wording and phrases such as “membrane adherent" may be used in relation to the nanoparticle. Such words and phrases are used for ease of description and should not be taken to be limiting or as confirmation that the nanoparticles touch, attach or are integral with the membrane.

[0100] The preformed liposomes used as the starting material to make liposomes of the invention comprising an amphipathic agent nanoparticle may be defined herein as having an internal concentration of ammonium ions which is equivalent to a specified concentration of an ammonium salt (for example from approximately 200 to approximately 600mM of an ammonium salt). Skilled persons should readily appreciate the intended meaning of this and how to calculate a desired concentration of ion or salt using known methods. However, by way of example for ammonium sulfate, a concentration of approximately 300mM is equivalent to an ammonium ion concentration of approximately 600mM (as ammonium sulfate has two ammonium ions per molecule). For ammonium citrate, a concentration of approximately 300mM is equivalent to an ammonium ion concentration of approximately 900mM (as ammonium citrate has three ammonium ions per molecule).

[0101] Method for making liposomes

[0102] The inventors have designed methods for the production of liposomes comprising an amphipathic agent nanoparticle which have unique characteristics and properties. The amphipathic agent nanoparticle contained within the liposomes made by the methods is positioned asymmetrically within the liposome, proximal to an area of an internal face of the liposomal membrane. In preferred embodiments, the nanoparticle is positioned proximal to an area of an internal face of the liposomal membrane such that a region of the liposomal membrane partially wraps the nanoparticle and is distended relative to the curve of adjacent regions of the membrane. The liposomes are stable but have a level of acoustic sensitivity which makes them particularly useful for the delivery of agents to a subject for therapeutic or other purposes. This may assist in treatment methods which require targeted or controlled delivery of agents, for example.

[0103] The methods of the invention utilise an ion gradient across a liposomal membrane (inside to outside the liposome) to load one or more amphipathic agent into a liposome. Loading methods of this type are described in US Patent 5,316,771. However, the inventors have surprisingly identified that use of certain conditions allows for the production of liposomes which have the unique properties described herein.

[0104] Generally, the methods of the invention comprise at least the steps of: a) Providing a suspension of liposomes, wherein and the concentration of ammonium ions inside the liposomes is the equivalent of from approximately 200 to approximately 600mM of an ammonium salt and is greater than the concentration of ammonium ions outside the liposomes, and, b) Adding one or more amphipathic agent to the suspension of liposomes, wherein on adding, the one or more amphipathic agent is taken into the liposomes to a final concentration within the liposomes which is greater than outside the liposomes and forms a nanoparticle inside the liposome.

[0105] In one preferred embodiment, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of from approximately 300mM to approximately 500mM of an ammonium salt. In one particular embodiment, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of approximately 300mM of an ammonium salt. In one particular embodiment, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of approximately 500mM of an ammonium salt.

[0106] In a preferred embodiment, in step a) the ratio of the concentration of ammonium ions inside the liposomes is substantially greater compared to the centration of ammonium ions outside the liposomes. In preferred embodiments, any medium outside of the liposomes, for example, the medium in which the liposomes are suspended, contains substantially no, or trace amounts of, ammonium ions. In other embodiments, such media comprise: pharmaceutically acceptable preservative agents, for example but not limited to: sodium metabisulfide and / or sodium ascorbate, and / or pharmaceutically acceptable antimicrobial including, but not limted to, kanamycin, gentamicin, lincomycin, tylosin tartrate, and chelating agents, including but not limited to ethylenediaminetetraacetic acid or a pharmaceutically acceptable salt thereof, and citric acid or a pharmaceutically acceptable salt thereof; and buffering agents and buffer formulations, for example, but not limited to, BES (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid) Buffered Saline, Bicine (2-(Bis(2-hydroxyethyl)amino)acetic acid), Carbonate-Bicarbonate, CHES (N-Cyclohexyl-2- aminoethanesulfonic acid), Diethanolamine, EBBS (Earle's Balanced Salt Solution), Glycine-Sodium Hydroxide Buffer, HEPES ((4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), HBSS (Hank's Balanced Salt Solution), HEPPSO (4-(2-Hydroxyethyl)piperazine-l-(2-hydroxypropanesulfonic acid) hydrate), HHBS (Hank's Buffer with HEPES), Imidazole-HCI, Maleic Acid, MES (2-(N-morpholino) ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PBS (Phosphate Buffered Saline), Sodium Borate Buffer, TAE Buffer (Tris Base, Acetic Acid, EDTA), TBS (Tris Buffered Saline), TE Buffer (Tris EDTA), Tricine (N-(2-Hydroxy-l,l-bis(hydroxymethyl)ethyl)glycine), TRIS (tris(hydroxymethyl)aminomethane) and / or Trizma (2-Amino-2-(hydroxymethyl)-l,3-propanediol).

[0107] In a preferred embodiment, the liposomes provided in step a) are from approximately 200nm to approximately 300nm in diameter. In one embodiment, the liposomes provided in step a) are from approximately 200nm to approximately 300nm in diameter. In a preferred embodiment, the liposomes provided in step a) are approximately 200nm in diameter.

[0108] In one embodiment, the liposomes comprise a single amphipathic agent nanoparticle.

[0109] In one embodiment, the methods result in an amphipathic agent nanoparticle which is approximately 10% to approximately 60% of the size of a liposome once loaded. In one embodiment, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of approximately 300mM of an ammonium salt and the amphipathic agent nanoparticle is from approximately 10% to approximately 30% of the size of a liposome once loaded. In another embodiment, the concentration of ammonium ions inside the liposomes provided in step a) is the equivalent of approximately 500mM of an ammonium salt and the amphipathic agent nanoparticle is approximately 40% to approximately 50% of the size of a liposome once loaded.

[0110] The liposomes to be loaded with one or more amphipathic agent in the methods of the invention are preformed and provided in the form of a liposome suspension. The starting liposomes can be formed using any one of a number of standard methods known in the art. However, by way of example, the thin-film rehydration method, methods exemplified in US Patent 5,316,771, or the methods described in the Examples section herein after, may be used.

[0111] In accordance with the invention, the preformed liposomes will be prepared according to such known techniques in the presence of a composition comprising one or more ammonium salt; so that they contain an appropriate ammonium ion concentration in their inner volume. Any appropriate ammonium salt(s) may be used to provide the desired ammonium ion concentration and gradient. Skilled persons will be able to readily identify appropriate ammonium salts having regard to the nature of the amphipathic agent(s) to be loaded into the liposomes and the end use to which the liposome is to be put. In a preferred embodiment, the ammonium salt is suitably safe for administration to a subject for therapeutic or other purposes, for example. In a preferred embodiment the ammonium salt is chosen from the group: ammonium sulfate, ammonium oxalate, ammonium citrate, ammonium trimesate, ammonium hydroxide, ammonium carbonate, ammonium bicarbonate and / or carboxyfluorescein ammonium salt.

[0112] Once formed, any composition comprising ammonium salt used to prepare the liposomes residing outside of the liposomes may be removed and replaced with an appropriate suspension medium such that the liposomes have an internal ammonium ion concentration greater than that outside of the liposomes. Skilled persons will readily appreciate a variety of suitable suspension media having regard to the nature of the ammonium salt used, the desired ammonium ion salt gradient, and the nature of the amphipathic agent(s) to subsequently be loaded into the liposomes, for example. Preferably, the suspension medium will have an osmotic concentration substantially equivalent to that inside the liposomes to help retain the integrity or prevent disruption of liposome membranes. By way of example, media described in US Patent 5,316,771 or in the Examples section herein after may be used. In certain embodiments, the suspension medium comprises one or more of: sodium chloride, sucrose and / or phosphate buffered saline, an antioxidant or preservative agent such as ascorbic acid, sodium ascorbate, sodium metabisulfite. In a preferred embodiment the suspension medium comprises phosphate buffered saline, sodium ascorbate. In a preferred embodiment, the suspension medium comprises phosphate buffered saline, sodium metabisulfite. In a preferred embodiment, the suspension medium comprises sodium chloride, phosphate buffer, sodium ascorbate. In a preferred embodiment the suspension medium comprises, phosphate buffer, sodium chloride, sodium metabisulfite.

[0113] In preferred embodiments, the one or more amphipathic agent is added to the suspension of liposomes in a loading medium having an osmotic concentration substantially equivalent to that inside the liposomes (i.e. substantially iso-osmotic with the osmotic concentration within the liposome). This helps to prevent the liposomes from being disrupted during the loading process. Any appropriate loading medium may be used. Skilled persons will readily appreciate appropriate loading mediums which may be used having regard to the nature of the ammonium salt / ion concentration inside the liposomes and the nature of the drug to be loaded into the liposomes. However, by way of example, the loading mediums described in US Patent 5,316,771 or as exemplified in the Examples section herein after, may be used. By way of further example, the loading medium may comprise one or more salts, buffers or other agents such as antioxidants. In a preferred embodiment, the loading medium comprises sodium chloride, sucrose and / or phosphate buffered saline. In other preferred embodiments, the suspension medium comprises one or more of: sodium chloride, sucrose and / or phosphate buffered saline, an antioxidant or preservative agent such as ascorbic acid, sodium ascorbate, sodium metabisulfite. In a preferred embodiment the suspension medium comprises phosphate buffered saline, sodium ascorbate. In a preferred embodiment, the suspension medium comprises phosphate buffered saline, sodium metabisulfite. In a preferred embodiment, the suspension medium comprises sodium chloride, phosphate buffer, sodium ascorbate. In a preferred embodiment the suspension medium comprises, phosphate buffer, sodium chloride, an antioxidant or preservation agent such as sodium ascorbate, sodium metabisulfite. In a preferred embodiment the suspension medium comprises phosphate buffered saline, sodium ascorbate. In a preferred embodiment, the suspension medium comprises phosphate buffered saline, sodium metabisulfite. In a preferred embodiment the suspension medium comprises sodium chloride, phosphate buffer, sodium ascorbate. In a preferred embodiment the suspension medium comprises phosphate buffer, sodium chloride, sodium metabisulfite.

[0114] The pH of a loading medium and / or suspension medium may be adjusted to alter the rate of loading of the one or more amphipathic agents into the preformed liposomes according to known principles. Skilled persons will appreciate how this may be achieved having regard to the pKa of the agents(s) to be loaded. However, generally, increasing the pH in the environment external to the liposome inner volume will increase the rate of loading compared to the use of the lower pH.

[0115] In one embodiment the pH of the loading medium is about 5.5 to about 10. In a preferred embodiment, the pH of the loading medium is about 6 to about 9. In a preferred embodiment, the pH of the loading medium is about 6.3 to about 8, or about 6.3 to about 7.5.

[0116] Any appropriate amphipathic agent may be used in the present invention. Skilled persons will readily appreciate such agents having regard to the description provided herein and the nature of the use they intend to put the liposomes to. However, by way of example, an amphipathic agent may be a drug or diagnostic agent, in one embodiment to be administered to a subject for therapeutic or other purposes. In certain embodiments, an amphipathic agent comprises an amine group. In a preferred embodiment, the at least one amphipathic agent is a neuromodulatory agent. In one embodiment, the neuromodulatory agent(s) is chosen from the dopamine group comprising but not limited to: apomorphine, bromocriptine, carbergoline, ciladopa, dihydrexidine, disnapsoline, doxanthrine, epicriptine, fenoldopam, lisuride, pergolide, piribedil, pramipexole, propylnorapomorphine, quinagolide, ropinirole, rotigotine, roxindole, sumanirole, and / or SKF38393. In one embodiment, the neuromodulatory agent(s) is chosen from the dopamine antagonist group comprising but not limited to: amisulpride, aripiprazole, azaperone, benperidol, bromopride, chlorpromazine, clopenthixol, clozapine, domperidone, droperidol, eticlopride, fluphenazine, flupentixol, fluspirilene, haloperidol, quetiapine, lurasidone, metoclopramide, olanzapine, paliperidone, penfluridol, perazine, perphenazine, pimozide, prochlorperazine, promazine, raclopride, risperidone, remoxipride, SCH23390, sertindole, sulpiride, suitopride, thiethylperazine, thiothixene, thioridazine, tiapride, trifluperidol, triflupromazine, trifluoperazine, ziprasidone. In one embodiment, the neuromodulatory agent(s) is selected from the group of anti-epileptic agents comprising but not limited to: muscimol, perampanel, levetiracetam, gabapentin, pregabalin, progabide, vigabatrin. In one embodiment, the neuromodulatory agent(s) is chosen from the group of serotonin modulators comprising but not limited to: alosetron, dolasetron, granisetron, ondansetron, palonosetron, topisetron, almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan, zolmitriptan, buspirone, citalopram, clozapine, desipramine, fluoxetine, m- chlorophenylbiguanide mCPG, m-chlorophenylguanidine mCPP, m-chlorophenylpiperazine, imipramine, olanzapine, propranolol, quetiapine, risperidone, tranylcypromine, trazodone, ziprasidone, zotepine. In one embodiment, the neuromodulatory agent(s) is selected from the group of cholinergic system modulators comprising but not limited to: acetylcholine, methacholine, carbachol, bethanechol, tacrine, muscarine, pilocarpine, cevimeline, nicotine, varenicline tartrate, galantamine hydrobromide, suxamethonium chloride, epibatidine, oxotremorine, atropine, benztropine mesylate, clidinium, cyclopentolate, darifenacin, dicylomine, fesoterodine, flavoxate, glycopyrrolate, homatropine hydrobromide, hyoscyamine, orphenadrine, oxybutynin, propantheline, scopolamine, methscopolamine, solifenacin, tiotropium, tolterodine, trihexyphenidyl, trospium. In preferred embodiments, the neuromodulatory agent(s) is chosen from apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride .

[0117] Skilled persons will readily appreciate liposome structures of use in the present invention. In a preferred embodiment, the liposomes are unilamellar liposomes comprising a single lipid bilayer sequestering an inner volume. The lipid bilayer can be any arrangement of amphiphilic lipid molecules characterized by a hydrophilic part (hydrophilic moiety) and a hydrophobic part (hydrophobic moiety) arranged into two dimensional sheets in which hydrophobic moieties are oriented inward while hydrophilic moieties are oriented outward.

[0118] Skilled persons will readily appreciate appropriate amphiphilic lipid molecules which are suitable to form liposomes of use in the present invention. However, by way of general example, lipids of synthetic or natural origin or biocompatible lipids may be used. The liposomes may include different ratios and combinations of two or more different types of appropriate lipids as will be appreciated by persons skilled in the art and may be formulated with other components such as permeability modifying agents (for example sterols such as cholesterol, or polyethylene glycols (PEGs), which may be further modified, for example DSPE-PEG2000). The liposomes may further comprise amounts of other molecules including, for example, surface ligands, proteins, or nanoparticles that may be of use in certain applications, such as for attaching to specific ligands, cells or tissues in use, or to direct liposomes towards a specific cell, tissue, or organ.

[0119] Examples of lipid molecules of use in liposomes of the invention include those described in US Patent 11,071,713, US Patent 9,968,583, US Patent 7,744,920 , US Patent Application 20150004219, and US Patent 5,316,771, for example. In certain embodiments, phospholipids such as phosphatidylcholine, and sphingolipids, such as sphingomyelin are used. Other specific examples of lipid molecules of use in the liposomes of the invention include DSPC (l,2-distearoyl-sn-glycero-3- phosphocholine), cholesterol, egg yolk sphingomyeline, bovine sphingomyelin, PEG conjugated phospholipids such as DSPE-PEG2000, and HSPC (hydrogenated soy L-a-phosphatidylcholine), SPC (soy L-a-phosphatidylcholine), DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine), sphingomyelin, DOPE (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine). In addition, suitable lipids and lipid mixtures for the preparation of liposomes may be purchased from a variety of manufacturers. Alternatively, they may be prepared according to published methods.

[0120] Skilled persons will readily appreciate the nature and amount of each type lipid or other molecules to include in a liposome formulation having regard to the use to which the liposome will be put, the nature of the amphipathic agents to be loaded, and the desired stability of the liposome formulation, the release profile of the amphiphilic agent and known principles and published methods. However, by way of example only, those described in the Examples hereinafter may be used. In one embodiment, the liposomes contain a low to medium amount of cholesterol (5-40%). In one embodiment, the liposomes contain a phospholipid characterised a phase transition temperature of greater than or equal to 35 degrees Celcius. In one embodiment, the liposomes are prepared from a composition comprising DSPC, cholesterol, sphingomyelin, and DSPE-PEG2000 in an approximately 100:5:5:4 mole ratio. In one embodiment, the liposomes are prepared from a compositing comprising HSPC, cholesterol and DSPE-PEG2000 in an approximately 55:40:5 mol% ratio. In one embodiment the liposomes are prepared form a composition comprising DOPE, DSPC, DSPE-PEG2000, and cholesterol in an approximately 25:27:8:40 mol% ratio.

[0121] Liposomes

[0122] As noted herein before, the invention provides liposomes comprising an amphipathic agent nanoparticle, wherein the amphipathic agent nanoparticle is positioned proximal to an area of an internal face of the liposomal membrane. In a preferred embodiment, the nanoparticle is positioned proximal to an area of an internal face of the liposomal membrane such that a region of the liposomal membrane partially wraps the nanoparticle and is distended relative to the curve of adjacent regions of the membrane.

[0123] The liposomes are preferably from approximately 80 nm to approximately 400 nm in diameter. In one embodiment, the liposomes are from approximately 200nm in diameter or approximately 300nm in diameter.

[0124] In certain embodiments, the amphipathic agent nanoparticle(s) comprise approximately from 10% to approximately 60% of the volume of the liposome.

[0125] The liposomes of the invention are sensitive to acoustic signals and can be activated to release one or more amphipathic agent from the interior of the liposome when an acoustic signal is applied. Preferably, the acoustic signal is ultrasound. Further details of the acoustic signal parameters adapted to activate the release of one or more amphipathic agent from the liposomes are provided herein after.

[0126] In one preferred embodiment, the liposomes comprising an amphipathic agent nanoparticle of the invention are prepared using the methods described herein before.

[0127] The liposomal membrane of a liposome comprising an amphipathic agent nanoparticle of the invention may comprise one or a combination of the molecules suitable for forming and inclusion in or on a lipid-bilayer as herein before described and the agent nanoparticle may comprise one or more amphipathic agent as herein before described. In a particularly preferred embodiment, the one or more amphipathic agent is chosen from apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride.

[0128] The liposomes of the invention may include any appropriate amount of the one or more amphipathic agent. However, in the case of apomorphine for example, the total dose of amphipathic agent may range from about 10 to about 40 mg of apomorphine per mL of liposome suspension

[0129] The liposomes comprising an amphipathic agent nanoparticle of the invention are acoustically sensitive and can be activated to release the one or more amphipathic agent contained therein. In one embodiment, the liposomes comprising the invention are acoustically activated by application of therapeutic frequency ultrasound. In one embodiment, the ultrasound frequency applied to the liposomes comprising the invention is between 0.3 - 50 MHz. In one embodiment, the ultrasound frequency applied to the liposomes comprising the invention is between 0.5 - 2 MHz. In one embodiment, the liposomes comprising the invention are acoustically activated by application of therapeutic frequency ultrasound with an acoustic intensity greater than or equal to 0.01 W cm'2. In one embodiment, the liposomes comprising the invention are acoustically activated by application of therapeutic frequency with an acoustic intensity greater than or equal to 0.1 W cm'2. In one embodiment, the liposomes comprising the invention are acoustically activated by application of therapeutic frequency with an acoustic intensity greater than or equal to 1 W cm'2. In one embodiment, the liposomes comprising the invention are acoustically activated by application of therapeutic frequency with an acoustic intensity between 0.01 W cm'2and 10 W cm'2. In one embodiment, the liposomes comprising the invention are acoustically activated by application of therapeutic frequency with an acoustic intensity between 0.1 W cm'2and 5 W cm'2. In one embodiment, the liposomes comprising the invention are acoustically activated by application of therapeutic frequency with an acoustic intensity between 0.5 W cm'2and 3 W cm'2. In one embodiment, the liposomes comprising the invention are acoustically activated by application of therapeutic frequency with an acoustic intensity between 0.7 W cm'2and 1.5 W cm'2. In one embodiment, the liposomes comprising the invention are acoustically activated by application of continuous frequency ultrasound. In one embodiment, the liposomes comprising the invention are acoustically activated by application of pulsed frequency ultrasound. In one embodiment, the liposomes comprising the invention are acoustically activated by application of pulsed frequency ultrasound with a pulse repetition frequency of pulse repetition frequency of 1 Hz to 1000 Hz and duty cycle range of 0.01% to 90%. Typical embodiment is therapeutic frequency of 0.5 to 1 MHz, acoustic intensity applied to tissue of 1 to 3 W cm'2, pulse repetition frequency of 100 Hz, duty cycle 5 to 10%, application time 5 to 30 seconds."

[0130] Compositions comprising liposomes

[0131] The invention also provides compositions comprising liposomes comprising an amphipathic agent nanoparticle of the invention and one or more suitable diluent, carrier, and / or excipient. In one embodiment, the compositions are intended for delivery to a subject and the one or more diluent, carrier and / or excipient are pharmaceutically acceptable. In certain preferred embodiments, the compositions are intended for use in the diagnosis or treatment of a condition in a subject. In one embodiment, the compositions are intended for use in the treatment of a neurological condition in a subject, particularly a neurological condition in the brain of a subject. In certain embodiments, the compositions are intended for the treatment of Parkinson’s disease.

[0132] Compositions of the invention may optionally include one or more additional ingredients which may be of benefit to include in a composition, having regard to its intended use. For example, where the composition is to be administered to a subject, it may comprise other agents or ingredients which may be of benefit to the subject. For example, they may include one or more additional active agent which is of benefit in treating a particular condition or disease or which is otherwise of benefit to the health or cosmetic appearance of a subject.

[0133] Skilled persons will readily appreciate a variety of suitable carriers, excipients and diluents of use in formulating compositions comprising liposomes, having regard to the nature of the intended use for the liposomes / composition and information published in standard texts and handbooks. By way of example, the information in The Handbook of Pharmaceutical Excipients, Sixth edition, 2009, editors Raymond C Rose, Paul J Sheskey and Marian E Quinn (http: / / pharmama.info / wp- content / uploads / 2018 / 10 / Excipients.pdf) may be referred to.

[0134] The compositions are preferably formulated so that the integrity of the liposomes is retained (ie substantially retain the one or more amphipathic agent inside the liposomes) or to prevent or at least slow premature leaking of the one or more amphipathic agent form the liposomes until such time that release is suitable or required. In one embodiment, this may be achieved through the use of carriers, excipients and / or diluents or other ingredients or agents that help to ensure the liposomes are contained in a medium having an osmotic concentration substantially equivalent to that inside the liposomes (i.e. substantially iso-osmotic with the osmotic concentration within the liposome). Skilled persons will readily appreciate appropriate diluents, carriers, excipients and / or other ingredients or agent to assist with this having regard to the information contained herein and known methods for formulating liposomes for use. However, by way of example liposome suspensions may be formulated containing saline, phosphate buffered saline, sucrose or other sugars, antioxidants such as ascorbic acid or sodium metabisulfite, or viscosity modifying polymers for example, but not limited to, hyaluronic acid or derivatives thereof, chitosan or derivatives thereof, alginic acid or derivatives thereof, or other agents such as sucrose and trehalose. In another embodiment, an alkali agent, for example sodium hydroxide or potassium hydroxide may be used to neutralise the final preparation to an acidity of pH 6 - 8 to help prevent pH-adverse reactions. For example subcutaneous injection of apomorphine is currently performed with an acidic apomorphine solution containing a pH of about 3 - 5 that results in local reactions including subcutaneous nodules, induration, erythemia, tenderness, panniculitis, irritation, itching, bruising and pain. The inventors contemplate that the higher pH afforded by liposome encapsulation may mitigate some of these adverse reactions experienced by current methods. In one embodiment, the drug-loaded liposome suspensions and excipients are formulated to be substantially devoid of ammonium ions.

[0135] Where compositions of the invention are to be administered to a subject, they may be formulated for any appropriate means of delivery including systemic or local delivery, for example. In a preferred embodiment, they are formulated for systemic delivery. Skilled persons will readily appreciate types of compositions suitable for systemic administration, including parenteral and enteral administration, such as injectable compositions, pills, capsules, gels, suspensions, oils, emulsions, sprays, powders, liquids and the like. In a preferred embodiment, a composition is formulated for parenteral administration, for example as an injectable composition. The injectable composition may be suitable for intravenous, intramuscular or subcutaneous injection. In one embodiment, the composition is formulated for oral, intranasal, intrathecal, or direct local administration. Compositions for administration to a subject may be made in accordance with standard techniques as may be found in such standard references as Liposomes, Methods and Protocols (Methods in Molecular Biology, by Gerard G. M. D'Souza), or Liposomes, A Practical Approach (by Volkmar Weissig and Vladimir Torchill in). In preferred embodiments, the formulations and methods described and exemplified herein may be used.

[0136] Skilled persons will readily appreciate that the compositions may include any appropriate amount of the liposomes / one or more amphipathic agent of the invention having regard to intended use for the liposomes / one or more amphipathic agent, the nature of the one or more amphipathic agent contained within the liposomes, the desired amount or dose of one or more amphipathic agent to be used in a method (for example, the desired amount to be administered or delivered to a subject), the route of administration to a subject, and the like. However, by way of example, in the case of an injectable composition for administration to a subject for treatment of a neurological condition of the brain, using an agent such as apomorphine, dihydrexidine, ropinirole, SCH23390, and / or SKF38393, and / or sulpiride, the composition may comprise 50 to 1000 pg / ml of drug packaged inside liposomes and a volume of liposomes administered of 5 to 200 ml.

[0137] Methods of delivery and treatment

[0138] The invention also provides methods for delivering an effective amount of one or more amphipathic agent to a subject. In one embodiment the methods generally comprise at least administering liposomes or a composition comprising liposomes of the invention to the subject. The methods may be performed for any purpose including therapeutic or diagnostic purposes, for example.

[0139] The liposomes or a composition comprising liposomes of the invention may be administered to a subject by any appropriate means. Skilled persons will readily appreciate appropriate administration routes having regard to the intended purpose of delivery, for example the nature and location of a particular condition to be treated, and the composition to be administered. For example, the liposomes or a composition of the invention may be administered systemically or locally. In one embodiment, the liposomes or compositions are administered systemically, for example parenterally or enterally. In one embodiment, the liposomes or composition are administered by subcutaneous, intramuscular, intrathecal, intravenous, or local injection. In another embodiment, the liposomes or compositions are administered intranasally or by inhalation. In a preferred embodiment, the liposomes or compositions comprising the liposomes are administered intravenously. ..

[0140] In a preferred embodiment the methods comprise the steps of: a) administering liposomes or a composition comprising liposomes of the invention to the subject; and, b) applying an effective acoustic signal to the subject to release the one or more amphipathic agent from the liposomes. The acoustic signal is applied to the subject on or at an area of the body in effective proximity to a target site of delivery. In one embodiment of the invention, the target site of delivery is the brain or an area or region of the brain of a subject. In this embodiment, the acoustic signal is applied to or at an area of the head of the subject in effective proximity to a target area or region of the brain.

[0141] In one embodiment the one or more amphipathic agent is a therapeutic agent and the method is for the treatment of a condition or disorder within a subject. In one particular embodiment, the methods are for the treatment of a neurological condition in a subject. In this embodiment, the one or more amphipathic agent may be a neuromodulatory agent. In certain embodiments the neuromodulatory agent is chosen from ropinirole, apomorphine, and / or dihydrexidine, however, other neuromodulatory agents may be used. In a particularly preferred embodiment, the methods are for the treatment of a neurological condition of the brain of a subject. In certain embodiments, the neurological condition of the brain is Parkinson’s disease, restless legs syndrome, clinical depression, or hyperprolactinaemia, however other neurological, neurodegenerative or neuropsychiatric disorders may be treated.

[0142] In a preferred embodiment the acoustic signal is ultrasound.

[0143] The dose of an active agent or composition comprising liposomes of the invention administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the size and / or severity of the condition to be treated, the stage of development of the condition, the type of agent or composition to be administered, the administration route, size of a unit dosage, the kind of excipients, carriers and the like used, the age and / or general health of a subject, and other factors well known to those of ordinary skill in the art. A skilled person will be able to determine the appropriate dose having regard to such factors and the information contained herein, for example.

[0144] Administration may include a single daily dose or administration of a number of discrete divided doses as may be appropriate.

[0145] In certain embodiments, the methods of the invention employ administration of an acoustic signal (eg ultrasound) to activate the liposomes to release amphipathic agents therefrom. Any ultrasound signal having parameters effective for releasing the amphipathic agent from the liposomes may be used in the methods of the invention. However, the inventors contemplate that given the sensitive nature of the liposomes of the invention to acoustic signals that ultrasound having a shorter duration, lower intensity, and / or duty cycle may be used compared to the ultrasound parameters used in conventional techniques, such as ultrasound for the delivery of agents to tissues including the brain. This may allow for an increase in delivery of agents at the same level of intensity, frequency, duration and / or duty cycle of ultrasound used in such techniques, or the same level of delivery of agents using a lower level of intensity, duration and / or duty cycle compared to that used in such techniques. Being able to use a low or lower intensity, duration and / or duty cycle may also provide the advantage of reducing the potential for damage to tissue and other negative side effects of the application of ultrasound to a subject.

[0146] Skilled persons will readily appreciate appropriate ultrasound parameters of use in activating the liposomes of the invention having regard to the nature of the methods in which the liposomes will be used, the teaching herein and known ultrasound dose and safety calculation methods including but not limited to mechanical index (Ml = peak negative pressure amplitude estimated in situ divided by square root of frequency) and thermal index (Tl = ratio of the total acoustic power to the acoustic power required to raise the tissue temperature by 1 degree C). However, by way of example only, factors such as the nature of the composition or tissue in which the liposomes are present, the composition of the liposomes, the distance of the ultrasound source from the liposomes or area in which one or more agent is to be released and the amount of energy required for release are considered and the frequency and intensity of the ultrasound signal, pulse ratio or duty cycle, and / or the total duration of application of the ultrasound adjusted.

[0147] By way of further example, the ultrasound signal may be provided at an intensity of about 0.01 to about 10 W cm'2. In certain embodiments, from about 0.5 to about 5 W cm'2. By way of further example, the ultrasound signal may be provided at a frequency of between about 20 kHz to about 100 MHz. In an embodiment, the ultrasound signal is provided at a frequency of between about 200 kHz and about 50 MHz. In a certain embodiment the frequency is from about 500 kHz to about 4 MHz. In preferred embodiments, the ultrasound signal is provided at a frequency of between about 200 kHz to about 1 MHz or from about 500 kHz to about 3 MHz.

[0148] By way of further example, continuous or regular or irregular pulsed application of ultrasound signal may be used. In one embodiment, a pulsed signal is used to provide a controlled step-wise release of an amount of an agent from the liposomes to the liposomes' surrounding. By way of example, when the agent-containing liposomes are inside the blood vessels of the brain and acoustically activated at a specific defined region of the brain, they release their agents at that specific defined region where the agents are then able to cross the blood brain barrier and elicit a therapeutic response. Then following such acoustic activation, and after an increment of time, the blood flow in the blood vessels causes the specific region of the brain to be replenished with intact agent-containing liposomes, which can then be acoustically activated. It is through repeated cycles of acoustic activation and replenishment that pulsed release of the agent can be caused in a specific defined region of the brain. The signal pulse series may be optimised to control the levels of drug agent in the tissues as to mitigate or prevent the occurrence of adverse drug reactions and undesired side effects while prolonging the therapeutic response.

[0149] In one preferred embodiment, the methods of the invention relate to the delivery of one or more amphipathic agent to the brain of a subject. In this embodiment, following delivery of liposomes or a composition comprising the liposomes of the invention to a subject, an ultrasound signal is applied to an area of the head of the subject in effective proximity to a target area of the brain of a subject to which it is desirable to administer the amphipathic agents. By way of example, the method may be performed for the treatment of a neurological condition of the brain such as Parkinson's disease. In this example, the liposomes comprise one or more amphipathic drug such as apomorphine, dihydrexidine and ropinirole (or any other agent suitable for treatment of the disease) and the liposomes or a composition comprising the liposomes are administered to the subject systemically. The liposomes or composition comprising the liposomes may be administered intravenously, intrathecally, subcutaneously, locally via direct injection, or intranasally to a patient. Preferably, the liposomes or compositions comprising the liposomes are administered intravenously or intrathecally.

[0150] A period of minutes to hours after administration, an ultrasound signal is applied intracranially by positioning the signal (via an ultrasound transducer) on the head of the subject aimed for a target area containing receptors that will be activated or inhibited by the agent released from the liposomes. By way of example, the target area could include a nucleus of the basal ganglia such as the caudate and / or the putamen, but could involve the cerebral cortex, diencephalon, and / or brainstem. In this preferred embodiment, the ultrasound signal is applied using the following parameters: intensity of 0.5 to 5 W cm'2, frequency of 0.5 to 1 MHz, duty cycle of 0.001 to 10%.

[0151] EXAMPLES

[0152] Through a series of investigations, the inventors have identified methods and formulations that can produce stable, acoustically sensitive liposome formulations, optimized for the encapsulation of neuromodulatory drugs.

[0153] Example 1: General description of materials and methods.

[0154] General method for liposome preparation

[0155] Liposomes were prepared using a variation of the thin-film rehydration method and adapted for active drug loading. Briefly, liposomes were prepared using a lipid composition comprised of DSPC, cholesterol, sphingomyelin, and DSPE-PEG2000 in a 100:5:5:4 mole ratio by combining the appropriate volumes of the membrane components dissolved in chloroform (Table 1) and removing the solvent in vacuo to form a lipid film. The dry lipid film was subsequently rehydrated with a solution containing the desired internal gradient salt, for example an ammonium salt, at the appropriate concentration (50 to 2000 mM), without any additional buffering agents. To ensure dispersion of the lipid film, the suspension was heated to approximately the phase transition temperature of the lipid bilayer (approximately 50 °C) and vortexed for five minutes, and subsequently extruded 15 times through 200 nm polycarbonate membranes at 60 °C.

[0156] Table 1: Lipid concentrations and volumes used in liposome preparation.

[0157]

[0158] The external an ammonium salt was removed via dialysis against 20 mM sodium phosphate buffer at pH 7.4 containing the appropriate amount of sodium chloride to ensure an approximate osmotic balance (i.e. 30 mM or 280 mM sodium chloride to balance 50 mM or 300 mM an ammonium salt). Dialysis was performed with 3 x 600 mL buffer changes at room temperature over 12 hours. After dialysis is complete, a 4 mM solution of the desired therapeutic agent, for example apomorphine, is prepared containing the appropriate amount of sodium chloride to balance the intraliposomal an ammonium salt concentration, and 2 mg mL1ascorbic acid as an antioxidant, with 20 mM sodium phosphate at pH 6.3. A 1 in 5 dilution of the liposome suspension is made using the drug solution (400 pL liposomes to 1.6 mL drug solution) in a 2 mL glass HPLC tube and loaded at room temperature for 12 hours. Any residual unloaded drug was subsequently removed by dialysis against phosphate buffer (20 mM) at pH 7.4 containing ascorbic acid (2 mg mL1) and sodium chloride (300 mM).

[0159] Apomorphine fluorescence standard curve

[0160] 10 pM and 100 pM apomorphine solutions were prepared containing 20 mM sodium phosphate at pH 7.4 with 2 mg mL1ascorbic acid and serially diluted to give the final apomorphine concentrations between 0 and 9pM and 10 and 90 pM respectively. Fluorescence intensity from 100 pL aliquots of the solution was subsequently recorded on a CLARIOstar’ fluorescence plate reader with an excitation and emission wavelength of 320 nm and 435 nm respectively using Nunc™ 96-well microtiter fluorescence plates. Additionally, the effect of liposomes and lysing surfactant myristyltrimethylammonium bromide (MTAB) was investigated through the addition of 10 pL of a 10 mM liposome suspension (aforementioned lipid formulation), or the addition of 10 pL of a liposome suspension with an additional 10 pL of 25 mM MTAB.

[0161] General procedure for TEM and Cryo-TEM experiments

[0162] Negative stain TEM was performed using a Philips CM100 BioTWIN transmission electron microscope (Phillips / FEI Corporation, Eindhoven, Netherlands) combined with a LaBg emitter, fitted with a MegaView III Olympus digital camera. Samples were prepared by depositing 10 pL of a diluted liposome suspension onto a 3 mm plasma-ionised carbon-coated copper specimen grid and removed by capillary wicking using Whatman’ filter paper after 60 seconds. Subsequently 10 pL of 1% phosphotungsted acid solution is deposited onto the grid and immediately removed by capillary wicking, and the grid is dried under a heat lamp. Cryo-TEM was performed using a JEOL 2200FS field emission scanning electron microscope (STEM) with an omega energy filter (JEOL Ltd., Tokyo, Japan), fitted with a TVIPS F416 CMOS camera (TVIPS GmbH, Germany) and Direct Electron DE-20 detector (Direct Electron LP, California, USA). Specimen holders used included a standard JEOL holder with a single tilt axis (JEOL Ltd., Tokyo Japan), and a Gatan model 914 high tilt cryo holder (Gatan Inc., California, USA). Images were acquired using SerialEM software (University of Colorado, Boulder, USA) in conjunction with TVIPS EM-Menu and DE Imaging Manager. Image analysis was performed using the IMOD software package (Boulder Laboratory for 3-D Microscopy, Colarado, USA).

[0163] General procedure for ultrasound release experiments

[0164] Liposome suspensions were diluted 1 in 20 with phosphate buffered saline (20 mM sodium phosphate, 100 mM sodium chloride) at pH 7.4 containing 2 mg mL1ascorbic acid. Aliquots of 750 pL were removed in triplicate and ultrasonicated in a quartz fluorimeter cuvette at 2.4 W cm'2for 0, 15, 30, 45, or 60 seconds. An additional aliquot was removed in triplicate and 75 pL of MTAB (100 mM) added to obtain the total fluorescence, and hence the total amount of encapsulated apomorphine. Fluorescence intensity was measured in a Nunc™ 96-well microtiter plate. The apomorphine concentration was subsequently calculated using the apomorphine standard curve.

[0165] Example 2: Auto-fluorescence of apomorphine.

[0166] Apomorphine, a non-selective dopamine agonist, was investigated as a neuromodulator drug candidate and a useful fluorescent probe for subsequent development and optimisation investigations. The aim of this investigation was to assess the fluorescence behaviour of apomorphine and its suitability for fluorescence detection in liposome drug release experiments.

[0167] Apomorphine is a weak amphipathic base with physiochemical properties suited for active loading against an an ammonium salt gradient. Apomorphine is fluorescently active, with excitation and emission wavelengths of approximately 320 and 435 nm respectively {Kent Van Tyle, 1971 #1870; Smith, 1983 #1941}.

[0168] The fluorescence of apomorphine was investigated by serial dilution of a 0.1 mM solution at pH 7.4. A linear increase in fluorescence intensity was observed between 10 and 100 pM apomorphine indicating the appropriate concentration range for subsequent ultrasound induced release studies. It is also apparent that the fluorescence intensity linearly declines between concentrations of 100 and 1000 pM, indicating a degree of self-quenching behaviour (Figure 1 A). Therefore, it was believed that apomorphine fluorescence should provide an adequate method for quantifying the degree of apomorphine released in response to ultrasound and liposome lysis, as the high intraliposomal concentration of apomorphine is diluted into the external medium.

[0169] The effects of liposomes and the lysing surfactant myristyltrimethylammonium bromide (MTAB) on the fluorescence intensity of apomorphine were investigated (Figure 1 B). Little effect on the fluorescence intensity of apomorphine was observed with addition of 200 nm liposomes. However, the fluorescence intensity was markedly enhanced by the addition of MTAB to the apomorphine liposome suspension. In all cases, the relationship between fluorescence intensity and apomorphine concentration is linear in the 10 to 100 pM range. Therefore, fluorescence intensity will firstly be converted to apomorphine concentration in subsequent ultrasound release studies using the standard curves (Figure 1 B), taking into account the enhancement effect MTAB has on fluorescence intensity, and used to calculate the percentage of apomorphine release using the following equation:

[0170] [Apomorphine]t— [Apomorphine]0

[0171] %Release =

[0172] [Apomorphine] , — [Apomorphine]0where [Apomorphine]o is the initial concentration of apomorphine prior to the application of ultrasound, [Apomorphine]tis the concentration of apomorphine after a period of ultrasound exposure, and [Apomorphine] , is the concentration of apomorphine in the suspension upon liposome lysis with MTAB.

[0173] It was concluded that apomorphine is useful proxy for performing encapsulation and release experiments due to its significant auto-fluorescence and concentration-dependant self-quenching behaviour.

[0174] Example 3: Active loading of apomorphine in response to an ammonium gradient.

[0175] For in vivo and clinical applications, liposomes should ideally contain a maximal amount of therapeutic agent per volume of liposome, while maintaining minimal drug leakage. The inventors looked to optimise liposomes for the encapsulation of apomorphine using an active loading approach against an ammonium ion concentration gradient.

[0176] Apomorphine was initially actively loaded against an an ammonium salt gradient. It was assumed that loading would occur as a complex equilibrium process between dissolved ammonium and ammonia within the liposome, and protonated and unprotonated apomorphine external to the liposome, where the uncharged species can freely cross the liposome membrane resulting in the intraliposomal formation of insoluble apomorphine sulfate (Figure 2). Liposomes were prepared containing an ammonium salt without any additional pH adjustment or the inclusion of buffers. Additionally, as apomorphine has a p / <aof 7.2, loading was initially performed at pH 6.3 to ensure an adequate proportion of neutral apomorphine, and at an apomorphine concentration of 4 mM.

[0177] The active loading process was investigated with 200 nm liposomes containing a range of an ammonium salt concentrations from 50 to 2000 mM and incubated for 12 hours in a solution of apomorphine, buffered to pH 6.3 with sodium phosphate (20 mM). After the removal of any residual, unencapsulated apomorphine, the baseline fluorescence of the liposome suspension was measured, indicating a relatively consistent baseline of approximately 14000 ±1000 fluorescence units regardless of the initial concentration of an ammonium salt used (Figure 3). A dramatic increase in the fluorescence intensity was observed upon lysis of the liposomes with MTAB, demonstrating a sufficient degree of encapsulation when an internal an ammonium salt concentration of 300 mM or greater is used, with maximal fluorescence intensity achieved using a concentration of 500 mM (Figure 3). Interestingly, the total apomorphine fluorescence of the lysed suspension decreased when the internal concentration of an ammonium salt was increased above 500 mM, possibly due to the high osmotic stress across the liposomal membrane as a result of the high internal salt concentration leading to liposome rupture or partial loss of the internal gradient.{Levin, 2004 #1943}.

[0178] In summary, apomorphine was successfully encapsulated within a liposome suspension using an active loading approach against an an ammonium salt gradient. Furthermore, an initial internal concentration of an ammonium salt of between about 300 mM to about 1000 mM is optimal for the gradient-driven loading of apomorphine.

[0179] Example 4: Effect of osmotic stress on apomorphine liposome loading.

[0180] Osmotic stress is a factor which can influence the behaviour and stability of liposomal drugs. In this example, the inventors studied the influence of osmotic imbalance on the drug loading process, and optimisation of the conditions of manufacture to maximally encapsulate neuromodulator drugs, for example apomorphine.

[0181] The effect of osmotic stress on liposome loading was investigated using apomorphine liposomes loaded using a 2000 mM an ammonium salt gradient, with and without an equivalent concentration of sodium chloride in the loading medium (Figure 4). Little change in fluorescence intensity was observed for a solution of apomorphine, or apomorphine with control liposomes containing no ammonium salt in the presence of 2000 mM sodium chloride over a period of 120 minutes. The addition of liposomes containing 2000 mM an ammonium salt to the apomorphine solution with and without the inclusion of sodium chloride resulted in a significant decrease in fluorescence intensity, indicating the active accumulation of apomorphine within the liposome interior. However, a significant increase in the degree of loading was observed with addition of 2000 mM sodium chloride (Figure 4), demonstrating that the inclusion of an equivalent concentration of sodium chloride to the intraliposomal an ammonium salt concentration within the loading medium leads to a far superior loading outcome. The large degree of error in the fluorescence intensity recorded during the loading of apomorphine without the inclusion of sodium chloride is likely the result of osmotic stress induced liposome disruption.

[0182] As the pH of the environment controls the ratio of protonated to deprotonated apomorphine (Figure 5 A), and as deprotonated apomorphine is the predominantly species able to freely cross the liposome membrane, the inventors postulated that the rate of active loading could be controlled by the external pH of the loading medium. Using liposomes containing 300 mM an ammonium salt, as these were demonstrated previously to have the highest encapsulation efficiency, the effect of the loading pH on the active accumulation of apomorphine was investigated. An ammonium salt liposomes were dispersed with a 4 mM apomorphine solution containing an equivalent concentration of sodium chloride and buffered between pH 5.5 and 7.5, with the fluorescence intensity monitored over a period of 120 minutes (Figure 5 B). In comparison to an apomorphine solution without the addition of liposomes (Figure 5 C), a decrease in the fluorescence intensity was observed in all cases, indicating that active liposome loading of apomorphine occurs under all the pH ranges evaluated. However, a significant increase in the rate of change in the fluorescence intensity is observed with increasing pH. This demonstrates that as the equilibrium between protonated and deprotonated apomorphine is shifted to increase the proportion of the uncharged species the rate of liposome loading can be dramatically increased.

[0183] In summary, both pH and osmotic stress are factors critical to the manufacture of liposomal neuromodulators. Optimal drug loading is achieved when osmolality, or the salt concentration of the internal liposome volume and external medium, are approximately equal. Furthermore, the rate of drug loading is primarily controlled by the pH of the medium, with higher pH resulting in a greater rate of drug loading and encapsulation.

[0184] Example 5: Effect of an ammonium salt gradient magnitude on loading capacity.

[0185] The apomorphine loading capacity of liposomes prepared from increasing an ammonium salt gradients was investigated. In this example, the inventors investigated the range of internal an ammonium salt gradients capable of maximally encapsulating apomorphine.

[0186] The apomorphine loading capacity was defined as the ratio of apomorphine to phospholipid concentration and was determined via analysis of the total phospholipid concentration and the total apomorphine concentration upon complete lysis of the liposomes. The phospholipid concentration within the liposome suspensions was estimated using the Stewart assay; {Stewart, 1980 #1527}. A linear relationship was confirmed between phosphatidylcholine and ammonium ferrothiocyanate (Figure 6 A), which was used to quantify the concentrations in the liposome suspensions post-dialysis after apomorphine loading. In all cases, the phospholipid concentration detected was below the initial preparation concentration of 2 mM. However, a decrease in detectable phospholipid was observed with increasing initial an ammonium salt gradient (Figure 6 B). Without wishing to be bound by theory, the inventors believe this may result from osmotically-induced destruction of the liposomes with higher internal concentrations during the loading process, and subsequent removal of a portion of the phospholipid content via dialysis.

[0187] As with liposomes loaded in the absence of external sodium chloride (Figure 3), a similar trend in the total concentration of apomorphine encapsulated within the liposome suspension was observed, with an increasing encapsulated concentration observed with increasing initial an ammonium salt gradient up to 500 mM, and subsequently decreasing for greater initial an ammonium salt concentrations (Figure 6 C). However, when normalising for the decreasing phospholipid concentration by considering the total apomorphine to phospholipid concentration ratio, no difference in the loading efficiency for 300 mM, 500 mM and 1000 mM an ammonium salt liposomes was apparent (Figure 6 C).

[0188] In summary, the initial concentration of an ammonium salt encapsulated within the liposome determines the magnitude of apomorphine encapsulation after the loading process. Higher initial gradients generally lead to higher apomorphine encapsulations, with optimal loading occurring with an initial an ammonium salt concentration of about 300 mM to about 1000 mM. Initial gradient concentrations beyond these parameters are seen to diminish the magnitude of apomorphine encapsulation.

[0189] Example 6: Liposome-encapsulated nanoparticulate apomorphine sulfate

[0190] The inventors studied the relationship between loading conditions and the final morphology of liposomes containing apomorphine. This was the first demonstration that liposomal nanostructures containing apomorphine are produced using the optimised active loading conditions previously described herein. Therefore, the experiments described critically demonstrate methods of producing nanoparticulate apomorphine which is stably confined within the aqueous compartment of a liposome.

[0191] Negative stain transmission electron microscopy (TEM) was performed on the apomorphine liposomes loaded using 300 mM an ammonium salt gradient at pH 5.5 to 7.0 in order to investigate any pH-dependent change in liposome morphology. In all cases, solid apomorphine was evident within the liposome structures (Figure 7 A-D). A smaller proportion of nanoprecipitate liposomes were evident in the micrographs of liposomes loaded at pH 5.5, which is consistent with the lesser degree of loading observed by fluorescence spectroscopy. Interestingly however, similar morphologies were observed between liposomes loaded at all pHs. Loaded liposomes tended to contain one spherical precipitate per particle which appeared to form at one side of the liposome as opposed to centrally within the aqueous core.

[0192] The effect of an ammonium salt gradient magnitude on the morphology of apomorphine loaded liposomes was investigated by increasing the intraliposomal concentration of an ammonium salt from 50 to 2000 mM. Negative stain TEM of apomorphine liposomes prepared against 50 and 100 mM an ammonium salt gradients indicated spherical liposomes with small solid apomorphine precipitates, generally located proximal to the liposome membrane (Figure 8 A and B). The presence of much smaller vesicles was also evident in all samples (Figure 8 A - G), although this may be an artefact of the staining technique, where the sample is evaporated on a carbon-coated copper grid.

[0193] A more interesting and unexpected observation was apparent in apomorphine liposomes prepared against 300 mM and 500 mM an ammonium salt gradients. The majority of species present in these samples generally displayed a single intraliposomal apomorphine sulfate nanoparticle which had grown to occupy approximately 20-30% and 40-50% of the liposome volume for liposome prepared from 300 mM and 500 mM an ammonium salt liposomes respectively. Furthermore, the apomorphine nanoparticle was unexpectedly attached or positioned next to the liposome membrane, leading to a unique deformation of the lipid bilayer and inducing regions of high curvature and curvature inflection at the liposome-nanoparticle interface (Figure 8 C and D).

[0194] The inventors observed that the apomorphine nanocrystal imparts structural membrane deformation as the initial an ammonium salt gradient increases, resulting in larger apomorphine sulfate nanoparticles and a greater nanoparticle to liposome volume ratio. Without wishing to be bound by theory, the inventors believe that the distended apomorphine liposomes observed in Figure 8 C and D are stable, partially wrapped intermediates in the nanoparticle membrane wrapping process.

[0195] Cryo-TEM studies of apomorphine liposomes confirm partial wrapping of the apomorphine sulfate nanoparticle and that the nanoparticle occupies approximately 30% of the liposome volume in all cases (Figure 9 A). Furthermore, DLS of the apomorphine liposomes in comparison to the initial 300 mM an ammonium salt liposomes indicates a small increase in the size distribution (Figure 9 B). The inventors believe that the presence of much smaller, sub 100 nm liposomes and apomorphine sulfate nanoparticles present in negative stain TEM images of samples prepared from all an ammonium salt gradients (Figure 8 A - G) may be a consequence of the dehydration of the sample perturbing the membrane wrapping process towards the fully wrapped state and subsequent budding of the lipid coated nanoparticle from the liposome, as these smaller particles are not evident by cryo-TEM or DLS.

[0196] Increasing the initial an ammonium salt gradient to 1000 mM surprisingly resulted in the formation of dual apomorphine sulfate nanoparticles at opposite ends of the liposome (Figure 8 E) as opposed to a larger single encapsulated nanoparticle. Increasing the internal an ammonium salt further to 1500 mM and 2000 mM resulted in an increase in the size of these opposing particles, ultimately filling the entire liposome volume (Figure 8 F and G). As the initial internal an ammonium salt concentration is increased, the inventors believe that the size of the apomorphine nanoparticle also increases, which increases the degree of bilayer distortion and bending strain imposed on the liposome membrane. Therefore, without wishing to be bound by theory, the inventors postulate that the secondary opposing nanoparticle is formed to alleviate the bending strain of the liposome membrane imposed to the wrapping behaviour of the nanoparticle and providing a lower energy alternative with a smaller degree of membrane distortion to fully budding the nanoparticle (Figure 10). As the internal concentration is further increased, to alleviate membrane bending energy and distortion the nanoparticles dually grow to fully occupy the internal volume of the liposome, producing a spherical membrane coated nanoparticle.

[0197] In summary, methods for producing and characterizing liposomal structures encapsulating a nanoparticulate form neuromodulator drug, apomorphine, have been demonstrated. The nanoparticulate drug can be produced with size and shape control by actively or remotely loading the drug against a concentration gradient (such as an ammonium salt). The degree of nanoparticulate growth can be controlled in the range of about 15 nm to about 200 nm, constrained by the size of the liposome construct. Futhermore, the nanoparticulate drug can be external to the liposome as a lipid coated drug nanoparticle, or confined within the liposome, where the size of the nanoparticle can be controlled by initial internal concentration of for example ammonium sulphate. For nanoparticles confined within the liposome, the nanoparticle can be created at sizes which partly or wholly occupy the internal volume of the liposome, and at certain sizes can impart membrane imperfections / deformation by creating inverse curvature to the liposome.

[0198] Example 7: Acoustically-sensitive liposomal nanostructures.

[0199] The inventors investigated the relationship between liposomal nanostructure morphology and ultrasonically-triggered drug release. The investigations surprisingly revealed that maximal ultrasound-induced release occurs with liposomal nanostructures which display a distended morphology, where the internal nanoparticulate drug is next to or contacts the liposome membrane. The experiments described herein detail methods of sensitising liposomal drugs to acoustic stimulation.

[0200] The effect of ultrasound on the release of apomorphine from liposomes actively loaded against an ammonium salt gradients with internal concentrations from 100 mM to 2000 mM was investigated. Ultrasound was applied to apomorphine liposomes prepared from liposomes encapsulating increasing concentrations of an ammonium salt from 100 mM to 2000 mM, and the absolute concentration and percentage of apomorphine release was measured by fluorescence intensity. Maximal absolute release was observed for liposomes prepared from a 300 mM internal an ammonium salt concentration, with a decreasing magnitude of release observed for increasing initial an ammonium salt concentrations from 500 mM to 2000 mM (Figure 11 A). However, in terms of percentage release (Figure 11 B), an equivalent maximal release is observed from liposomes prepared from both 300 mM and 500 mM internal an ammonium salt concentrations. Additionally, the percentage of apomorphine released from liposomes prepared using 1000 mM and 2000 mM an ammonium salt gradients also appear identical, which may be a function of the decreased loading capacity of the 2000 mM liposomes.

[0201] Interestingly, maximal release was achieved from the liposome populations which possessed a distended morphology where the membrane has partially wrapped around the internal apomorphine sulfate nanoparticle, as opposed to liposomes containing the maximal internal apomorphine sulfate concentration. Therefore, due to their maximal sensitivity to acoustic stimulation, apomorphine liposomes prepared from 300 mM an ammonium salt (herein referred to as Aposomes) were used for subsequent studies. The effect of both the frequency and duty cycle of applied ultrasound on apomorphine release was investigated.

[0202] To assess the suitability of the Aposome system for in vivo applications, the effect of duty cycle on the release of apomorphine as well as the degree of passive release at physiological temperature was investigated. During in vivo and clinical investigations, lower acoustic duty cycles are preferred to reduce the likelihood of initiating cavitation and tissue heating. Little difference in the percentage of apomorphine released was observed when ultrasound was applied at 50% and 100% duty cycles with an equivalent energy input (i.e. for 120 seconds and 60 seconds exposure respectively) - Figure 12. Furthermore, a small degree of passive release was observed at a rate of 0.6% per hour upon incubation of an Aposome suspension at 37 °C, reaching a maximum of 7.5% release over the investigated 12-hour period (Figure 12 C).

[0203] Ultrasound was applied to an Aposome suspension with increasing intensity between 0 and 2.4 W cm'2. No release was observed upon application of ultrasound for 60 seconds with an intensity between 0 and 0.4 W cm'2. However, an approximately linear increase in the percentage of apomorphine released was observed with increasing acoustic intensity between 0.6 W cm'2and 2 W cm'2(Figure

[0204] 13 A), thus demonstrating that an energy threshold must be met in order to acoustically stimulate apomorphine release and that onset of apomorphine release occurs at acoustic intensities above 0.6 W cm'2. Interestingly however, a more prominent threshold is evident, with a far greater onset of apomorphine release occurring at acoustic intensities between 2 W cm'2and 2.4 W cm'2(Figure 13 A). The derivative of percentage apomorphine release with respect to ultrasound intensity was therefore determined to ascertain the greatest rate of change in apomorphine release. Little change in percentage release with respect to ultrasound intensity was observed between 0 and 2 W cm'2, with the greatest rate of change occurring at 2.4 W cm2(Figure 13 B).

[0205] The morphology of the Aposome nanostructures after 300 seconds of cumulative ultrasound exposure at the energy threshold of 2.4 W cm'2(ensuring adequate release) was investigated by cryo-TEM. Prior to the application of ultrasound, liposomes of approximately 255 nm ±9 nm (n = 30) were present in the suspension (Figure 14 A). The majority of liposomes present within the population appeared to contain a single internal apomorphine sulfate nanoparticle with a size of approximately 90 nm ±4 nm (n = 30), therefore occupying approximately 35% ±3% of the internal volume. Additionally, in virtually all cases the nanoparticles were located proximal to the liposome membrane, inducing an asymmetrical distention as a result of partial membrane wrapping of the nanoparticle surface. Interestingly, a small number of rod-shaped nanoparticles were also evident accounting for approximately 4% of the population (Figure 14 A, black arrow; 14 out of 359 particles counted). These rod-shaped nanoparticles closely resemble that found with the intraliposomal crystallisation of doxorubicin, which has been determined to form a crystalline as opposed to amorphous morphology by solution x-ray scattering and high-sensitivity differential scanning calorimetry.{Schilt, 2016 #1938;Wei, 2016 #1939}. Assuming no contamination of the sample, and that apomorphine sulfate is present in liposomes of both morphologies, while not wishing to be bound by any particular theory, this likely suggests that apomorphine sulfate present in the distended liposomal nanostructures is amorphous as opposed to crystalline in nature.

[0206] After the application of ultrasound, a shift in the morphology of the liposome population was apparent, producing a substantial number of smaller liposomes approximately 169 nm ±8 nm in diameter (n = 49 particles), with a “wrinkled" morphology and without an internal nanoparticle (Figure

[0207] 14 B; dashed arrows). Prior to the application of ultrasound, a small population of these vesicles were present (approximately 4%). However, post-ultrasound application these vesicles account for 57% of the total liposome population, demonstrating a significant shift in the morphology of the liposome population. Post-application of ultrasound, a large number of lipid discs are also evident with a length of 58 nm ±4 nm and width of 8 nm ±0.3 nm (n=35) (Figure 14 B; light arrows), as well as small, spherical nanoparticles approximately 8.5 nm ±0.3 nm (n = 36) (Figure 14 B; bold arrows).

[0208] Intriguingly, post-application of ultrasound a number of liposome species are present with an extremely distended lipid membrane induced by the protrusion of the internal apomorphine sulfate nanoparticle (Figure 15B) in comparison to pre-ultrasound liposome (Figure 15A). These liposomal nanostructure species appear to be intermediate structures theoretically present during budding of an internal nanoparticle leading the inventors to postulate that drug release is a function of acoustically accelerated budding of the internal apomorphine sulfate nanoparticle (Figure 16), which subsequently dissolves leaving empty liposomes, lipid discs, and smaller residual lipid-coated nanoparticles evident by cryo-TEM (Figure 14 B).

[0209] The formation of lipid discs suggests that release of the apomorphine sulfate nanoparticle occurs prior to complete membrane wrapping. Therefore, without wishing to be bound by theory, the inventors postulate that increased bending strain during the wrapping process leads to a breakage at the points of inflection where bending strain is highest (Figure 24; dashed lines). A comparison of the average surface area occupied by the 58 nm ±4 nm lipid discs ("'2,600 nm2), with the surface area of spherical cap of the protruding apomorphine sulfate nanoparticle ("'10,050 nm2) (Figure 16; light line) was made. The diameter of a spherical liposome with a surface area equivalent to that of the spherical surface cap would be approximately 56 nm, well below the diameter of spontaneously formed liposomes containing 5 mol% DSPE-PEG2000 ("'100 - 200 nm).{Szleifer, 1998 #1952}. Therefore, as the surface area of the spherical cap is below that required to form spontaneous and stable liposome structures, and is approximately 3-4 times greater than that of the average lipid disc, it is likely that multiple lipid discs are formed during membrane rupture and release of the apomorphine sulfate nanoparticle.

[0210] In summary, the morphology of liposomal nanostructures encapsulating nanoparticulate apomorphine controls the sensitivity to acoustic stimulation. The internal nanoparticle can be created at sizes which partly or wholly occupy the internal volume of the liposome, and at certain sizes and nanoparticle volume to liposome volume ratios which impart membrane imperfections and deformations which create inverse curvature within the liposome membrane, and confers a degree of sensitivity to acoustic energy. This surprising phenomenon was most evident when liposomal nanostructures were manufactured from liposomes containing an ammonium salt at an initial concentration of about 300 mM to about 500 mM. Furthermore, these liposomal nanostructures are stable under physiological temperature, with minimal passive leakage of apomorphine over a prolonged period of time. Therefore, these experiments demonstrate an entirely new approach to sensitising a liposomal nanodrug to acoustic stimulation by using the internal drug nanoparticle as a mechanism to confer acoustic sensitivity through liposome membrane-nanoparticle interactions.

[0211] Example 8: Morphology and Ultrasound-induced release from liposomal nanostructures with alternative liposome compositions.

[0212] A phase transition gel to fluid phase transition is present in the lipid composition used in the preparation of Aposome suspension, occurring at approximately 42 - 44 °C. However, clinically used liposome formulations commonly include high levels of cholesterol to significantly depress membrane phase transitions to increase in vivo stability. Therefore, the inventors investigated the influence of high cholesterol formulations on the morphology of liposomal nanostructures and the corresponding ultrasound-release behaviour. The results broadly demonstrate the formation of acoustically-sensitive liposomal nanostructures containing nanoparticulate apomorphine with alternative and clinically- used liposome membrane compositions.

[0213] Liposomes containing 300 mM an ammonium salt were prepared using the Doxil’ lipid composition containing HSPC, cholesterol, and DSPE-PEG2000 in a 55:40:5 mol% ratio; as the high cholesterol content in this lipid formulation has been demonstrated to completely depress the gel to fluid phase transition temperature. {Abraham, 2005 #1565} Actively loading the liposomes against apomorphine in an analogous manner to the previous lipid composition resulted in a similar liposome morphology where a single nanoparticle resides within the liposome distending the membrane (17 A).

[0214] Upon application of ultrasound at an intensity of 2.4 W cm'2, a linear increase in the percentage of apomorphine released was observed (17 B).

[0215] In summary, acoustically-sensitive liposomal nanostructures containing nanoparticulate apomorphine can be prepared using alternative liposome membrane compositions, such as that used in commercially-available liposomal doxorubicin (DoxiT). Using this phospholipid composition, which includes a high percentage of cholesterol to depress the lipid membrane gel-liquid phase transition, and an initial an ammonium salt concentration of about 300 mM, the resulting liposomal nanostructures maintain the previously observed morphology of a single apomorphine nanoparticle which is located directly next to or contacts and distends the liposome membrane. While the cholesterol-rich liposomal nanostructures maintain their sensitivity to acoustic stimulation, the overall magnitude of apomorphine release in response to an equivalent acoustic energy input is diminished compared to low-cholesterol liposome formulations which maintain a phase-transition. This provides a critical and novel insight into the design of acoustically-sensitive liposomal nanostructures. Example 9: Liposomal nanocrystals with alternative counter-ions, and using DI and D2 specific agonists.

[0216] Due to the success of acoustically-stimulated release from liposomal nanostructures containing apomorphine sulfate, the inventors investigated the formation of acoustically-sensitive liposomal nanostructures using alternative drug candidates, as well as alternative ammonium salts in the loading process. This is the first demonstration that the acoustically-sensitive liposomal nanostructure system, which uses an internal drug nanoparticle to produce sensitivity to ultrasound stimulation, was broadly applicable to multiple drug candidates and salts.

[0217] The effect of counter anion on the morphology of actively loaded apomorphine liposomes was investigated by negative-stain TEM. Liposomes were prepared containing either ammonium citrate (300 mM), carboxyfluorescein ammonium salt (300 mM), ammonium oxalate (250 mM), or ammonium trimesate (300 mM) (Figure 18 A), to form the initial ammonium gradient and incorporate a range of structurally diverse counterions. Interestingly, little difference in the morphology of the resulting liposomes was observed when actively loaded with apomorphine, with single nucleated apomorphine nanoparticles evident by negative stain TEM which were seemingly adherent to the liposome membrane in each case (Figure 18 B - D). Therefore, it is likely that the intraliposomal drug crystallisation or precipitation has a greater dependence on the physical and structural characteristics of the drug candidate than on the counterion.

[0218] The effect of structural diversity on the intraliposomal crystallisation of a drug in response to an ammonium gradient was further investigated using structurally diverse drug candidates dihydrexidine and ropinirole (Figure 19 A). Dihydrexidine and ropinirole were chosen due to their relevance in the treatment of Parkinson’s disease, as selective dopaminergic DI- and D2-type receptor agonists respectively.{Salmi, 2004#1953;Coldwell, 1999#1955;Parvez, 2010 #1954} In the context of a therapy for Parkinson’s disease, the encapsulation of these selective agents was of great interest to the inventors for the potential to create a system capable of releasing patient-specific ratios of DI and D2 agonists, customised to optimise the pharmacological prescription to the patients response.

[0219] The encapsulation of dihydrexidine was successfully achieved in response to both 300 mM and 500 mM an ammonium salt gradients, resulting in the formation of a single, spherical nanoparticle similar to that achieved with apomorphine and inducing membrane deformation at the membrane- nanoparticle interface (Figure 19 B). The active loading process required to achieve nanoparticle formation was much slower for dihydrexidine than for apomorphine, occurring over a period of 7 days as opposed to 12 hours; likely due to the higher p / <aof dihydrexidine and consequentially a lower concentration of the uncharged species which can freely cross the lipid bilayer. Intraliposomal nanoprecipitation of ropinirole in response to an ammonium salt gradient was unsuccessful. However, intraliposomal nanoprecipitation of ropinirole could be achieved in response to a 250 mM ammonium oxalate gradient, resulting in active loading and nanoparticle formation. Multiple nanoparticles per liposome were generally observed when loading occurred at pH 6.0, with little adherence or distention to the liposome membrane (Figure 17 C), and single membrane distending nanoparticles were apparent when loaded at pH 7.5 (Figure 19 D); thus, suggesting that the loading pH plays a crucial role in the formation of single, membrane adherent nanoparticles.

[0220] In summary, these studies confirm that the scope of this unique liposomal nanostructure system can be extended beyond the encapsulation and release of apomorphine. Drug loading and nanoparticulate formation can be achieved using multiple ammonium salts. Furthermore, a cationic neuromodulator drug, such as apomorphine, dihydrexidine, and ropinirole, can be loaded against an ammonium salt of a fluorescent dye, such as carboxyfluorescein, creating a liposomal nanostructure construct which may be useful in both therapeutic and diagnostic applications.

[0221] Example 10: In vitro ultrasound-triggered release of apomorphine and dihydrexidine from liposomal nanostructures.

[0222] The inventors studied the in vitro release of dihydrexidine and apomorphine from liposomal nanostructures using low duty cycle, biologically-safe, levels of ultrasound. Results are shown in Figure 20.

[0223] . Liposomes were prepared as previously described in Example 3. A solution containing dihydrexidine (4 mM), ascorbic acid (2 mg / mL), phosphate buffer (pH 7.4) and an equivalent concentration of sodium chloride to that of encapsulated ammonium sulphate was used as the loading medium. The ammonium sulphate liposome suspension (0.4 mL) was diluted with the dihydrexidine loading solution (1.6 mL) and incubated at 25 degrees Celsius for 72 hours with stirring. Excess (unencapsulated) dihydrexidine was removed via dialysis against phosphate-buffered saline (pH 7.4).

[0224] Example 11: In vivo ultrasound-triggered release of apomorphine from liposomal nanostructures. The inventors translated the measurement of apomorphine and dihydrexidine to in vivo application. In order to determine that they were getting release of the neurochemical without the confounding effect of endogenous monoamines such as dopamine, one side of the brain was first depleted using a toxin 6-hydroxydopamine to create a Parkinson's-like lesion and this was verified behaviorally using a step test and a cylinder test to ascertain that the depletion was complete. With the animals anaesthetized a stimulating electrode was placed into the dopamine cell layer on both sides of the brain, the signal in both striata was measured and it was verified that electrically-evoked dopamine release could be measured only in the dopamine intact striatum. A cannula was then placed into the jugular vein of the dopamine deficient side and the electrochemical signal for apomorphine or dihydrexidine was measured at baseline, after the liposomes were injected intravenously and following ultrasound. It was found that ultrasound applied through the skull for only about 5 to 10 seconds would generate release of about 1 pM of apomorphine (Figure 21). This is sufficient to activate the low affinity DI receptors for dopamine in the target area, the striatum.

[0225] In conclusion, release of apomorphine and dihydrexidine can be achieved from liposomes comprising the present invention in response to short periods of ultrasound application, producing localised concentrations of dopaminergic agonist at levels known to induce a biological or therapeutic response.

[0226] Example 12: Non-invasive in vivo release of dihydrexidine from liposomal nanostructures in an awake animal evokes a behavioral response.

[0227] Behaviourally, animals were operated on to fit the rat hat collet ultrasonic transducer, to allow observations of activity in an awake animal model using rats fitted with the miniaturized, custom-built rat hat ultrasound transducer (Figure 22 left). In some animals (for apomorphine) dopamine was again depleted on one side of the brain, and in others (for dihydrexidine) the dopamine system was left intact. For dihydrexidine liposomes, a single dose dihydrexidine liposomal nanostructures was administered intravenously and the animal monitored for a change in the frequency of mouth and facial movements. An increase in behavioural activity was observed with short applications of ultrasound (5-10 seconds), which occurred repeatedly over a period of days (Figure 22 right) and was not observed in the absence of ultrasound or dihydrexidine liposomal nanostructures, or in the presence of ultrasound alone. A decrease in the frequency of mouth movements with ultrasound application was also observed over a period of days, which correlates with the expected elimination rates of liposomes formulated with this size and composition

[0228] For apomorphine, the use of sufficient dose of apomorphine liposomal nanostructures followed by an application of ultrasound was associated with the induction of tight contraversive turning, as described for apomorphine injections previously.

[0229] With the observation of an electrochemical signal in combination with a behavioral effect after ultrasound application to target areas containing the neurochemical-loaded liposomes, the inventors have successfully demonstrated release of neurochemical-like drugs from liposomes non-invasively along with observed therapeutic effects in vivo. The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0230] Titles, heading, or the like are provided to enhance the reader’s comprehension of this document and should not be read as limiting the scope of the invention.

[0231] The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference. However, the reference to any applications, patents, and publications in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0232] REFERENCES

[0233] Kent Van Tyle, W.; Burkman, A. M., Spectrofluorometric Assay of Apomorphine in Brain Tissue. Journal of Pharmaceutical Sciences 1971, 60 (11), 1736-1738.

[0234] Smith, R. V.; De Moreno, M. R., Determination of apomorphine and N-n- propylnorapomorphine in plasma using high-performance liquid chromatography and fluorescence detection. Journal of Chromatography B: Biomedical Sciences and Applications 1983, 274, 376-380.

[0235] Levin, Y.; Idiart, M. A., Pore dynamics of osmotically stressed vesicles. Physica A: Statistical Mechanics and its Applications 2004, 331 (3-4), 571-578.

[0236] Stewart, J. C. M., Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Analytical Biochemistry 1980, 104 (1), 10-14

[0237] Schilt, Y.; Berman, T.; Wei, X.; Barenholz, Y.; Raviv, U., Using solution X-ray scattering to determine the high-resolution structure and morphology of PEGylated liposomal doxorubicin nanodrugs. Biochimica et Biophysica Acta (BB A) - General Subjects 2016, 1860 (1, Part A), 108-119.

[0238] Wei, X.; Cohen, R.; Barenholz, Y., Insights into composition / structure / function relationships of Doxil® gained from “high-sensitivity” differential scanning calorimetry. European Journal of Pharmaceutics and Biopharmaceutics 2016, 104, 260-270.

[0239] Szleifer, I.; Gerasimov, O. V.; Thompson, D. H., Spontaneous liposome formation induced by grafted poly(ethylene oxide) layers: Theoretical prediction and experimental verification. Proceedings of the National Academy of Sciences 1998, 95 (3), 1032-1037.

[0240] Abraham, S. A.; Waterhouse, D. N.; Mayer, L. D.; Cullis, P. R.; Madden, T. D.; Bally, M. B., The Liposomal Formulation of Doxorubicin. In Methods in Enzymology, Academic Press: 2005; Vol. Volume 391, pp 71-97.

[0241] Salmi, P.; Isacson, R.; Kull, B., Dihydrexidine — The First Full Dopamine DI Receptor Agonist. CNS Drug Reviews 2004, 10 (3), 230-242.

[0242] Coldwell, M. C.; Boyfield, I.; Brown, T.; Hagan, J. J.; Middlemiss, D. N.,

[0243] Comparison of the functional potencies of ropinirole and other dopamine receptor agonists at human D2(long), D3 and D4.4 receptors expressed in Chinese hamster ovary cells. British Journal of Pharmacology 1999, 127 (7), 1696-1702. Parvez, S.; Winkler-Stuck, K.; Hertel, S.; Schonfeld, P.; Siemen, D., The dopamine- D2- receptor agonist ropinirole dose-dependently blocks the Ca2+-triggered permeability transition of mitochondria. Biochimica et Biophysica Acta (BBA) - Bioenergetics 2010, 1797 (6-7), 1245-1250.

Claims

CLAIMS1 . A method for producing acoustically activatable liposomes comprising an amphipathic agent nanoparticle, the method comprising:(a) providing a suspension of liposomes, wherein the concentration of ammonium ions inside the liposomes is from approximately 200 mM to approximately 1000 mM of an ammonium salt and is greaterthan the concentration of ammonium ions outside the liposomes; and(b) adding one or more amphipathic agents to the suspension, wherein the amphipathic agent is taken up into the liposomes to a final concentration greater than outside the liposomes and forms a nanoparticle within the liposome.

2. The method of claim 1 , wherein the ammonium salt is selected from the group consisting of ammonium sulfate, ammonium oxalate, ammonium citrate, ammonium trimesate, and carboxyfluorescein ammonium salt.

3. The method of claim 1 or claim 2, wherein the ammonium salt is ammonium sulfate.

4. The method of any one of claims 1 to 3, wherein the concentration of ammonium ions inside the liposomes is from approximately 300 mM to approximately 500 mM.

5. The method of any one of claims 1 to 4, wherein the liposomes are from approximately 80 nm to approximately 400 nm in diameter.

6. The method of any one of claims 1 to 5, wherein the amphipathic agent nanoparticle comprises approximately 10% to approximately 60% of the liposome size.

7. The method of any one of claims 1 to 6, wherein the amphipathic agent is added in a loading medium having an osmotic concentration substantially equivalent to that inside the liposomes.

8. The method of claim 7, wherein the loading medium comprises sodium chloride, sucrose, or phosphate-buffered saline.

9. The method of any one of claims 1 to 8, wherein the amphipathic agent comprises a drug or therapeutic agent.

10. The method of claim 9, wherein the amphipathic agent comprises a positively ionizable functional group.11 . The method of claim 10, wherein the functional group is selected from the group consisting of a primary or secondary amine, imine, isoxazole, or nitrogen-containing heterocycles including pyridine, pyrrole, and quinoline.

12. The method of claim 11 , wherein the amphipathic agent is a neuromodulatory agent.

13. The method of claim 12, wherein the neuromodulatory agent is selected from the group consisting of apomorphine, dihydrexidine, ropinirole, SCH23390, SKF38393, and sulpiride.

14. An acoustically activatable liposome comprising an amphipathic agent nanoparticle, wherein the liposome is produced by the method of any one of claims 1 to 13.

15. A composition comprising liposomes according to claim 14.

16. The liposome of claim 14, wherein the amphipathic agent nanoparticle is positioned proximal to an internal face of the liposomal membrane such that a region of the membrane is partially wrapped around the nanoparticle and distended relative to adjacent regions.

17. The liposome of claim 14 or claim 16, wherein the liposome is activatable by an ultrasound signal to release the amphipathic agent.

18. The method of delivering an amphipathic agent to a subject, comprising: a) administering a composition according to claim 14, 16 or 17 to the subject; and b) applying an ultrasound signal to release the amphipathic agent from the liposomes.

19. The method of claim 18, wherein the ultrasound has a frequency of about 200 kHz to 1 MHz and an intensity of about 0.01 to 10 W / cm2.

20. The method of claim 18 or claim 19, wherein the amphipathic agent is delivered to the subject’s brain.21 . A method for treating a neurological disorder in a subject, the method comprising administering a composition according to claim 15 and applying an effective ultrasound signal to release a neuromodulatory agent.

22. The method of claim 21 , wherein the neurological disorder is Parkinson’s disease.

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