Patent Application
20090098044
The present invention relates to a process for controlled destabilisation of microemulsions to form solid particles.
Controlled delivery of drugs, for example for treatment of tumours, has been achieved by various methods. One method is to encapsulate the drug into a solid particle, which can be delivered to the site of action, such as the tumour, and there release the drug at a controlled rate.
In order to be effective, such particles must be of an appropriate size for lodging at a tumour site. Particles that are above approximately 300 nm diameter will commonly be trapped by the lungs, liver, spleen and other organs, and will thus not preferentially lodge at the site of action. Particles that are below approximately 50 nm diameter are capable of penetrating through the walls of blood vessels, and are therefore distributed throughout the body. Thus for this application, it is desirable to have a particle of approximately 50 to 300 nm diameter. In order to have an appropriate release rate of an encapsulated drug, the particles should have nanoscale pores. The size of the pores governs the release rate. In order to achieve an appropriate release rate, the pores should be below approximately 2 nm, and preferably should be about 1.5 nm diameter. A further requirement is that the conditions of manufacture of the drug-laden particles should be such that they do not significantly degrade the drug. Commonly anti-cancer drugs, for example doxorubicin, are unstable in basic solution, and so it is preferable for any particles to be produced under neutral or acidic conditions.
Silica nanoparticles possess several intrinsic advantages as drug carriers for in vivo applications. In particular, they are biologically inert, intrinsically hydrophilic (which reduces their detection by the reticuloendothelial system) and provide extended shelf life to their payload. Moreover, it has been established that spherical particles in the 50-250 nm diameter range possessing the appropriate physico-chemical properties can be selectively distributed into tumour masses from the general circulation over a period of one to two days after intravenous injection.
Silica nanoparticles may also be used for controlled release of other substances, for example catalysts, enzymes etc.
The preparation of silica nanoparticles using base-catalysed sol-gel chemistry in microemulsions has been extensively investigated. However, this base catalysis chemistry poses two disadvantages for the encapsulation of bioactive species:
In contrast, acid catalysis yields microporous particles, which exhibit much slower release of the encapsulated species. However, due to the intrinsic mechanisms of particle evolution in acid environment, the size of silica particles formed by acid catalysis is either less than 30 nm or larger than one micron.
There is therefore a need for a method to produce particles in the 50-500 nm range with sustained release characteristics. The particles may have both appropriate particle size (between approximately 30 nm and 1 μm, optionally between approximately 50 and 250 nm, in diameter) and pore size (i.e. microporous) for passive targeting of tumours and may be made under conditions that do not degrade a drug encapsulated in the particles.
It is the object of the present invention to overcome or substantially ameliorate at least one of the above disadvantages. It is a further object to at least partially satisfy the above need.
In a first aspect of the invention there is provided a process for making a particulate substance comprising:
The particulate substance may be seeded by the nuclei or otherwise derived from the nuclei.
The nuclei may be primary particles. The nuclei may be nuclei for formation of primary particles. The nuclei may be nuclei for formation of solid particles (including porous solid particles) or gel particles. The process may comprise the step of forming primary particles from the nuclei. The particles of the particulate substance may be derived from the droplets. The particles of the particulate substance may be derived from the primary particles (e.g. seeded by the primary particles), which may in turn be derived from the nuclei (e.g. seeded by the nuclei) or correspond to the nuclei. The nuclei may be solid nuclei, or may be gel nuclei or a combination thereof. The nuclei may comprise polymeric silicate molecules. The nuclei may comprise pre-ceramic polymers. The pre-ceramic polymers may be capable of being converted into a ceramic material e.g. silica. The step of at least partly destabilising the droplets may comprise at least partly coalescing the droplets. It may comprise combining a coalescing liquid and the emulsion. The particles of the particulate substance may be formed or derived from the nuclei by association, coalescence or agglomeration of the nuclei or of the primary particles. At least some of the droplets may comprise a condensable species. The condensable species may be capable of reacting with the primary particles or the nuclei, and may be capable of coalescing or agglomerating the primary particles or nuclei. The condensable species may be derived from a tetraalkoxysilane (e.g. tetramethoxysilane or tetraethoxysilane) or an alkyltrialkoxysilane (e.g. aminopropyltrimethoxysilane), or a mixture thereof, or it may comprise a silicate or a polysilicate or a mixture thereof. The droplets may comprise a releasable substance, and the process may make a particulate substance comprising the releasable substance. The droplets may also comprise one or more non-releasable substances and the process may make a particulate substance comprising the releasable and non-releasable substances. The nuclei and the primary particles (if present) may or may not comprise the releasable substance (and optionally the non-releasable substance). The releasable substance may be releasable from the particles. The releasable substance may comprise a single releasable substance, or may comprise two or more individual releasable substances.
The process may additionally comprise the steps of:
The step of providing an emulsion may comprise providing a microemulsion wherein at least some of the droplets comprise nuclei. The nuclei may be preformed or may be formed in situ.
The step of providing the emulsion may comprise:
The step of forming the nuclei may comprise ageing the precursor emulsion for sufficient; time for formation of the nuclei from the condensable species.
The process of providing the precursor emulsion may comprise the steps of:
The hydrolysable species may comprise a tetralkoxysilane, for example tetramethoxysilane or tetraethoxysilane.
In an embodiment there is provided a process for making a particulate substance comprising a releasable substance, comprising:
The plurality of particles may be seeded or otherwise derived from the nuclei. The plurality of particles may be derived from the droplets.
The releasable substance may be at least partially immobilised in and/or on the particulate substance, and may be releasably immobilised therein and/or thereon. The releasable substance may be an organic compound or an organometallic compound and may be a drug. It may be an anti-cancer drug for example doxorubicin. The releasable substance may be a fluorescent dye, a radiopharmaceutical, an enzyme, a hormone, a biocide or some other substance. The releasable substance may be releasable into water or an aqueous fluid or some other solvent. It may be releasable on exposure of the particulate substance to water or the aqueous fluid or other solvent, or on immersion of the particles in water or the aqueous fluid, or on agitation of the particles in water or the aqueous fluid or other solvent.
In another embodiment there is provided a process for making a particulate substance, said particulate substance comprising a first and a second releasable substance, said method comprising:
The particles may be seeded by the nuclei or otherwise derived from the nuclei. The combining may comprise mixing, swirling, agitating, homogenising etc. The first and second emulsions may be combined in any desired ratio. They may be combined in a ratio such that the particulate substance comprises the first and second releasable substances in a desired ratio.
In another embodiment there is provided a process for making a particulate substance comprising:
The step of combining may comprise adding the coalescing liquid to the emulsion, or it may comprise adding the emulsion to the coalescing liquid. It may comprise stirring, swirling, sonication or otherwise agitating either the emulsion or the coalescing liquid or both. The step of combining may destabilise the droplets and/or emulsion. The step of combining may lead to formation of the particulate substance. The particulate substance may comprise a plurality of particles, each of the particles being formed from one or more of nuclei, for example by coalescence of a plurality of the nuclei or by growth of the nuclei. The step of combining may comprise allowing sufficient time for formation of the particulate substance. The sufficient time may be up to about 10 hours. The formation of the particulate substance may comprise at least partial condensation, polycondensation or crosslinking, of the condensable species. The formation of the particulate substance may comprise reaction of the condensable species with the nuclei. The particles of the particulate substance are porous and may be microporous and/or mesoporous. They may have pores between about 0.5 and 5 nm diameter. They may be spherical, or may be an irregular shape or some other shape. The particles may have a particle size between about 30 and about 5000 nm, or between about 30 and about 1000 nm or between about 50 and about 300 nm diameter. The coalescing liquid may be miscible with the continuous liquid phase, and may comprise a destabilising liquid and may also comprise a non-polar liquid. The destabilising liquid may be a polar liquid, and may be acetone, or ethanol, or a mixture of acetone and ethanol.
In another embodiment, the droplets which comprise nuclei have a pH greater than 7, and the step of providing the emulsion is followed by the step of acidifying the droplets and adding a condensable substance or a precursor thereto, such that during the step of at least partially destabilising the droplets, the condensable substance condenses in order to form the particles. The precursor may comprise a hydrolysable silane, for example a tetraalkoxysilane, as described herein. The step of providing the emulsion may comprise combining (optionally mixing or agitating) a surfactant, a hydrophobic liquid, a basic aqueous liquid and a condensable substance or precursor thereto, and allowing the condensable material to condense to form the nuclei (or allowing the precursor to form the condensable substance and then allowing the condensable substance to condense to form the nuclei). The emulsion may be a microemulsion. The precursor or the condensable substance for the nuclei may be the same as or different to the precursor or condensable substance for the particles, as described above. Typical precursors comprise hydrolysable silanes, which may hydrolyse to form at least partially hydrolysed and/or partially condensed silanes, which are the condensable substances which may condense to form the nuclei, or to form the particles.
In another embodiment the process of providing the emulsion comprises the steps of:
The process may also comprise one or more of the steps of:
In another embodiment, the emulsion droplets comprise a releasable substance, and the process at least partly immobilises the releasable substance in and/or on the particulate substance. The releasable substance may be temporarily or releasably immobilised in and/or on the particulate substance. That is, the releasable substance may be immobilised on the particulate substance, but be capable of being at least partially released therefrom when subjected to appropriate release conditions, e.g. immersed in a liquid capable of releasing the releasable substance. The liquid may be for example a solvent for the releasable substance. The releasable substance may be an organic compound or an organometallic compound and may be a drug. It may be an anti-cancer drug for example doxorubicin. The releasable substance may be stable to the conditions pertaining in the emulsion droplets before and during the process.
In another embodiment, there is provided a process for making a particulate substance, comprising the steps of:
In another embodiment, there is provided a process for making a particulate substance comprising a releasable substance, comprising the steps of:
In another embodiment, there is provided a process for making a particulate substance comprising a releasable substance, comprising the steps of:
In another embodiment there is provided a process for making a particulate substance comprising:
The condensable species may be a silicate, and may be a soluble silicate for example sodium silicate or potassium silicate. The process may also comprise adding a metal oxide with the condensable species. The metal may be a transition metal for example titanium, zirconium, iron, zinc, vanadium, chromium or hafnium. Other oxides, such as those of tin, aluminium, germanium, calcium or phosphorous may also be used. The oxide may be added in a ratio to the condensable species of between about 0 and 80% on a molar or w/w basis, or between about 0 and 75%, 0 and 60%, 0 and 50%, 0 and 40%, 0 and 30%, 0 and 20%, 0 and 10%, 20 and 80%, 50 and 80%, 50 and 75%, 25 and 75% or 25 and 50%, and may be added in a ratio of about 0, 10, 20, 30, 40, 50, 60, 70 or 80% on a molar or w/w basis. Biodegradable particles and/or particles suitable for promoting apatite formation, e.g. for orthopaedic applications, may be made in this manner.
In a second aspect of the invention there is provided a process for making a particulate substance comprising:
The present invention also provides for a particulate substance, or a particulate substance comprising a releasable substance, when made by the processes of the first or the second aspect of the invention. The releasable substance, if present, may be at least partly immobilised, and may be at least partly releasably immobilised.
In a third aspect of the invention there is provided a microporous particulate substance comprising a releasable substance. The releasable substance may be releasably immobilised on and/or in the microporous particulate substance. The particles of the particulate substance may be between about 30 and about 5000 nm or between about 30 and about 1000 nm or between about 50 and about 300 nm mean particle diameter. The particulate substance may have a mean pore size of between about 0.5 and 50 nm in diameter. It may have micropores of less than about 1 nm diameter, together with mesopores of between about 1 and 50 nm, for example between about 1 and 10 nm. The particles of the particulate substance may comprise nuclei associated together. They may comprise agglomerates of nuclei. The nuclei may have a mean diameter of between about 1 and 50 nm. The particulate substance may be made by the process of the first or second aspect of the invention.
The releasable substance may be unstable in a basic environment, and may be stable in an acidic environment, and may be a drug, for example a drug for treatment of cancer. The releasable substance may be a fluorescent dye, a radiopharmaceutical, an enzyme, a hormone, a biocide or some other substance. The releasable substance may be releasable into water or an aqueous fluid or some other solvent. It may be releasable on exposure of the particulate substance to water or the aqueous fluid or other solvent, or on immersion of the particles in water or the aqueous fluid or other solvent, or on agitation of the particles in water or the aqueous fluid or other solvent. The releasable substance may be releasable without substantial degradation, or dissolution or erosion of the particles of the particulate substance. Over an extended period e.g. over about 6 months, some dissolution of the particles may take place. This may influence or contribute to the release profile. Release of the releasable substance may occur by diffusion out of particles of the particulate substance.
In a fourth aspect of the invention there is provided a method for treating a condition in a mammal, for example a human, comprising administering to the mammal a therapeutically effective quantity of a particulate substance according to the present invention, said particles comprising a releasable substance, said releasable substance being indicated for the condition. The releasable substance may be a drug, and the drug may be an anti-cancer drug. The condition may be a disease. The condition may be for example cancer, diabetes, hormonal dysfunction, hypertension, pain (for example pain treatable by morphine and/or opiates), or asthma.
There is also provided a particulate substance according to the present invention when used for the manufacture of a medicament for the treatment of a condition in a mammal, for example a human, said particulate substance comprising a releasable substance, said releasable substance being indicated for the condition. The condition may be for example cancer, diabetes, hormonal dysfunction, hypertension, pain (for example pain treatable by morphine and/or opiates), or asthma.
There is further provided the used of a particulate substance according to the invention for the treatment of a condition in a mammal, for example a human, said particles comprising a releasable substance, said releasable substance being indicated for the condition. The condition may be for example cancer, diabetes, hormonal dysfunction, hypertension, pain (for example pain treatable by morphine and/or opiates), or asthma.
In a fifth aspect of the invention there is provided a method for delivering a releasable substance, said method comprising exposing a particulate substance according to the present invention to a medium capable of releasing said releasable substance, said particles comprising the releasable substance. The exposing may comprise immersing the particles in the medium, and may additionally comprise one or more of stirring, shaking, swirling or otherwise agitating the medium having the particles therein. Alternatively the exposing may comprise passing the medium past and/or through the particles. The medium may be a fluid, and may be a liquid. The medium may be a biological fluid such as blood. It may be an aqueous fluid, such as water or an aqueous solution. The medium may be capable of dissolving the releasable substance. The releasable substance may be for example a fluorescent dye, a radiopharmaceutical, a drug, an enzyme, a hormone, a biocide or some other substance, or it may be a mixture of any two or more of these. The exposing may be under conditions suitable for release of the releasable substance into the medium. The method may also comprise the step of allowing the releasable substance to release into the medium.
A preferred form of the present invention will now be described by way of example with reference to the accompanying drawings wherein:
The present invention describes a process for making particles of a desired size. In one aspect, the process comprises initially producing an emulsion, for example a microemulsion, with well-controlled particle size through well-known processes, infusing a hydrolysable species into the emulsion droplets of the emulsion, hydrolysing the hydrolysable species in the emulsion droplets to form a condensable species and condensing the condensable species within the emulsion droplets using well-known sol-gel chemistry. The resulting particles are then coalesced by initiating a controlled destabilisation of the emulsion to produce a particulate substance with the desired particle size. The chemistry required for making particles having controlled pore sizes is known, and by combining that technology with the present process for controlled destabilisation, it is possible to produce a particulate substance comprising particles of a desired mean particle size with controlled pore size. Particular combinations of particle size and pore size are thus achievable that were hitherto difficult to produce. If a releasable substance such as a drug is incorporated into the emulsion droplets of the initial microemulsion, then that releasable substance may be releasably immobilised in and/or on the particulate substance, which may then (if the releasable substance is a drug) be used for therapeutic purposes. By appropriate control of the particle size, the particulate substance may be targeted at particular parts of a patient's body, and by appropriate control of the pore size, the release rate of the releasable substance may be controlled to a desired rate.
The process of the present invention may be used to make a microporous particulate substance, which may comprise a releasable substance, for treatment of conditions such as cancer in a mammal, for example a human. The releasable substance may be releasably immobilised on and/or in the microporous particulate substance. The microporous particulate substance may comprise particles that are between about 30 and about 10000 nm in diameter, or may be between about 30 and 500 or about 30 and 100 or about 50 and 1000 or about 100 and 1000 or about 500 and 1000 or about 50 and 500 or about 50 and 300 or about 50 and 250 or about 100 and 400 or about 100 and 300 or about 100 and 250 or about 150 and 250 mm, and may be about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 nm in diameter. The particles of the particulate substance may be nanoparticles. The particles of the particulate substance may be microporous (that is they may have a pore diameter of less than about 1.7 nm) and/or mesoporous. They may be both microporous and mesoporous. They may have a mean pore diameter of less than about 50 nm, or less than about 40, 30, 20, 10, 5, 4, 3, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6 or 0.5 nm. They may have a mean pore diameter of between about 0.5 and 5 nm, or between about 0.5 and 2 nm or about 0.5 and 1 nm or about 1 and 5 nm or about 2 and 5 nm or about 1 and 2 nm or about 4 and 5 nm or between about 5 and 50 nm, 10 and 50 nm, 20 and 50 nm, 10 and 20 nm, 5 and 20 nm or 5 and 10 nm, and may have a mean pore diameter of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 17, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nm. They may have mesopores between about 1 and about 50 nm and micropores below about 1 nm. The pore size may be tailored by adjusting the conditions under which the particles of the invention are made. In the process of the present invention, these particles are produced from nuclei. The nuclei may be primary particles. The nuclei may have a mean particle diameter about 1 and 50 nm, or between about 1 and 20 or about 1 and 10 or about 1 and 5 or about 1 and 2 or about 2 and 50 or about 5 and 50 or about 10 and 50 or about 20 and 50 or about 2 and 20 or about 2 and 10 or about 5 and 10 nm, and may have a mean particle diameter of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45 or 50 nm The weight of the releasable substance may depend on the nature of the drug and the nature of the particulate substance, and may be between about 0.01 and 100 mg per gram of particulate substance, or between about 0.01 and 20 mg or 0.01 and 10 mg or 0.01 and 5 mg or about 0.01 and 1 mg or about 0.01 and 0.5 mg or about 0.01 and 0.1 mg or about 0.01 and 0.05 mg or about 1 and 100 mg or about 10 and 100 mg or about 50 and 100 mg or about 1 and 10 mg or about 5 and 10 mg or about 0.1 and 1 mg or about 0.1 and 0.5 mg per gram of particulate substance, and may be about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg per gram of particulate substance, or may be less than 0.01 or greater than 100 mg per gram of particulate substance.
The process of the invention may comprise providing a precursor emulsion having a condensable species in the emulsion droplets, wherein the emulsion droplets comprise the discontinuous phase of the emulsion. The emulsion may be a w/o microemulsion, so that the condensable species is located in the aqueous emulsion droplets (“water pools”). The emulsion may be made by:
The hydrolysable species may be added to an emulsion, and may infuse into the emulsion droplets. Commonly a material in an emulsion may partition between the two phases of the emulsion, the partitioning being dependent on the relative affinities of the material for the two phases. Thus a highly hydrophilic material will be predominantly located in the aqueous phase, whereas a highly hydrophobic material will be predominantly in the hydrophobic phase. If the material partitions into the aqueous phase, and reacts there, then further material may partition into the aqueous phase. Addition of a hydrolysable species to the continuous (hydrophobic) phase may lead to partitioning of the hydrolysable species into the aqueous phase (the emulsion droplets), where conditions may pertain which promote hydrolysis of the hydrolysable species to form the condensable species, and subsequent formation of nuclei. The formation of nuclei may comprise at least partial condensation of the condensable species. The addition of the condensable species may or may not be accompanied by at least some agitation or swirling. Brownian motion may provide sufficient energy for mixing without externally applied mixing.
The process of the present invention may comprise hydrolysing the hydrolysable species and condensation of the resulting condensable species to form nuclei within the emulsion droplets. If the hydrolysable species is an alkoxysilane, and the emulsion droplets comprise an acidic fluoride solution, then the step may comprise allowing sufficient time for the alkoxysilane to be hydrolysed and for the resulting condensable species to condense to form nuclei.
Alternatively, nuclei and/or primary particles may be formed under basic conditions and then acidified, and the resulting emulsion may be destabilised to form the particulate substance.
Alternatively the nuclei and/or primary particles may be preformed. For example the nuclei may be particles of colloidal or fumed silica, of some other colloidal material or may be some other particles of appropriate size. The condensable species may be capable of forming the particles of the particulate substance from the nuclei, for example by agglomerating the nuclei by condensing in the presence of the nuclei. The condensable species may be compatible with the nuclei and may be capable of reacting with the nuclei.
In one embodiment of the invention, the process of providing the emulsion comprises the steps of:
The basic emulsion may be a water in oil emulsion and may be a microemulsion. It may comprise a surfactant, e.g. NP9, and may additionally comprise a cosurfactant, e.g. 1-pentanol. The continuous liquid may be a hydrocarbon e.g. cyclohexane. The first and second hydrolysable species may comprise alkoxysilanes, for example tetraalkoxysilanes, as described elsewhere herein. They may be the same or may be different. The pH of the basic emulsion may be between about 8 and 13, or between about 8 and 10, 8 and 9, 9 and 13, 11 and 13 or 9 and 11, and may be about 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5 or 13 or may be greater than 13. After addition of the first hydrolysable species, the emulsion may be aged for sufficient time to at least partially hydrolysing the first hydrolysable species within the emulsion droplets to form the condensable species, and to form nuclei, for example primary particles. The time of ageing may be between about 5 and 100 hours, or between about 5 and 80, 5 and 60, 5 and 40, 5 and 20, and 100, 20 and 100, 50 and 100, 10 and 50, 20 and 5 or 40 and 50 hours, and may be about 6, 12, 18, 24, 30, 36, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 60, 66, 72, 78, 84, 92 or 96 hours. The time may depend on the temperature of ageing, which may be between about 15 and 95° C. or some other temperature, for example room temperature or ambient temperature. The temperature may be for example between about 20 and 80, 50 and 80, 10 and 50 or 30 and 70° C., and may be about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90° C. After acidification of the emulsion, the acidified emulsion may be stirred or aged for at least about 1 hour, or at least about 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours, for example between about 1 and 10 hours or between about 1 and 8, 1 and 6, 1 and 4, 2 and 10, 5 and 10, 2 and 8 or 4 and 6 hours, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. The ageing may be at between about 30 and 90° C., or between about 30 and 70, 30 and 50, 50 and 90, 70 and 90, 40 and 80 or 30 and 70° C., for example about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90° C. The addition of the second hydrolysable species may be conducted during or ageing this latter step of stirring or ageing. Before or concurrently with addition of the second hydrolysable species, a surfactant, optionally the same as the surfactant used in making the basic emulsion, together with a solvent and optionally a cosurfactant (which may be the same as or different to that used in the basic emulsion), may be added. The solvent may be miscible with, and may be the same as, the continuous liquid phase. The ratios of solvent, surfactant and cosurfactant (if present) may be the same as or different to the ratios in the basic emulsion. After addition of the second hydrolysable species, the acidified emulsion may be aged for sufficient time to at least partially hydrolyse the second hydrolysable species. This may be at least about 24 hours, or at least about 30, 36, 42, 48, 54 or 60 hours, may be between about 24 and 60 hours, or between about 24 and 48, 48 and 60, 36 and 54 or 40 and 50 hours, and may be about 30, 36, 42, 48, 54 or 60 hours.
Following the step of forming the nuclei, the processes of the invention comprise the step of combining a coalescing liquid with the emulsion to form the particulate substance. The step of combining may comprise adding the emulsion to a coalescing liquid, or may comprise adding the coalescing liquid to the emulsion. The adding may comprising dropping, pouring or otherwise adding, and may be accompanied by agitation, swirling, mixing, stirring etc. and may be accomplished rapidly or slowly. The addition may be at a rate of between about 1 and 1000 ml/min, or between about 1 and 500, 1 and 200, 1 and 100, 1 and 50, 100 and 1000, 500 and 1000, 10 and 500 or 100 and 500 ml/minute, and may be at a rate of about 1, 2, 5, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 ml/minute, or may be at some other rate. Following the addition, further time may be allowed for coalescence of the nuclei to proceed. The further time may be up to about 10 hours, or up to about 5, 2 or 1 hour, or up to about 30, 20, 10, 5, 2, 1 or 0.5 minutes, and may be about 0, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40 or 50 minutes, or about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. The coalescing liquid may be a liquid that is miscible with both the continuous phase and the emulsion droplets. It may comprise a polar liquid, and may be for example acetone or ethanol. It may comprise a mixture of liquids. It may for example comprise a mixture of a destabilising liquid, such as a polar liquid, and a non-polar liquid. The non-polar liquid may be the same as the continuous phase of the emulsion, or it may be different. For example the coalescing liquid may be a mixture of acetone and cyclohexane or a mixture of ethanol and cyclohexane. The ratio of destabilising liquid to non-polar liquid may be between about 1:3 and 3:1 w/w or v/v, and may be between about 1:3 and 2:1, 1:3 and 1:1, 1:3 and 1:2, 1:2 and 3:1, 1:1 and 3:1 or 2:1 and 3:1, and may be about 3:1, 2:1, 1:1, 1:2 or, 1:3 w/w or v/v. The amount of coalescing liquid may be sufficient to cause formation of the particulate substance. It may be sufficient to cause coalescence of the nuclei. The coalescing liquid may be in a quantity that does not lead to formation of a gel. The amount of coalescing liquid may be between about 2 and 6 ml per ml of emulsion, and may be between about 3 and 5 or 2 and 4 or 4 and 6 ml per ml of emulsion, and may be about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6 ml per ml of emulsion, and may be some other amount depending on the nature of the continuous phase and the coalescing liquid.
The process may also comprise one or more of the steps of:
The separating may comprise one or more of settling, centrifuging, decanting, ultracentrifuging, filtering, microfiltering or some other suitable separation process. The washing may comprise exposing the particulate substance to a suitable solvent, optionally agitating the solvent, and then separating the solvent from the particulate substance. It may comprise suspending the particulate substance in the solvent, or it may comprise passing the solvent through the particulate substance. The washing may at least partially remove surfactant from the particulate substance. The solvent may be a solvent for the surfactant. The solvent for particle washing may depend on the solubility and molecular size of dopant. If the dopant is highly soluble in polar solvents such as acetone or less polar solvents such as chloroform, non-polar solvents may used for particle washing. As the HLB of the surfactant is commonly around 10, the surfactant is generally soluble in either polar or non-polar solvents. The drying may comprise exposing the particulate substance to a gas, for example air, nitrogen, carbon dioxide or mixtures thereof. The gas may be a dry gas, and may be dried before use. The exposing may comprise passing the gas over or through the particulate substance, and may comprise sucking the gas through the particulate substance. Alternatively the drying may comprise exposing the particulate substance to a partial vacuum. The partial vacuum may have an absolute pressure of less than about 0.5 bar, or less than about 0.2, 0.1, 0.05, 0.01, 0.005 or 0.001 bar, and may have an absolute pressure of about 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.02, 0.01, 0.005, 0.002 or 0.001 bar. The drying may be conducted at a temperature below the degradation temperature of the releasable substance, and may therefore depend on the nature of the releasable substance. The drying may be conducted for example at less than about 100° C., or less than about 90, 80, 70, 60, 50, 40, 30 or 20° C., and may be conducted at about 20, 30, 40, 50, 60, 70, 80, 90 or 100° C. or at some other suitable temperature.
Releasable substance that has not been incorporated into particles may be recovered from the coalescing liquid and reused if required.
An example of a process for producing the particulate substance is summarized in
The emulsions parameters (surfactant/water ratio, TMOS/water ratio) as well as the nature, the volume and the way the coalescing liquid is added may control the final particle size and distribution.
The growth of solid precursor particles within the droplets of an emulsion is governed by the same mechanisms as in a colloidal suspension: nucleation and growth by either ripening aggregation or coagulation.
As in colloidal suspension, nucleation in water-in-oil emulsions takes place when the concentration of condensable species present in the droplets (the discontinuous phase) exceeds the nucleation threshold [Cn]. The only difference between this and classical colloidal suspension is that, in emulsions, the solution is compartmentalised in small droplets. Thus, the concentration of condensable species may change from water pool to water pool (ie from droplet to droplet). In other words, in emulsions the number of molecules of condensable species increases with their average supersaturation until the average concentration per pool is larger than [Cn].
Based on these considerations, the number of nuclei increases with increasing concentration of condensable species and decreases with the number of droplets. Since the number of molecules of condensable species is proportional to the amount of free water available per micelle, the number of nuclei increases with increasing free water content. In other words the number of nuclei increases with the water to surfactant ratio. (Free water may be contrasted with bound water i.e. the water solvating the surfactant hydrophilic head and which does not participate in the hydrolysis of the hydrolysable species, e.g. silicon alkoxide. The amount of free water depends on the nature of the surfactant (size and nature of the hydrophilic head) and the water to surfactant ratio.)
Another specific characteristic of emulsions is that the emulsion droplets are capable of exchanging their cores during collision. An increase in the rate of inter-micellar exchange can induce a redistribution of the condensable species before the supersaturation reaches the nucleation threshold. Thus increasing the collision of micelles can lead to a decrease in nucleation rate.
As with aqueous colloidal suspensions, growth of solid particles in emulsions follows nucleation. To a first approximation, the water pools (droplets of discontinuous phase) may be viewed as micro-reactors in which particle growth takes place by addition of condensable species to the nuclei. Thus, the larger the number of nuclei, the smaller the nuclei. Similarly, for a fixed number of nuclei, the higher the supersaturation in the water pool, the larger the resulting nuclei.
As mentioned above, emulsions are dynamic systems and the emulsion droplets collide constantly with one another, exchanging the content of their aqueous core in the process. Growth of the droplets may then take place by collision of pools containing solid nuclei or nuclei with under-saturated pools containing only condensable species. In other words, growth can take place by consumption of unsaturated micelles (in a process akin to coalescence). Thus the final particle size of the droplets increases with the number of collisions or micellar exchanges.
Base catalysis of sol-gel reactions promotes both hydrolysis/condensation and, more is importantly, dissolution. When combined with micro-emulsions, base catalysed sol-gel chemistry leads to a rapid nucleation due to the high hydrolysis rate. The rapid condensation leads to a rapid consumption of all the condensable species inside the micelles and further growth takes place by ageing and scavenging of other micellar pools. High dissolution rates ensure the production of spherical particles by ripening of the aggregates formed during the collision of droplets containing nuclei (see
Acid catalysis promotes hydrolysis but hinders both condensation and dissolution (except at very low pH i.e. pH<0). Consequently, for the emulsion synthesis of silica particles using acid catalysis, many nuclei are generated but very little growth is observed. In fact, no solid particles were observed in acid catalysed emulsions even after 48 h ageing. A condensation catalyst such as fluoride must be introduced to produce solid particles. Even in this case, nuclei remain very small (i.e. about 10 nm) as revealed by direct TEM analysis of the emulsions prior to destabilisation (see
The basis of the present invention is the destabilisation of an emulsion using a polar liquid as a coalescing liquid, or as a component thereof, to induce coalescence of the aqueous emulsion droplets and thus initiate growth and aggregation of the nuclei.
Addition of water (a highly polar solvent) is known to swell emulsion droplets and to produce larger particles. This works up to a certain amount of water after which multiphase domains are formed (e.g. water and emulsion phase). In the case of acetone, SANS experiments reveal that addition of a small quantity of acetone leads to a significant swelling of the droplet cores (see Table 1). This suggests that acetone does induce coalescence of the small droplets into larger droplets. In contrast to water, addition of a larger quantity of acetone, does not lead to the formation of two distinct phases (e.g. water and reverse emulsion). In fact what is observed when the destabilisation is successful is the slow precipitation of particles out of this single-phase (i.e. acetone+cyclohexane+water+surfactant) system.
The destabilisation of the emulsion system appears to go through the following steps:
It is important to note that this destabilisation process is also a kinetic process with a competition between the emulsion droplet coalescence rate (and the resulting formation of open channels) with the sot-gel reaction rate (i.e. silica production rate).
Using the mechanistic model outlined above, it is possible to rationalise the influence of the different processing parameters on the destabilisation of the emulsion and the final particle morphology.
After addition of the coalescing liquid, four different types of behaviour may be observed:
Increasing the surfactant concentration is known to increase the stability of micro-emulsions. Correspondingly, for a fixed quantity of coalescing liquid (e.g. acetone), emulsions with increasing surfactant concentrations are more difficult to destabilise and result in slow gelation of nuclei (see pictures c and d in
As expected, an increase in the amount of silicon precursor leads to an increase in particle size. At acid pH the rate of hydrolysis of the alkoxide is high and the rate of condensation is low, which means that the number of monomer molecules is high and the number of nuclei remains low. As the amount of silicon precursor is increased, the number of hydrolysed precursor molecules increases significantly more than the number of new nuclei. When destabilisation occurs this leads to a higher proportion of monomer to nuclei and thus larger particles.
Apart from the case of Triton X-100, which forms particles prior to destabilisation, two surfactants that produced submicron particles were NP5 and NP6, which have intermediate HLB's around 10. An HLB of 10 indicates a strong amphiphile nature (i.e. balanced hydrophilicity and hydrophobicity) and denotes a medium strength molecular interaction between the surfactant polar head and water from the droplet. Such a medium interaction leads at high water content to the coalescence of the droplets and the formation of open channels as surfactant with weaker or stronger interaction with water remain as dispersed droplets. This reinforces the importance of the formation of water channels during destabilisation, to provide a path for the unreacted hydrolysed precursor to migrate from their original micellar “prison” towards nuclei and thus provide materials for particle growth. For HLB<10, no particles were detected suggesting that the addition of acetone does not succeed in destabilising the emulsion. In contrast, for HLB>10, destabilisation is often too fast and the nuclei aggregate to form a gel before growth can occur, although Tween 21 (HLB 13.3) has been found to be usable. Thus an HLB of between about 10 and about 14 may be a suitable guideline for the surfactant. It also appears that surfactants having between about 4 and 6 oxyethylene units in their polar head group may be suitable for use in the invention.
When the concentration of surfactant is increased, a similar trend is observed, with the difference that destabilisation by Brij 30 produces a colloidal gel with fused particles, suggesting a partial destabilisation and coalescence of water droplets. The important reduction in the average particle size obtained with NP6 may be explained by an increase in emulsion stability due to an increase in the surfactant concentration.
TMOS hydrolyses more rapidly than TEOS and consequently for an identical concentration of alkoxide inside a droplet, the TMOS system may reach the nucleation threshold faster, thus leading to production of more nuclei. This larger number of nuclei in the TMOS emulsion system leads after destabilisation to the production of smaller particles (since more nuclei for the same amount of hydrolysed monomer provides for more and smaller particles).
A wide range of organic liquids with different dielectric constants and polarities were used, either by themselves or in combination with cyclohexane, to destabilise an NP5 emulsion and produce submicron particles (see Table 2, which shows the liquids according to their dielectric constant). The effect on the emulsion system and the final morphology differs drastically depending on their polarity and their respective miscibility in both water and cyclohexane.
They may be classified into three different categories:
As described above, the key to a successful destabilisation is the formation of a single phase system which will allow the content of the droplet core to participate freely in colloidal growth and consequent production of submicron particles. Phenomenologically, this may be done either by a substantial swelling of the polar phase channel and the formation of a bi-continuous phase or simply by destruction of the emulsion into a classical solution. In other words, to produce submicron particles, the addition of the coalescing liquid should form a single phase (or bi-continuous) system. This requires the coalescing liquid to be miscible in both water and cyclohexane.
It is important to stress that this miscibility requirement is necessary but not sufficient. The amount used, as well as the dielectric constant of the destabilising liquid (which is mixed with a non-polar liquid to generate the coalescing liquid) is important for control of the formation of submicron particles. Two destabilising liquids (ethanol and acetone) have been successfully used to produce submicron particles, although seven different liquids (see Table 2) were tested and found to produce a single-phase emulsion after addition. This underlines the fact that the miscibility in both the continuous phase and water, as well as the production of a single phase system after destabilisation, is not sufficient to ensure the production of submicron particles.
The inventors hypothesise that the polarity or dielectric constant of the destabilising liquid plays a key role in the successful destabilisation of the emulsion and production of the submicron particles.
Phenomenologically, it appears that the water phase channels formed during the destabilisation are not large enough to permit the content of micelles to flow freely and participate in particle growth. In other words, as the destabilising power decreases, the mobility of the hydrolysed precursor become lower than their condensation rate and thus gelation takes place rather than particle growth, leading to the gelation of the content of the water channel or the bi-continuous phase. This hypothesis is further confirmed by the very open structure of the gel observed in TEM (
On the other hand, when acetone is replaced by ethanol, the morphology progressively evolves towards dense aggregates of submicron and micron-size particles. This trend may be explained by an increase in the destabilisation speed and a further reduction of the condensation rate by a re-esterification of the hydrolysed silicates in the presence of excess ethanol. This decrease in condensation rate might lead to the production of “soft” non-fully condensed submicron particles that aggregate due to Brownian motion into dense clumps.
Following the concept of “destabilising power” discussed above, it appears that the amount of destabilising power (i.e. a combination of the volume and dielectric constant) needs to be high enough to fully destabilise the emulsion, thus freeing the contents (i.e. hydrolysed alkoxides) of the emulsion droplet to allow them to participate in particle growth.
If insufficient coalescing liquid is introduced, a gel is gradually formed by collision of nuclei. If too much coalescing liquid is used then the system can form a gel (as in
As can be seen from Table 3 and
It is important to note that the coalescing liquid contained 100 ml of cyclohexane thus shifting the average polarity of the oil phase upon addition.
It is important to note that the surface area of the particles corresponds to the surface of dense silica particles around 6 nm in size. This confirms that surface area is related to the internal surface of the particles and consequently that the submicron particles are highly porous. Furthermore, the proportion of microporous volume decreases with an increase in pH by titration of the droplets prior to destabilisation. It is known that addition of base may promote condensation inside the droplets, thus increasing the number of solid particles. This will, after destabilisation, increase the proportion of mesopores. This provides an example of control of the particles internal structure and thus release kinetic of the encapsulated molecules.
An additional consequence of the destabilisation of the emulsion and colloidal growth by migration of the content of the core through polar phase channels is the low encapsulation efficiency of the releasable substance, for example a drug (see
In summary, submicron particles may be produced by destabilisation of acid catalysed sol-gel emulsions. The resulting particles may be used to encapsulate and release small molecules in a controlled fashion over extended periods of time (up to 6 months). The internal structure of the submicron silica particles, and thus the release rate, may be controlled by initial sol-gel parameters such as pH. The encapsulation efficiency appears to be dependent on the solubility of drug in destabilising mixture.
The conditions necessary to achieve successful destabilisation appear to be:
The process of the present invention is capable of producing sub-micron particles in an acid environment by use of a sol-gel mechanism in a microemulsion. Acid catalysis is important in the production of microporous particles that can encapsulate small molecules such as doxorubicin and release them in a controlled fashion. The present invention uses a novel approach for building sub-micron (i.e. large nano) particles in emulsion. Contrary to earlier work (WO1/62332 Barbé & Bartlett “Controlled release ceramic particles, compositions thereof, processes of preparation and methods of use”) in which it was necessary to screen surfactant/solvent couples to obtain an emulsion with the correct droplet size to generate the desired final particle size, the method of the present invention starts with a stable microemulsion having droplets of about 5 to 10 nm diameter, and then destabilises the microemulsion to produce particles of between about 30 and 100 nm, or about 50 and 500 nm or about 100 and 400 nm.
The particulate substance of the invention having a drug releasably immobilised therein and/or thereon may be used in the treatment of a condition in a mammal. The mammal may be selected from the group consisting of human, non-human primate, equine, murine, bovine, leporine, ovine, caprine, feline and canine. The mammal may be selected from a human, monkey, ape, horse, cattle, sheep, dog, cat, goat, llama, rabbit and a camel, for example. The condition may be a disease. The condition may be for example cancer, diabetes, hormonal dysfunction, hypertension, pain (for example pain treatable by morphine and/or opiates), or asthma.
Doxorubicin can be encapsulated inside the sub-micron particles and release gradually in PBS (phosphate buffer saline) solution.
The present invention may also be used for controlled release of other substances, such as fluorescent dyes, radiopharmaceuticals, enzymes, catalysts, hormones, biocides and/or other substances. Applications may include diagnostics, radiodiagnostics, radiotherapy, biotechnology, bioreactors, imaging etc.
The following general experimental procedure was used in the experiments detailed below for preparing nanoparticles containing doxorubicin, with the variations from this general procedure detailed for the individual experiments:
Particles were synthesized using the following experimental parameters: water (pH1) 60 mmol, F− 0.06 mmol, TMOS 6 mmol, cyclohexane (at step 1) 50 mL, ageing 48 h and destabilisation with a mixture of 100 ml acetone/100 ml of cyclohexane. The surfactant (NP-5) concentration at step 1 was varied from 0.2 mol/L to 0.8 mol/L. TEM micrographs of the resulting particles are shown in
Result: The particle size decreased with increasing surfactant concentration. Above 0.4 mol/L, no coalescence was observed.
Particles were synthesized using the following experimental parameters:
Surfactant NP-5 0.4 mol/L, F−/TMOS molar ratio 0.01, water at pH1, cyclohexane (at step 1) 50 mL, ageing 48 h and destabilisation with a mixture of 100 ml acetone/100 ml of cyclohexane. The TMOS concentration at step 3 was increase from 4 mmol to 12 mmol while keeping the water to TMOS molar ratio at 10. TEM micrographs of the resulting particles are shown in
Result: The particle size was found to increase with TMOS content. The coalescence/fusing of the particles at [TMOS]=12 mmol is significant.
The following surfactants were used in place of NP-5 at step 1: (a) Brij 30 (HLB=9), (b) NP-5 (HLB=10), (c) NP-6 (HLB=10.9) (d) Triton X-114 (HLB=12.4) with 1-pentanol 20 mmol (e) NP-9 (HLB=13) with 1-pentanol 20 mmol (f) Triton X-100 (HLB=13.5) with 1-pentanol 20 mmol. Particles were synthesized using the following experimental parameters: Surfactant 0.2 mol/L, water (pH1) 60 mmol, F− 0.06 mmol, TMOS 6 mmol, in 50 mL cyclohexane (at step 1), ageing 48 h and destabilisation with a mixture of 100 ml acetone/100 ml of cyclohexane. TEM micrographs of the resulting particles are shown in
Result: The only systems to form submicron sized spheres were those with NP-5 and NP-6. No destabilisation took place in sample (a). The “coalesced droplet” morphology of sample (f) was the results of the unstable nature of the microemulsion used in its synthesis.
The same experiments were conducted as at low concentration (above), increasing the surfactant concentration at step 1 to 0.4 mol/L. TEM micrographs of the corresponding particles are shown in
Result: The only systems to form submicron spheres at this surfactant concentration were those with NP-5. All the others with the exception of those with NP-6 produced particles of diameter less than 20 nm, suggesting that the micro-emulsion are not destabilised, and the particles do not coalesce, on addition of acetone. The slight growth observed for systems with NP-6 suggests a reduced destabilisation.
Particles were synthesized using the following experimental parameters:
Water (pH1) 60 mmol, F− 0.06 mmol, TMOS 0.6 mmol, cyclohexane (at step 1) 50 mL, ageing 48 h and destabilisation with a mixture of 100 ml acetone/100 ml of cyclohexane. The surfactant at step 1 was: (a) NP-5 0.2 mol/L; (b) NP-5 0.4 mol/L; (c) NP-5 0.2 mol/L, 1-pentanol 0.2 mol/L. TEM micrographs of the corresponding particles are shown in
Result: No visible improvement was caused by adding a co-surfactant (i.e. surfactant) to the emulsion. Increasing the surfactant concentration (experiment 4(b)) was more effective in narrowing the particle size distribution.
Destabilised using 100 mL Acetone/100 mL Various Non-Polar Solvents:
Particles were synthesized using the following experimental parameters: NP-5 10 mmol (0.2 mol/L), water (pH1) 60 mmol, F− 0.06 mmol, TMOS 6 mmol, a non-polar solvent (at step 1) 50 mL, ageing 48 h and destabilisation with a mixture of 100 ml acetone/100 ml of the non-polar solvent. Several solvents were used as the continuous phase for forming the microemulsions: (a) petroleum benzine, 1-hexanol 20 mmol; (b) hexane, 1-hexanol 20 mmol; (c) octane, 1-hexanol 20 mmol; (d) decane, 1-hexanol 20 mmol; (e) dodecane, 1-hexanol 40 mmol; (f) toluene. Hexanol was added as a cosurfactant in order to obtained stable micro-emulsions. TEM micrographs of the corresponding particles are shown in
Result: Some submicron particles were obtained for experiments 6d and 6e, suggesting that a partial controlled destabilisation (ie particle coalescence) was achieved for emulsions synthesized in decane and dodecane.
The same emulsions as described in experiment 6 were prepared and destabilised using a 50 mL ethanol/100 mL cyclohexane mixture at step 5. TEM micrographs of the corresponding particles are shown in
Result: a) petroleum benzine/1-hexanol 20 mml and b) hexane/1-hexanol 20 mmol systems formed submicron particles. Coalescence of the octane/1-hexanol 20 mmol (experiment c) resulted in a gel and no coalescence took place in decane or dodecane (experiments d and e).
The following surfactants were used in place of NP-5: (a) NP-5, (b) Triton X-114 and (c) NP-9. Particles were synthesized using the following experimental parameters: surfactant 20 mmol (0.2 mol/L), water (pH1) 40 mmol, F− 0.06 mmol, TMOS 6 mmol, toluene 100 mL (at step 1 in place of cyclohexane), ageing 24 h and destabilisation with a mixture of 100 ml acetone/100 ml of cyclohexane. TEM micrographs of the corresponding particles are shown in
Result: During the synthesis, silica particles were isolated from the emulsions with time and they eventually formed a gel with average particle sizes between 10-50 nm.
Particles where synthesized using the following experimental parameters: NP-5 10 mmol (0.2 mol/L), water (pH1) 60 mmol, F− 0.06 mmol, TMOS 6 mmol and cyclohexane 50 mL. This emulsion was aged for: (a) 24 h, (b) 48 h, (c) 120 h, (d) 168 h, before being destabilised with a mixture of 100 ml acetone/100 ml of cyclohexane. TEM micrographs of the corresponding particles are shown in
Result: The ageing time had no significant influence on the final particle size.
Particles were synthesized using the following experimental parameters: NP-5 10 mmol (0.2 mol/L), TMOS or TEOS 6 mmol, water (pH1) 60 mmol, F− 0.06 mmol, cyclohexane (at step 1) 50 mL, ageing 48 h and destabilisation with a mixture of 100 ml acetone/100 ml of cyclohexane. TEM micrographs of the particles before and after destabilisation are shown in
Result: A comparison of the particles before and after destabilisation clearly shows that addition of acetone, which on the macroscopic scale results in a crashing of the stable micro-emulsions into a white precipitate, induces on the microscopic scale substantial particle growth. Final particles exhibit a slightly larger particle size when TEOS is used as the hydrolysable species.
Particles were synthesized using the following experimental parameters: NP-5 10 mmol (0.2 mol/L), water (pH1) 60 mmol, F− 0.06 mmol, TMOS 6 mmol, cyclohexane (at step 1) 50 mL, ageing 48 h.
The destabilisation of step 5 was then conducted in different ways.
(a) by pouring 50 mL acetone into the microemulsion; (b) by pouring the microemulsion into 50 mL acetone; (c) by pouring the microemulsion into 100 mL acetone; (d) by pouring the microemulsion into a mixture of 100 mL acetone and 100 mL cyclohexane. TEM micrographs of the corresponding particles are shown in
Result: Although submicron particles are obtained in all cases, pouring the microemulsion into a diluted solution of acetone (experiment 11-1d) appears to have produced the most homogenous and less aggregated samples.
by pouring the microemulsion: (a) into a mixture of 50 mL acetone and 50 mL cyclohexane; (b) into a mixture of 100 mL acetone and 100 mL cyclohexane; (c) into a mixture of 150 mL acetone and 150 mL cyclohexane; (d) into a mixture of 200 mL acetone and 200 mL cyclohexane. TEM micrographs of the corresponding particles are shown in
Result: Although the destabilisation with a mixture of 50 ml of acetone/50 ml of cyclohexane (experiment 11-2a) produced submicron particles, the particles were largely aggregated and fused together. Increasing the volume of the destabilising mixture leads to a lower aggregation but an increase in polydispersity.
Particles were destabilised using a 100 ml acetone/100 ml cyclohexane mixture. The destabilisation (step 5) was performed by pouring the microemulsion in: a) a 1 litre beaker stirred with a rod shape magnetic stir-bar; b) a 250 ml beaker stirred with a cross-shape magnetic stir-bar; c) a 250 ml beaker stirred with a rod shape stir-bar; d) in a 250 ml beaker ultrasonicated for 5 minutes; and e) with NaCl (0.2M) added. In e), 0.135 mL 1 mol/L NaCl solution was added into the microemulsion, which was then shaken until clear, and then destabilised by pouring into a stirred mixture of acetone and cyclohexane in a 250 ml beaker stirred with a rod-shaped stir-bar stirring at moderate stirring speed. TEM micrographs of the corresponding particles are presented in
Result: The container volume, magnetic stir-bar shape, ultrasonication, or salt addition had no significant effect on final particle size.
Particles were synthesized using the following experimental parameters: NP-5 10 mmol (0.2 mol/L), water (pH1) 60 mmol, F− 0.06 mmol, TMOS 6 mmol, cyclohexane (at step 1) 50 mL, ageing 48 h. The pH of the water droplets was adjusted from 1.5 to 7 by addition of base (1M aqueous NaOH solution), before destabilisation in a mixture of 100 ml acetone/100 ml cyclohexane. TEM micrographs of the corresponding particles are shown in
Result: No significant effect of the pH on the final particle morphology was observed.
Particles were synthesized using the following experimental parameters: NP-5 10 mmol (0.2 mol/L), water (pH1) 60 mmol, F− 0.06 mmol, TMOS 6 mmol, cyclohexane (at step 1) 50 mL and ageing 48 h. The microemulsion was then destabilised at step 5 using a mixture of 100 mL cyclohexane and different amounts of acetone: (a) 10 mL, (b) 20 mL, (c) 50 mL, (d) 100 mL and (e) 200 mL. TEM micrographs of the corresponding particles are shown in
Result: In experiments 10-1a and 10-1b (10 or 20 ml acetone), no immediate precipitate was observed, however a gel gradually formed with time. In experiment 10-1c (50 ml acetone), the gel formed immediately. In experiments 10-1d and 10-1d (100 ml or 200 ml acetone), spherical submicron particles were formed.
Particles were synthesized using the following experimental parameters: NP-5 10 mmol (0.2 mol/L), water (pH1) 60 mmol, F− 0.06 mmol, TMOS 6 mmol, cyclohexane (at step 1) 50 mL, ageing 48 h. The microemulsion was then destabilised at step 5 using a mixture of 100 mL cyclohexane and different amount of ethanol: (a) 5 mL; (b) 10 mL; (c) 20 mL; (d) 35 mL; (e) 50 mL; (f) 80 mL; (g) 100 mL. TEM micrographs of the corresponding particles are shown in
Result: When the quantity of ethanol was 5 or 10 ml (experiments 10-2a bd 10-2b), no immediate precipitate was observed, however a gel gradually formed with time. With 20 ml ethanol (experiment 10-2c) a gel formed immediately after pouring the emulsion into the cyclohexane/ethanol mixture. From 35 ml to 80 ml ethanol (experiments 10-2d to 10-2f), spherical submicron particles were formed although aggregation increased with the amount of ethanol, giving rise to a mixture of particles and gel for the sample destabilised with 80 ml of ethanol (experiment 10-2f). A further increase in the quantity of ethanol (experiment 10-2g) led to gelation.
Particles were synthesized using the following experimental parameters: NP-5 10 mmol (0.2 mol/L), water (pH1) 60 mmol, F− 0.06 mmol, TMOS 6 mmol, cyclohexane (at step 1) 50: mL, ageing 48 h. The microemulsion was then destabilised at step 5 using a mixture of: a) 100 mL cyclohexane, 80 mL acetone, 20 mL ethanol; b) 100 mL cyclohexane, 60 mL acetone, 40 mL ethanol; c) 100 mL cyclohexane, 40 mL acetone, 60 mL ethanol; d) 100 mL cyclohexane, 20 mL acetone, 80 mL ethanol. TEM micrographs of the corresponding particles are presented in
Result: Submicron particles were formed with a mixture cyclohexane, acetone and ethanol. The aggregation increases with increasing proportion of ethanol, giving rise to a gel structure for more than 20/80 ethanol to acetone volume ratio (experiments 10-3b to 10-3d).
Particles were synthesized using the following experimental parameters: NP-5 10 mmol (0.2 mol/L), water (pH1) 60 mmol, F− 0.06 mmol, TMOS 6 mmol, cyclohexane (at step 1) 50 mL, ageing 48 h. The micro emulsion was then destabilised at step 5 using a mixture of 100 mL cyclohexane and different amount of the following solvents:
(a) iso-propanol: 10 mL, 25 mL, 50 mL, 100 mL
(b) 1-propanol: 25 mL, 50 mL, 100 mL, 200 mL
(c) Methylethyl ketone: 25 mL, 50 mL, 100 mL, 200 mL
(d) chloroform: 25 mL, 50 mL, 100 mL.
(e) toluene: 100 mL
(f) benzene: 100 mL
(g) tetrahydrofuran (THF): 100 mL
(h) dimethylformamide (DMF): 100 mL
(i) pyridine: 100 mL
(j) 1-butanol: 100 mL
(k) acetonitrile: 2 mL, 5 mL, 10 mL, 20 mL
(l) methanol: 2 mL, 5 mL, 10 mL, 20 mL
(m) dimethylsulfoxide (DMSO): 2 mL, 5 mL, 10 mL, 20 mL
(n) tert-butanol: 100 mL
(o) 1-pentanol: 100 mL
(p) 1-hexanol: 100 mL
(q) dichloromethane: 100 mL
TEM micrographs of some of the corresponding particles are shown in
Results: Destabilisation using isopropanol and n-propanol led to the formation of gel with time. Destabilisation did not take place when acetonitrile or methanol was used: no particles were formed. Chloroform led to the formation of coalesced gel structure, and use of toluene, benzene or THF led to the production of fine gel structures.
Particles were synthesized using the following experimental parameters: 5 mmol NP-5, cyclohexane (at step 1) 25 mL, pH=1. HNO3 with water 30 mmol, F− 0.03 mmol, TMOS 3 mmol and aged 48 hrs. The micro emulsion was then destabilised using a mixture of 50 mL cyclohexane and different amount of the following solvents:
To illustrate, the morphology of LNK 752 to LNK 755 are represented in
In normal phosphate buffer, doxorubicin decomposes exponentially during the first ten days with half decay (T1/2) about 4.5 days, as shown in
A 0.2 M surfactant solution was prepared in 50 mL cyclohexane. 1.08 mL nitric acid 0.1M containing 0.06 mmol NaF was added to the surfactant solution and the resulting mixture was stirred for 20 minutes to produce a microemulsion. 6 mmol TMOS was then added into the above system which was stirred for 48 hours. The emulsion was then poured into a stirring mixture made of 100 mL acetone and 100 mL cyclohexane and was left stirring for 10 more minutes. After sedimentation, the particles were separated from organic phase and washed three times with acetone. The particles were then resuspended in about 10 mL of NaCl aqueous solution, washed further by decantation with chloroform, and finally freeze dried. The particles were found to be distributed homogeneously in NaCl matrix with silica and sodium chloride weight ratio at 85%. The particles could also be used directly in the form of an aqueous suspension after washing prior to freeze-drying. The process is shown schematically in
The particles were synthesised according to the typical procedure (see
Particles were synthesised according to typical procedure using a TMOS/TEOS mixture as silica precursors in place of a single alkoxide. The corresponding TEM micrographs are presented in
Two microemulsions were prepared according to the typical synthesis conditions. Emulsion-A contains dye-A and emulsion-B contains dye-b. Immediately before destabilisation, the two emulsions were briefly mixed and stirred for 10 minutes. (See
6) Destabilisation of an Emulsion Containing Both Nanoparticles Synthesised Using Base Catalysis (i.e. Seeds) and Monomer Hydrolysed in Acid Media
The silica nanoparticles produced using base catalysis were synthesised as follows. NP-9 (6 mmol), 1-pentanol (6 mmol) and cyclohexane (30 mL) were mixed together. 0.648 mL of aqueous ammonia NH4OH (1.333M) representing an equivalent 36 mmol water was combined to the previous solution to form a micro-emulsion. TEOS (1.2 mmol) was then added into the microemulsion and the system was aged for 48 hours. An acidic aqueous phase was then added to the micro-emulsion now containing 50 nm silica particles and the system was further stirred at 60° C. for 5 hours. NP-9 (12 mmol), 1-pentanol (12 mmol) and cyclohexane (60 mL) were then added, followed by addition of silicon alkoxide. The mixture was further aged for 48 hours and then destabilised using a mixture of acetone and cyclohexane. The corresponding TEM image are shown in
The original seed synthesised in base may also be made of hybrid materials (i.e. using a mixture of ORMOSILs and TMOS) A typical synthesis procedure for these seeds involved mixing of NP-5 (5 mmol) and cyclohexane (25 mL) and 0.54 mL 1.333M NH4OH followed by the addition of TMOS or a mixed silica precursor and ageing for 24 hours. The corresponding TEM micrographs of the seed particles are shown in
7) Destabilisation of a Two Emulsions System in which One Contains Nanoparticles (Synthesized in Base) and the Other One Contains Oligomers (Synthesized in Acid):
The synthetic procedure follows the scheme in
The nitrogen adsorption-desorption isotherms for two samples synthesised at two different pH's have been discussed above. In
The particle size distribution was measured by dynamic light scattering (DLS) using a Malvern HPPS instrument. The silica particle suspension was sonicated for 10 minutes in water bath. 0.5 mL of the resulting suspension was diluted by 5 mL of pH 9 NaOH solution and filtered through a 0.8 μm filter into a glass cuvette. Measurements were conducted at 25° C. The resulting particle size distribution is shown in
Silica nanoparticles containing doxorubicin were prepared according to the following procedure. 20 mmol of NP5 was mixed with 100 mL of cyclohexane, 2.16 mL of HNO3 at pH=1, 0.12 mmol of F− and 1.5 mg of doxorubicin (Doxorubicin-HCl purchased from Australian Pharmaceutical Ingredients Pty. Ltd). 12 mmol of TMOS was then added and the micro-emulsion was aged for 3 days. Then 0.432 mL of NaOH 0.5 M was added to bring the water pools to pH=7.
After the addition of the base, the samples were stirred for 2 more hours prior to destabilisation with 200 mL acetone and 150 mL cyclohexane. After settling of the particles at the bottom of the beaker they were washed with 100 mL acetone three times. Then the particles were resuspended in a solution composed of 4.80 g of NaCl dissolved in 20 mL of deionised water. The suspension was washed three times by decantation using 100 mL of chloroform each times and freeze-dried.
11) Characterisation of the Doxorubicin Release from the Silica Particles Using HPLC
All solvents used were HPLC grade and filtered through a 0.45 μm filter prior to degassing by sonication. The HPLC system consisted of a Waters 1525 binary HPLC pump, in combination with a Waters 717 plus autosampler, a Waters 2487 Dual λ absorbance detector (480 nm), and a Waters 2475 multi λ fluorescence detector (excitation λ: 363 nm, emission λ: 550 nm). The HPLC column used was a Waters Atlantic™ dC-18, 5 μm, 4.6 mm×150 mm column, with Waters Atlantic™ dC-18 guard column attached. The mobile phase consisted of 1% acetic acid in water, and acetonitrile, with a gradient of 5% to 95% acetonitrile over 20 minutes and a flow rate of 0.8 mL/min. Doxorubicin concentrations were determined by integration of the area under the curve and comparison to that of standard doxorubicin samples at 0.4, 0.8, 2, 4, and 10 μg/mL. The minimum acceptable R2 for the standard curve was 0.9. Processing of both absorbance and fluorescence HPLC spectra was performed using Waters Breeze™ software.
11.1) Release at pH<4
The release of doxorubicin was studied at pH<4 (pH ˜3.4) as the compound is most stable at this pH. Release experiments were performed using doxorubicin doped nanoparticles (1 g). The particles were suspended in 1% acetic acid solution in Milli-Q water (30 mL). Samples were incubated at 37° C. with stirring. Aliquots (100 μL) were taken after centrifugation at 5000 rpm for 5 minutes. Samples were analysed by HPLC as previously described.
11.2) Release at pH=7.4
Studies were undertaken of the release of doxorubicin at physiological pH (7.4). Release studies were performed in 0.02 M phosphate buffered saline (PBS) at 37° C. according to the same procedure used at pH<4.
The decomposition rate of doxorubicin at physiological pH (pH 7.4) versus its decomposition rate at pH<4 makes analysis of the system difficult.
To overcome this problem an alternative experimental procedure was developed. As opposed the previous methods where an aliquot of the liquid was isolated from the total volume, the new procedure involves complete removal of the supernatant for each time point. Doxorubicin doped nanoparticles (35 mg) were suspended in PBS buffer (1 mL) and shaken at room temperature. At the required time points (1 hour, 1, 2, 6 and 9 days) the samples were centrifuged (12,000 rpm, 30 minutes) and the supernatant removed. The particles were resuspended in fresh PBS buffer (1 mL) and agitation continued. The removed supernatant was then analysed using HPLC according the method described above.
Camptothecin comes in two forms: 1) the lactone form with a high anti-cancer efficacy but which is sparingly soluble in water and 2) the carboxylate form which is highly soluble in water but which is clinically inactive. The transformation from one form to the other is pH dependent. When pH is greater than 4, the lactone form is transformed into the hydroxy acid (carboxylate) form, and below pH 4 the reverse reaction occurs.
The present invention relates to production of a practical drug delivery system. In order to achieve a high therapeutic index, it is desirable to encapsulate the hydrophobic lactone form of camptothecin. However the present invention is designed to encapsulate only hydrophilic molecules. To overcome this limitation the inventors designed the following alternative encapsulation procedure:
Camptothecin was dissolved in 0.1 mol/L sodium hydroxide solution with concentration 2 mg/mL. 1.08 mL of the above solution was added into 10 mmol NP-5 mixed with 50 mL cyclohexane to produce a microemulsion with camptothecin in the carboxylate form located in the water pools. A second microemulsion was produced using the typical synthesis process described in paragraph 1 and
The release of camptothecin was analysed using HPLC. The HPLC system consisted of a Waters 1525 binary HPLC pump, in combination with a Waters 717 plus autosampler, and a Waters 2475 multi λ fluorescence detector (excitation λ: 363 nm, emission λ: 550 nm). The HPLC column used was a Waters Atlantic™ dC-18, 5 μm, 4.6 mm×150 mm column, with Waters Atlantic™ dC-18 guard column attached. The mobile phase was isocratic and consisted of 70% 0.075 M ammonium acetate buffer (pH 6.4) and 30% acetonitrile to which tertiary butyl ammonium phosphate (TBAP) was added to a final concentration of 5 mM. The flow rate used was 0.8 mL/min. Camptothecin concentrations were determined by integration of the area under the curve and comparison to that of standard Camptothecin samples at 0.4, 0.8, 2, 4, and 10 μg/mL. The minimum acceptable R2 for the standard curve was 0.9. Processing of HPLC spectra was performed using Waters Breeze™ software.
The initial release studies of Camptothecin were performed using a cumulative release approach. Samples of Camptothecin encapsulated nanoparticles (1 g) were suspended in HPLC mobile phase with no TBAP added (30 mL), and stirred continuously. As the time points required the samples were centrifuged and an aliquot (100 μL) of supernatant isolated. The supernatant was analysed using HPLC to determine the concentration of Camptothecin.
From the results shown in
Release with Aqueous Phase Replacement
With the knowledge that Camptothecin is only sparingly soluble in water, an experiment was conducted in which the aqueous phase was replaced at each time point, to prevent the saturation of Camptothecin caused by its limited solubility. Samples of Camptothecin (33 mg) were suspended in the HPLC mobile phase with no TBAP added. At the time periods required the particles were pelleted by centrifugation, and the supernatant completely removed and analysed for Camptothecin concentration using HPLC. The particles were then resuspended in fresh mobile phase (1 mL) and stirring continued for the required time.
The following molecules have been encapsulated into silica particles using the acid destabilisation process: orange II (O-II); rhodamine-B (R-B); rhodamine-6G (R-6G); methyl-violet (M-V); copper (II) phthalocyanine-tetrasulfonic acid tetrasodium salt (CuPC); (tris(2,2′-bipyridyl) dichlororuthenium(II) hexahydrate (Rubpy); rhodamine-β isothiocyanate (RBITC). The encapsulation efficiency of each material may depend on one or more of:
The syntheses were conducted with several alternative surfactants. The corresponding TEM micrographs are presented in
The incorporation of ORMOSIL into the particle structure is important because, in addition to providing alternative hybrid matrices, it offers a possible way to increase the encapsulation efficiency by changing the pore size and internal structure of particles as well as providing chemical anchors for the molecules to encapsulate (e.g. amino groups from APTMS which can react with carboxylate groups in a dopant). However, as shown in
The two successful systems have a hydrophilic organic group attached to the silicon: amino propyl and glycidoxypropyl. In acid, the epoxy ring of the glycidoxypropyl group is hydrolysed and opens up according to the reaction:
The fact that the alkyl and aryl substituted alkoxides (Methyl (MTMS), Phenyl (PTMS), Octyl (OTES)) did not produce any particles suggests that the organic group stabilises the emulsion (perhaps acting as a co-surfactant), thus preventing its destabilisation. The gel obtained using CHEETES suggests that the cyclohexenyl ethyl ligand decreases the stability of the emulsion, which results in a very rapid and uncontrolled destabilisation and the production of a dense gel.
The amount of organically modified silane influences the final morphology of the particles.
For APTMS, the particle size decreases with increasing amount of APTMS introduced. A possible explanation for this trend relates to the catalytic effect of the amino group on the sol-gel condensation, which leads to a faster nucleation (i.e. production of more nuclei) and hence less growth and smaller particles.
As expected there is no significant difference for morphology for particles synthesised with pure TMOS/TEOS or with a mixture thereof. During acid catalysed hydrolysis, whether starting from TEOS or TMOS, the same hydrolysed silica oligomers are produced.
Several active materials may be encapsulated inside silica particles produced by the present process. The actives may be introduced either by mixing them initially inside the water pool followed by the addition of the silicon precursor, or by mixing several emulsion systems in which different active molecules in which different active molecules have been incorporated. The destabilisation of both systems leads to the production of submicron particles containing the different dyes although it is not certain at what the scale the dyes are dispersed. It is not known whether the destabilisation of a multiple emulsion leads to a molecular mixing and homogeneous distribution of the actives inside the submicron particles or whether the submicron particles contain nano-domains of concentration corresponding to the initial water pools of each emulsion prior to destabilisation.
The results in
An alternative way to produce composite submicron particles containing mesoporous seeds dispersed inside a micorporous acid catalysed silica matrix is to mix two different emulsions, one containing the acid catalysed species and the other one the base catalysed particles, rapidly before destabilisation. As shown in
Two different factors are important to note: the previous analyses were performed using TEM, which is a poor method for analysing particle size distribution; and the ultrasound source used in the earlier experiment was a bath rather than the ultra-sound horn that that was used in the present experiment, which is much more powerful.
Camptothecin was encapsulated and released both in the lactone and carboxylate form. Although, due to the HPLC procedure used, it is not possible to determine accurately the exact proportion of the two forms of the drug encapsulated inside the particles, a substantial proportion of the encapsulated camptothecin was in the lactone form.
The comparison between the quantity released in
Tween 61 (HLB 9.6) and Tween 81 (HLB 10) produced unstable emulsions that generated submicron particles prior to destabilisation. Those submicron particles were of similar size to those obtained after acid destabilisation. The inventors hypothesize that in this case the submicron particle formation takes place prior to destabilisation. In the case of Tween 21(HLB 13.3) a relatively stable emulsion formed (i.e. approximately transparent as compared to opaque for Tweens 61 and 81). After reacting for 3 days, the particles were centrifuged at 12000 rpm for 20 min to recover a relatively small yield of product (relative to the normal yield after destabilisation). By TEM this product appeared to be large irregular aggregates and contained some gels. Only a few particles appeared spherical. The inventors hypothesize that when starting with a stable micro-emulsion, acid destabilisation is necessary to obtain submicron particles.
The inventors hypothesize that the number of oxyethylene units in the surfactant molecule may play an important role in controlling the production of submicron particles. In those surfactants which provided an acceptable product when used in the process of the invention, the number of PEG is relatively small: 4 for Tween 21, 5 for NP5 and 6 for NP6 compared to 9 for NP9 and 7.5 for Triton X-114 (the latter two surfactants having been found unsuitable for use in the invention). Furthermore the number of oxyethylene units, (which form the polar head of the surfactant molecule) may influence the interaction between the polar head group of the surfactant and water which may control the coalescence process.
Although the number of oxyethylene units in the surfactant molecule plays a critical role in controlling the production of submicron particles, it is not sufficient to ensure proper destabilisation of the emulsion. This is exemplified by Brij 30, which possesses 4 oxyethylene units units but forms stable emulsions that do not produce submicron particles after destabilisation. Moreover, as mention earlier, in order to achieve successful destabilisation, the surfactant should have medium strength molecular interaction between its polar head and the water pool. This molecular interaction may be characterised by the surfactant footprint (A), which can be calculated by dividing the surface area of the water droplet surface (II*d2, where d is the water pool diameter) by the surfactant aggregation number (N).
A=(II*d2)/N
Using values from the literature, the footprint was calculated for a range of the surfactant that we used. The results are listed below. A medium interaction corresponds to a footprint between 1.5 and 10 nm2 per molecule.
It appears that surfactants with HLB between about 9 and about 14 and having between about 4 and 6 oxyethylene units and having a foot print between 1 and 5 nm2 per molecules may be suitable for use in the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004926544 | Nov 2004 | AU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU05/01738 | 11/15/2005 | WO | 00 | 9/4/2007 |
