Materials and Methods Relating to Stabilised Polymeric Silicate
Compositions
Field of the Invention
The present invention relates to materials and methods relating
to stabilised polymeric silicate compositions, and in particular
to processes for producing stabilised polymeric silicate
compositions, to compositions obtainable using the processes and
their uses, in particular in therapy.
Background of the Invention
Silicon is an environmentally ubiquitous element and adult humans
in the Western world ingest about 15 to 50 mg per day currently.
Naturally it occurs as silicates wherein silicon links to oxygen
atoms. Silicic acid and silica are also terms used for such
structures. These range from the simplest mono silicic acid, also
termed ortho, to silica particles. Its precise biological role
is not yet understood but much evidence points to an important
role in connective tissue health (Jugdaohsingh et al., 2008) .
Whilst quintessential connective tissues include bone, joints,
blood vessels, skin, hair and nails, there is also notable
evidence for dietary, supplemental, or therapeutic benefit of
soluble or polymeric silicate in a wide array of medical
conditions that include, but are not limited to, osteoporosis,
osteopenia and other musculoskeletal and joint disorders, cancers
of all types, various skin conditions, vascular, cardiovascular
and coronary heart diseases, inflammatory diseases, autoimmune
diseases, Alzheimer's disease and varying forms of cognitive
impairment, infections of various types, wounds and ulcers,
gastrointestinal, liver, kidney and immune related disorders and
hormone related changes and disorders. Beneficial nutritional
and therapeutic effects of silicate appear to extend to other
animals, especially other mammals.
Silicate has been used as an oral nutritional supplement,
although achieving a formulation that allows effective
acquisit ion (absorption) following dosing is not straightforward.
Silicon in its naturally occurring inorganic form is soluble as
orthosilicic acid. However, its concentration, e.g. in drinking
water, needs to be relatively low ( 1 .7 mM) as, under natural
conditions, this is the maximum equilibrium solubility of aqueous
silicate at pHs < 9 to prevent the onset of irreversible
polymerisation of particles that gradually condense and/or
increase in size and then are not easily re-solubilized. This
behaviour has bedevilled the development of silicate supplements
as concentrated forms do not dissolve in the gut to enable
absorption, whilst dilute forms result in large quantities of
supplement (e.g. 20-100 ml/day) needing to be ingested.
Normally, certain chemical moieties such as ligands may be used
to bind and render soluble cations/anions that otherwise would
precipitate at physiological pH, but silicate is awkward because
the monomer typically has greater affinity for itself (i.e. to
undergo self-assembly) than for any other molecules, and the
higher the concentration of silicon the more difficult it becomes
to arrest its self-assembly in aqueous solution. This has led to
alternate strategies for producing bioavailable and
therapeutically useful silicate composition being pursued.
US 5,807,951 (Nippon Zoki Pharmaceutical Co., Ltd) describes a
process for producing silicate polymers by adding acid to a
silicate solution. Optionally a saccharide or sugar alcohol may
be added to the silicate compositions with the examples using
lactose or mannitol. However, the process described in US
5,807,951 requires a final drying step, between 150°C and 250°C,
to produce a dry lyophilised composition. However, as described
further below, the use of heating, and any other form of drying
to a powder, has the significant disadvantage of causing the
formation of condensed silicates with corresponding poor
resorbability .
US 2011/0229577 (Kerek) discloses silicic acid particles having
shell-like structures in which the particles condense under
conditions in which the pH of the reaction mixture is first
reduced and then increased, leading to a composition said to be
at a pH 2 .1 or a pH greater than 9.2.
Kim et al. (Macromolecules , 45: 4225-4237, 2012) describes the
production of nanocomposite melts formed from a mixture of
silicate nanoparticles and ethanol mixed with a defined mass of
PEG. The samples were heated in a vacuum oven to remove residual
ethanol. The vacuum oven was then purged several times with
nitrogen followed by evacuation of the chamber to remove oxygen,
yielding the polymer nanocomposites . Importantly, the silicate
nanoparticles were relatively large (44 ± 5 nm) . Also, these
were synthesized based on a base-catalysed hydrolysis and
condensation of tetraethylorthosilicate (TEOS) which yields
condensed silicates.
Gao et al (Colloids and Surfaces A : Physicochem. Eng. Aspects
350: 33-37, 2009) precipitated silicates in the presence of PEG
but the process employed required first calcination at 550°C,
producing highly condensed materials that are distinct from those
described herein.
Given the disadvantages of the prior art silicate composition
formulations, the conditions used to formulate them and the
manner in which they need to be administered, it remains an
unsolved problem in the art to improve the properties of
stabilised polymeric silicate acid compositions. It would be
advantageous to provide compositions in which the silicates are
resorbable, i.e. are capable of undergoing efficient dissolution
to provide bioavailable soluble silicic acid, and in which the
compositions do not tend to form condensed forms of silicates, as
occurs in the prior art when compositions are dried.
Alternatively or additionally, it would further be advantageous
if the other components of the stabilised polymeric silicate
compositions were suitable for administration to a human or
animal subject, e.g. without the dilution or other steps needed
with prior art silicate supplements. Alternatively or
additionally, it would be advantageous if the compositions were
capable of providing amounts of stabilised polymeric silicates
suitable for use in therapy, as compared to prior art
compositions that contain only low levels of bioavailable
silicate present as appropriately resorbable polymeric silicates.
Summary of the Invention
Broadly, the present invention addresses the need in the art to
provide stabilised and aquated polymeric silicates, to
compositions obtainable using the processes and their uses, that
are suitable for administration as therapeutic agents and
supplements. In particular, the present invention addresses the
provision of stabilised and aquated polymeric silicates that are
capable of intravenous delivery or for topical administration,
e.g. in the form of a solid or semi-solid ointment. These
formulations may be useful in the treatment of wounds.
As well known in the art, there is an equilibrium between soluble
silicic acids and increasingly condensed silicate compositions.
Accordingly, in the present invention, "stabilised polymeric
silicate composition" includes polymeric silicic acid and
nanosilicate particles having the properties described herein, as
well as soluble forms of silicic acid and polysilicic acid that
they are in equilibrium with in the composition or in a
formulation comprising it.
Evidence is emerging in the art that suggests that silicic acid
is beneficial for health and disease prevention or cure in humans
and other animals. In general, the compositions of the present
invention comprise polymeric silicate compositions in which the
natural tendency of polymeric silicates to grow to form higher
order polysilicates and silicate particles is inhibited by the
inclusion of substances such as organic compounds that are
capable of acting as growth retardants, i.e. which inhibit the
natural tendency of polysilicic acid to grow to form gels and
more condensed silicate particles or polymers and particles
larger than those of the desired size. Moreover, in some
aspects, the present inventors have found that this approach
means that the compositions are stable at physiological
acceptable pHs, especially neutral or mildly acidic pH or mildly
alkaline pH.
A further advantage of the method described herein is that
through the selective control of pH, silicon concentration, and
stabiliser concentration during the synthesis, the particle size
may be tailored from small polymers of less than 1 nm diameter up
to 20 nm diameter depending upon the desirable particle size and
that this may then be stabilised according to the invention
outlined to enable administration to a subject or animal at the
chosen particles size. It will be clear to those skilled in the
art that a particle size refers to a range of sizes and the
number quoted herein refers to the average diameter, most
commonly mean diameter of that range of particles.
The inventors have discovered that polyalkylene glycols such as
PEG and/or sugars such as sucrose are advantageous size
stabilisers and/or stability modulators of poorly condensed
nanosilicates that may then find use for oral, parenteral or
topical administration. Sucrose is especially advantageous for
oral or parenteral administration as it is a well known,
extremely safe molecule with a long history of use in intravenous
iron products, for example. In contrast, PEG is especially well
suited for topical delivery of silica as it forms a cream or an
ointment and is available in a range of different molecular
weights, allowing the tailoring of viscosity and other physical
parameters that may be desirable in the final ointment. The
application of the present invention to topical products has
therapeutic use for wound healing and as in anti-infective
compositions.
Accordingly, in a first aspect, the present invention provides a
process for producing a stabilised polymeric silicate composition
comprising polymeric silicic acid and nanosilicate particles
having mean diameters of 20 nm or less, the process comprising
the steps of:
(a) providing an aqueous solution of a soluble silicate at a
pH greater than or equal to 9.5;
(b) reducing the pH of the silicate solution to cause
polymerisation of the silicate to form polymeric silicic acid and
nanosilicate particles; and
(c) simultaneously or sequentially with steps (a) and/or (b)
adding to the silicate solution a stabilising agent that
comprises a polyalkylene glycol and/or a sugar thereby producing
a stabilised silicate composition in which the stabilising agent
inhibits formation of condensed silicates;
wherein the stabilised polymeric silicate composition is
aquated and wherein the process does not involve drying the
composition or heating it above 100°C.
In a further aspect, the present invention provides a stabilised
polymeric silicate composition comprising polymeric silicic acid
and nanosilicate particles having mean diameters of 20 n or less
as obtainable by the process of any one of the preceding claims.
Silicate nanoparticles that are transiently stable in vivo may
have suitable roles for re-activation of the immune system to
help treat infections and cancers, for example. Cancers include
melanoma, skin cancers, lung cancer, pancreatic cancer, colon
rectal and other splanchnic cancers, gastric cancer, breast
cancer, lymphomas, leukaemias, uterine cancers, prostate cancer,
oesophageal cancer, bone cancers, bladder cancers, cervical
cancer, endometrial cancer, brain cancer, eye cancers, ovarian
cancer, testicular cancer, liver cancer, renal cancer, head and
neck cancers and includes metastatic and primary cancers.
Infection includes, but is not limited to: infection with
viruses, retroviruses and bacteria such as mycobacteria, Gram
positive bacteria and Gram negative bacteria, as well as
helminths, parasites and other infectious agents.
The transiently stable silicate nanoparticles may also act as a
reservoir for the release of silicic acid that itself is
effective in enhancing connective tissue health and may be useful
in osteoporosis, fracture healing, joint diseases, skin diseases,
blood vessel disorders, or for nutritional supplementation to
ensure adequate supply of silicate.
As such, administration may be by topical application, oral
administration or parenteral administration, the latter
especially by intravenous administration.
In a further aspect, the present invention provides a stabilised
polymeric silicate composition comprising polymeric silicic acid
and nanosilicate particles having mean diameters of 20 nm or less
as defined herein for use in a method of treatment.
In a further aspect, the present invention provides a stabilised
polymeric silicate composition comprising polymeric silicic acid
and nanosilicate particles having mean diameters of 20 nm or less
as defined herein for use in a method of promoting wound healing
and/or treating or preventing bacterial infection, wherein the
composition is formulated for topical administration.
In a further aspect, the present invention provides a silicatecontaining
supplement comprising a stabilised polymeric silicate
composition comprising polymeric silicic acid and nanosilicate
particles having mean diameters of 20 nm or less as defined
herein for use in the delivery of silicic acid to a human or
animal subject. The composition may be employed in the treatment
of conditions ameliorated by administration of silicates.
In a further aspect, the present invention provides the use of a
stabilised polymeric silicate composition comprising polymeric
silicic acid and nanosilicate particles having mean diameters of
20 nm or less as defined herein in the manufacture of a
medicament for the treatment of a condition ameliorated by
administration of silicate.
In a further aspect, the present invention provides the use of a
stabilised polymeric silicate composition comprising polymeric
silicic acid and nanosilicate particles having mean diameters of
20 nm or less as defined herein as a silicate containing
supplement .
In a further aspect, the present invention provides a composition
comprising a stabilised polymeric silicate composition comprising
polymeric silicic acid and nanosilicate particles having mean
diameters of 20 nm or less as defined herein for use in therapy.
In a further aspect, the present invention provides a method of
treating a condition ameliorated by administration of silicic
acid, the method comprising administering to a subject in need of
treatment, a therapeutically effective amount of a composition
comprising a stabilised polymeric silicate composition comprising
polymeric silicic acid and nanosilicate particles having mean
diameters of 20 nm or less as defined herein.
In a further aspect, the present invention provides a silicatecontaining
supplement comprising a stabilised polymeric silicate
composition comprising polymeric silicic acid and nanosilicate
particles having mean diameters of 20 nm or less as obtainable by
the process of as described herein for use in the delivery of
transiently stable silicate polymers to a human or animal
subject.
In a further aspect, the present invention provides a stabilised
polymeric silicate composition for use in a method of treatment,
wherein the composition comprising polymeric silicic acid and
nanosilicate particles having mean diameters of 20 nm or less and
a stabilising agent comprising sucrose and/or a polyalkylene
glycol, wherein composition is formulated for intravenous (IV)
administration via an intravenous drip.
In a further aspect, the present invention provides a stabilised
polymeric silicate composition for use in a method of treatment,
wherein the composition comprising polymeric silicic acid and
nanosilicate particles having mean diameters of 20 nm or less and
a stabilising agent comprising a polyalkylene glycol, wherein
composition is formulated for topical administration, the
composition is for use in a method of promoting wound healing
and/or treating or preventing bacterial infection.
Embodiments of the present invention will now be described by way
of example and not limitation with reference to the accompanying
figures. However, various further aspects and embodiments of the
present invention will be apparent to those skilled in the art in
view of the present disclosure.
"and/or" where used herein is to be taken as specific disclosure
of each of the two specified features or components with or
without the other. For example "A and/or B" is to be taken as
specific disclosure of each of (i) A , (ii) B and (iii) A and B ,
just as if each is set out individually herein.
Unless context dictates otherwise, the descriptions and
definitions of the features set out above are not limited to any
particular aspect or embodiment of the invention and apply
equally to all aspects and embodiments which are described.
Brief Description of the Figures
Figure 1 . Schematic representation of particle size at various
stages of three theoretical synthesis processes shown by the
dashed line.
Figure 2 . Particle size of lactose containing silicates prior to
drying. Top panel shows lack of stability of materials after
synthesis (70 mM; pH8) and bottom panel shows lack of stability
in simulated physiological conditions (40 mM, pH 7.0) . The
materials reported in this figure were synthesized according to
US 5,807,951 for comparison with the stabilised polymeric
silicate compositions of the present invention.
Figure 3 . Dissolution rates of small amorphous nano-silicates
(SANS) after heating at 0 °C for various periods. Note that
heating did not lead to changes in particle size (not shown) .
Figure 4 . Dissolution rates for small amorphous nano-silicates
(SANS) before and after autoclaving (121°C for 15min) .
Figure 5 . A ) Dissolution rates for small amorphous nano-silicates
(SANS) and commercial condensed silicates (Ludox SM30®) in
water (pH 7.2 + 0.3) . B ) Dissolution rates of small amorphous
nano-silicates (SANS) , autoclaved SANS, and PEG-stabilised ultra
small amorphous nano-silicates (uSANS)as well as and nonstabilised
uSANS .
Figure 6 . Change in particle size upon raising a non-stabilised
suspension of ultra small amorphous nano-silicates (40mM) to pH
7.0.
Figure 7 . Change in particle size upon raising the pH of a nonstabilised
suspension of ultra small amorphous nano-silicates
(0.5 M ) to pH .0 .
Figure 8 . Transient particle size stability at pH 4.0 of a
suspension of ultra small amorphous nano-silicates (uSANS) (0 .5 )
stabilised with Sucrose (1.5) .
Figure 9 . Stability of ultra small amorphous nano-silicates
suspensions (1.4% Si; i.e. 0.5 M ) . Nanoparticulate silicates were
stabilised by various compounds at pH 3.5 (A) . The effect of pH
is also shown in (B) . The number of days required to form a gel
was used as a proxy for stability. The results shown at pH 3.5
include a comparison of sucrose stabilised and non-stabilised
materials .
Figure 10. Particle size stability of sucrose-stabilised ultrasmall
amorphous nano-silicates (uSANS) at physiological pH.
Figure 11. Size of small amorphous nano-silicates (SANS)
particles before and after freezing.
Figure 12. Silicon release from disperse and agglomerated small
amorphous nano-silicates (SANS) particles (both at 5mM) in a PEG
ointment .
Figure 13. Silicon release from small amorphous nano-silicates
particles (SANS; 5mM Si) and ultra small amorphous nano-silicates
(uSANS 40 and 60mM Si) in a PEG ointment as per Figure 12.
Figure 14. Silicon release from PEG 200-stabilised silicate at pH
3 in which adjustment to pH 7 was carried out at different stages
in the formation of the PEG ointment. Note that the silicate
material was first stabilised with PEG 200 and then PEG 400 and
PEG 3350 were added to form the ointment.
Figure 15. Reduction in peak wavelength with increase of [Si],
showing that DMHP was released from Fe-DMHP complex in the
presence of stabilised silicate materials. Dashed line indicates
the peak obtained for free DMHP in the absence of Fe .
Figure 16. Peak wavelength shift over time following iron
sequestration by stabilised and non-stabilised ultra small
amorphous nano-silicates (both at 1.5 mM) .
Figure 17: E.coli growth curves over time in the presence of
different Si ultra small amorphous nano-silicates.
Figure 18. Experiment to show the effect of drying lactose
containing silicates at 200°C as described in US Patent No:
5,807,951. Large black visible agglomerates were formed after 4
hours of drying, dramatically altering the material produced.
Figure 19. Particle size of small amorphous nano-silicates and
Ludox SM30® in water. SANS particle size was increased to
approach the particle size of Ludox SM30® so dissolution can be
compared independent of size. Size was increased by adding
concentrated saline (1-2 mL, 1.54 M ) to the nanosilica dispersion
immediately after pH adjusting. This was done in so that the
particle size of the Ludox SM30® was broadly similar to that of
the SANS so that dissolution was not a function of particle size.
The dispersion was incubated for ca . 2 4 h at room temperature
prior to use.
Figure 20. Dissolution rates as determined by the molybdate assay
for uSANS dispersions (500 mM Si, pH 1.5) before and after
spiking with soluble metal ions.
Figure 21. Dissolution rates as determined by the molybdate assay
for a uSANS dispersion (500 mM Si, pH 1.5) after various
incubation periods at pH 10.
Detailed Description
The biological role of silicon and the chemistry of silicates
Evidence suggests that silicic acids whether monomeric or
polymeric are beneficial for health and disease prevention or
cure in humans and other animals. However, as described above,
the fundamental problem in the art is that silicic acid, the
monomer of which is represented as Si (OH) , self assembles and at
pHs 9.0 and concentrations above the maximum solubility of
aqueous silicate (1.7 mM at 25°C, see Figure 1 of Jugdaosingh et
al ., supra) it forms insoluble species. A s is well known in the
art, there is an equilibrium between soluble silicic acids and
increasingly condensed silicate species, namely mono-, di- and
tri-silicic acids, polysilicic acids and silicate particles. The
process of growth from solutions of silicic acid involves
evolution where the single unit grows in size and becomes more
and more evolved (i.e. less labile, soluble and/or dissolvable)
and, thus, less able to return towards Si (OH) in the absence of
added alkali. Growth can include polymerisation, agglomeration,
aggregation or an increase in size due to surface deposition of
soluble species. The growth of polysilicic acids eventually
leads to gel formation under suitable conditions. These factors
make it extremely difficult to stabilise silicate compositions
above these concentrations of aqueous silicate and at
physiologically relevant pH .
The dosing of silicate is therefore a challenge because the
dosage must deliver silicon as required for a desirable effect in
terms of both concentration and chemical form, and at a pH that
is compatible with physiological health and in a manner that will
avoid persistent nanoparticles of silicate that may have adverse
effects to health. Of particular note is that during application
of a dosage, three notable changes generally occur due to the
physiological environment. Firstly, there will be dilution by
the physiological fluids, and secondly there will be a pH change,
and thirdly there will be a change in the ionic strength. The
net effect of these influences will determine the behaviour of
the dosed silicate. In these respects, the present inventors
have found that certain conditions can be achieved to generate
metastable silicate dosages, at compatible pHs for application to
humans or animals and that upon a change in the chemical
environment, as may be brought about by a physiologically
relevant system, desirable properties of the silicate dosage are
achieved, or retained.
Stabilised polymeric silicate compositions
The present invention provides processes for producing a
stabilised polymeric silicate composition comprising polymeric
silicic acid and nanosilicate particles, in particular particles
having mean diameters of 20 nm or less. In the processes of the
present invention, polymerisation of silicates and particle size
growth is controlled and the resulting particles are rendered
size stable through the combination of silicate concentration, pH
and/or stabiliser. This is shown schematically in Figure 1 . In
some embodiments, the compositions may additionally be doped with
metal cations as the present inventors have found that these may
induce particle size growth and may provide the compositions with
useful additional properties. Doping with copper (Cu + ) or silver
(Ag+) is preferred as this may provide the formulations with
antimicrobial properties.
The stabilised and aquated polymeric silicate compositions may
subsequently be formulated according to the application to which
the composition is intended. If intended for topical uses, the
compositions may be incorporated into an ointment (e.g. a PEG
cream) , which itself may confer extra stability. For intravenous
applications it may be advantageous to adjust the pH and
concentration of the stabilised suspension prior to
administration, e.g. by dilution in an i.v. buffer. In this
case, rather than long-term stability, the main goal may be size
stability under physiological conditions during the therapeutic
window during which the composition is administered to a subject.
In some circumstances, dilution may be used to offset some of the
loss in stability that the change in pH causes as silicic acid
compositions are more stable at low concentrations of silicon.
Accordingly, the stabilisation may provide sufficient time for
the material to be used by dilution at a point of use.
One preferred feature of the present invention is that
stabilisation and size control are achieved without the use of
high temperatures at any stage in the process. This may be
contrasted with the approach taken in US Patent No: 5,807,951 in
which drying the silicates at 200°C causes the formation of
condensed forms of silicates that are then less bioavailable .
Preferably, this means that the processes of the present
invention are carried out at less than 100°C and more preferably
less than 70°C. Alternatively or additionally, it is preferred
that stabilisation is achieved without the removal of solvent
(i.e. drying), since this also favours the formation of condensed
forms of silicate. There are processes known in the art to
produce stable colloidal silicates but these use a combination of
heat-induced ageing, and/or organic solvents, and/or drying
processes at temperatures exceeding 100°C, or even 200°C.
However, these strategies produce nanoparticles that are
relatively large (typically larger than 20 nm) and, importantly,
exhibit a high level of condensation. Overall, such high levels
of condensation result in more persistent particles, as compared
to the poorly condensed forms of stabilised polymeric silicates
compositions of the present invention, with the potential for
long-term toxicity.
Preferably, the polymeric silicates compositions of the present
invention have the property of being resorbable, that is that
they are poorly condensed amorphous silicates that are capable of
undergoing dissolution, within therapeutically useful timescales,
upon administration. The amorphous nature of polymeric silicate
acid compositions and different levels of condensation and the
corresponding structural arrangement of the solid phase that can
be exhibited by amorphous mineral phases, may be
indistinguishable by XRD analysis (or equivalent) . Accordingly,
in the present invention, the level of condensation can be
determined by appropriate in vitro dissolution assays, whereby
poorly condensed amorphous nanosilicates exhibit faster
dissolution rates as compared to condensed amorphous silicates of
equivalent particle size.
In one example a dissolution assay may involve taking a sample of
a polymeric silicate composition and diluting it in buffer. A
molybdic acid assay may be used to determine the concentration of
soluble silicate present in an aliquot of the buffer over time
course of the assay. A s shown in the examples, the composition
may be diluted in 10 mM HEPES buffer and adjusted to pH 6.7-7.0.
An exemplary molybdic acid assay employs 100 of the test
solution or standard (prepared from Sigma Aldrich Si ICP
standard, 1000 mg/L) and 200 ]i molybdic acid colouring solution
(0.6105 g NH4M07 4 20 , 15 mL 0.5 N 2S04, 85 mL H20 ) . The assay
solution is transferred to a well plate and mixed for 10 minutes.
After the incubation, the absorbance (405 nm) can be measured and
the concentration of soluble silicic acid determined using a
standard curve. By way of example, a "poorly condensed"
polymeric silicate composition will be resorbable, for example as
determined in an in vitro dissolution assay in which at least 25%
of the composition, and more preferably at least 35% of the
composition, more preferably at least 50% of the composition, and
more preferably at least 75% of the composition dissolves in 24
hours in HEPES buffer.
The polymeric silicic acid compositions of the present invention
comprise soluble polysilicic acid and nanoparticles of polymeric
silicic acid having mean diameters of 20 nm or less, and in some
cases mean diameters that are more preferably less than 10 nm,
more preferably less than 5 nm, 4 nm, 3 nm, 2 nm or 1 nm. In
some embodiments, the particles may range from about 1 nm to
about 2 nm, or from about 1 nm to about 3 nm, or from about 1 nm
to about 4 nm, or from about 1 nm to about 5 nm, or from about 1
nm to about 10 nm, or from about 1 nm to about 15 nm, or from
about 1 nm to about 20 nm, or from about 5 nm to about 20 nm, or
from about 5 nm to about 15 nm, or from about 5 nm to about 10
nm, or from about 10 nm to about 15 nm, or from about 10 nm to
about 20 nm, or from about 15 nm to about 20 nm.
The non-soluble nature of the polymeric silicic acid and/or
nanosilicate particles may be confirmed indirectly by the
molybdic acid assay mentioned above as this determines the
soluble silicic acid fraction. In general, the materials will be
in equilibrium with the soluble silicic acid, with typical
soluble silicic acid concentration being about <2 mM at pHO.O.
The polymeric silicate compositions of the present invention may
be contrasted with more condensed forms of silicates, including
larger nanoparticles (e.g. preferably having an average size
greater than 50 nm, and more preferably greater than 20 nm) ,
polysilicic acid gels and silicon dioxide (S1O2) the fully
condensed form of silicic acid, in which -OH groups are virtually
absent. The size of the particles of polysilicic acids can be
determined using dynamic light scattering and it is preferred
that the measurements are made on freshly prepared samples, if
not stabilised. A s will be understood by those skilled in the
art, the polysilicic acids will be in equilibrium with other
silicate species. For example, and depending on the precise
conditions present, this may include smaller amounts of soluble
silicic acid.
The polymeric silicic acid compositions of the present invention
are aquated, that is water is present throughout their synthesis
and, at least to some degree (e.g. at least 5 wt%, more
preferably at least 10 wt%, at least 20 wt water) , preferably
also in the final formulation, i.e. the materials are not dried
or significantly heated prior to formulation and subsequent
administration. It will be clear, however, that stabilisers or
other formulation agents may be used at such a high
concentrations that displaces water molecules from the silicate
particles. As such, the water may be displaced although the
formulation is not dried.
The stabilisation of the polymeric silicic acid compositions of
the present invention preferably extends from their synthesis to
their storage, formulation and/or administration (e.g. unwanted
lack of agglomeration) .
The polymeric silicate compositions of the present invention are
metastable, that is the compositions possess a stability that is
fit for the purpose of shelf-life of their intended use, and do
not grow to any significant extent. By way of illustration, it
is preferred that the polymeric silicate compositions of the
present invention are storage stable, for example being stable
for 3 months or more, more preferably for 6 months or more, more
preferably for 12 months or more, and more preferably 2 4 months
or more. Thus, the polymeric silicate compositions of the
present invention may be produced by partial condensation of
silicic acid (or silicate) molecules. These materials are
metastable as discrete, non-aggregated clusters or colloids.
In the present invention, the polymeric silicate compositions
include a stabilising agent, preferably a sugar and/or a
polyalkylene glycol. The sugars include oligosaccharides
composed of eight monosaccharides or fewer, such as monomeric,
dimeric or trimeric saccharides. A preferred sugar is sucrose.
The maximum number of monomeric units in the sugar is chosen such
that its administration does not elicit an immune response in the
subject on administration. Polyalkylene glycols are a family of
polyether compounds that include polyethylene glycol (PEG) and
polypropylene glycol. Examples of stabilising agents that are
sugars (saccharides) include monomeric, dimeric, trimeric and
polymeric sugars (saccharide), or the corresponding sugar
alcohols, such as glucose, fructose, mannose, sucrose, threitol,
erythritol, sorbitol, mannitol, galactitol or adonitol. In some
embodiments in which the stabilising agent is a sugar, it is an
oligosaccharide other than lactose. In some embodiments in which
a sugar alcohol is used, it is other than mannitol. The use of
sugars as stabilising agents for compositions that are
administered internally is preferred in the present invention as
they are safe for administration to human and animal subjects.
In some embodiments, it is possible to employ combinations of
more than one different sugar (s) or polyalkylene glycol (s), e.g.
two, three, four or five or more sugars or polyalkylene glycols,
e.g. by adding them in step (a) and/or (b) . Sugar and/or
polyalkylene glycol stabilising agents are generally added at a
concentration between 0.01 M and 3.0 M , and more preferably
between 0.03 and 2.0 M , and most preferably between 0.1 M and 1.5
M . The skilled person can carry out routine tests to determine
which combinations of sugars and/or polyalkylene glycols work
best in any given situation.
The stabilised polymeric silicate compositions of the present
invention may be distinguished from the compositions disclosed in
US Patent Publication No: 2003/0206967 (Choi et al.) which
describe a composition that comprises sodium metasilicate , borax,
sodium thiosulfate, potassium carbonate and refined sugar in
water. This results in a very alkaline composition having a pH
of about pH 13, in contrast to pHs of the stabilised polymeric
silicate compositions of the present invention, which are
preferably between pH 3.0 and 9.0, more preferably between 3.0
and 8.0 and more preferably between 5.5 and 7.5. The process
used to make the compositions o f Choi et al . differs from the
present invention as the present invention produces the
compositions b y lowering the pH to produce stable silicate
polymers. In view o f the above, it is preferred that the
stabilised polymeric silicate compositions o f the present
invention do not comprise one o r more o f sodium metasilicate ,
borax, sodium thiosulfate, potassium carbonate, and preferably do
not include borax.
In other aspects, the present invention may use carboxylic acids
as stabilizing agents and the carboxylic acid may b e a C2- 1
carboxylic acid, for example a dicarboxylic acid such as oxalic
acid, malonic acid, glutaric acid, tartaric acid, succinic acid,
adipic acid or pimelic acid, or ionised forms thereof (i.e., the
corresponding carboxylate) , such as adipate . O r for example a
monocarboxylic acid, such a s gluconic acid. Further examples o f
stabilizing agents are dicarboxylic acids, which may b e
represented b y the formula HOOC-Ri-COOH (or an ionised form
thereof) , where R i is an optionally substituted Ci-10 alkyl, Ci-10
alkenyl o r Ci-10 alkynyl group. In general, the use o f carboxylic
acids in which R i is a Ci-10 alkyl group, and more preferably is a
C2-6 alkyl group, is preferred.
In some embodiments, the polymeric silicic acid compositions may
b e contacted with metal cations, such a s Ca2+ , M g2+ , A g+, A l3+ ,
Cu2+ ,Fe 3+ and/or Zn2+ a s the inventors have found that this helps
to stabilise the compositions against dissolution, which may b e
advantageous in some applications, o r confer additional
functional benefits (e.g. antimicrobial action, for example b y
including A g+ and/or Cu2+ ) . Without wishing to b e bound b y any
particular theory, the present inventors believe that the cations
coat the nanosilicate particles via interaction with free silanol
groups (-OH) present in the materials. By way o f guidance, it is
preferred that the metal cation is added to provide a final
concentration between 0.01 M and 1.0 M and more preferably the
metal cation is added to provide a final concentration between
0.05 M and 0.5 . Preferably, the metal cation is added to
provide a Si to metal ratio of between 100:1 and 10:1, and
optionally to provide a Si to metal ratio of 20:1.
The present inventors also surprisingly found that polymeric
silicate acid compositions of the present invention may be
further stabilised by adding a non-aqueous solvent, such as an
alcohol. A preferred example of an alcohol is ethanol. By way
of illustration, the non-aqueous solvent may be added between 10
and 50% v/v, or between 20 and 50% v/v or between 10 and 20% v/v.
Furthermore, in some cases the present inventors found that the
combination of sucrose with alcohol was particularly effective
for stabilising the compositions.
In the following discussion of the steps of the processes of the
present invention, it will be apparent to those skilled in the
art that it may be possible to reorder some of the steps of the
above process and/or for some of the steps to take place
simultaneously. Others of the steps are optional as indicated
above and explained further below.
In the work leading to the present invention, the inventors found
that a number of factors contribute to the stability of the
polymeric silicate compositions including the rate at which the
pH of the starting alkaline silicate solution is lowered, the
inclusion of stabilisers, notably sugars or polyalkylene glycols,
the addition of metal cations and/or the addition of a non
aqueous solvent. Accordingly, the processes of the present
invention may employ these approaches, alone or in any
combination, to produce polymeric silicate compositions having
sufficient stability for use, e.g. as supplements or therapeutic
agents. The metal cations may also serve to provide
antibacterial properties to the compositions (e.g. by adding Ag+
or Cu2 ) and/or to inhibit dissolution of the composition as
demonstrated in Figure 20.
In some cases, in particular for the production of ultra small
particles of nanosilicates ("uSANS") , the rate at which the pH of
the alkaline silicate solution is lowered may have a significant
effect on the stability of the resulting polymeric silicate
compositions. Preferably, the pH is lowered (e.g. to a pH of
less than or equal to pH 4.0 or 3.0) over a period of less than
60 seconds, more preferably less than 30 seconds, more preferably
less that 10 seconds, or most preferably less that 5 seconds.
In step (a) , it is preferred that the concentration of the
alkaline silicate solution is between 0.05 M and 1.5 M , and more
preferably is between 0.03 and 2.0 M , and most preferably between
0.1 M and 1.0 M . The use of pHs that are higher than 9.5 is also
preferred in order to maintain the solubility of the silicates,
and preferably in step (a) the pH of the alkaline silicate
solution is about pH 10.5 or above, and still more preferably is
about pH 11.5 or above. In the final polymeric silicate
compositions, the concentration of silicon may be 2 .5mM or more,
5 .OmM or more, 25mM or more, 30 mM or more, 40 mM or more, 50 mM
or more, lOOmM or more, 250mM or more, 500mM or more. In the
final stabilised polymeric silicate compositions, the
concentration of silicon may be .5M or less, 1.0M or less, 500mM
or less, and ranges between these lower and upper limits.
In some embodiments, the reduced pH in step (b) has an effect on
the type of stabilised silicate nanoparticles that can be
produced. As shown in the examples, uSANS or very small
particles that have mean diameters of 5 n or less can be formed
by rapidly dropping the pH from pH greater than 10 to 3.0 or less
and enable concentrations of silicon up to 1 M to be used.
Alternatively, SANS or small nanoparticles have mean diameters of
lOnm of less and may be formed by reducing the pH to about 7.4.
In this case, lower concentrations of about 50 mM or less can be
used. Accordingly, the reduced pH may be 7.4 or lower, or pH 3.0
or lower. This enables the preparation of uSANS at a low pH, as
described, the pH raised to grow uSANS to SANS of a determined
particle size, and the size stabilised by dropping the pH again,
should this be required. Stabiliser is required at some stage in
this process. These processes are an important part of the art.
In some case, the pH may be lowered and/or the suspension diluted
for long term storage of stabilised aqueous suspensions.
Alternatively or additionally, upon long term storage at a nonphysiological
pH and prior to administration to a subject, the
nanosilicate suspension may be adjusted to a physiological pH,
and/or diluted and/or stabiliser added.
In situations in which the silicate compositions are formulated
in an ointment or cream, or where the suspension is diluted so
that it has a silicon concentration of lOOmM or less, it may be
preferred that the pH of the composition or a formulation
containing it is raised to a physiological pH, preferably to a pH
between 3.0 and 8.0, and more preferably to a pH of between 5.5
and 7.5. Conveniently, this may be done by adding a base, such
as sodium hydroxide or sodium carbonate. Generally the pH of the
composition should be suitable for administration, i.e. close to
physiological, for example pH 7.4± 1.5. The aim of this is so
that administration to a subject will not result in unintended
clinical outcomes, such as pain or inflammation. However,
depending on the route of administration, it may be acceptable if
the final formulation containing the stabilised polymeric
silicate compositions has a pH in the range between pH 3 and pH
9 .
The composition should be suitably stabilised, such that the
particle size of the nanosilicates will remain sufficiently
stable (<20 nm) for the intended application. For example, in
the case of a formulation for intravenous administration, the
particle size of the first storage solution (e.g. at pH <3 and
100 mM Si) will be stable for the duration of the storage period
and then once diluted with a buffered i.v. solution it will
remain stable first for the few hours before application and
then, once administered, it will not undergo agglomeration. PEG
stabilisation in topical applications would also mean that
particle size would be sufficiently constant in the ointment
during storage and upon application.
Formulation and Uses of Compositions
The stabilised polymeric silicate compositions of the present
invention may be formulated for use in a range of biologically
relevant applications, including formulation for use as
pharmaceutical or nutritional compositions, and in particular as a
silicate-containing supplement or therapeutic agents. The
compositions of the present invention may comprise, in addition to
one or more of the solid phase materials of the invention, a
pharmaceutically acceptable excipient, carrier, buffer, stabiliser
or other materials well known to those skilled in the art. Such
materials should be non-toxic and should not interfere with the
efficacy of the stabilised polymeric silicate compositions
depending on their intended use. A s well as having applications
for the treatment or prevention of conditions in human subjects,
the present invention has application in the veterinary field.
The present invention provides compositions suitable for a range
of different applications in which silicic acid is provided to a
su ect .
In one application, the stabilised polymeric silicate
compositions may be for use in oral delivery of silicic acid to a
subject via the gastrointestinal tract, in particular wherein the
stabiliser is a sugar. In this aspect of the invention,
preferably the composition generally is for direct administration
to a subject, i.e. there is no requirement for a dilution step to
be carried out by the subject prior to administration.
Preferably, the stabiliser is chosen from among the saccharides
set out herein. Typically, the sugar will be employed in an
amount between 0.01 M and 3.0 M , and more preferably between 0.03
and 3.0 M , even more preferably between 0.1 and 3.0 M , and most
preferably between 0.01 M and 1.5 M . More preferably, the
compositions have a pH between 1.0 and 6.0, 1.5 to 5.0, or 2.2 to
4.0, or 2.4 to 4.0. In other embodiments, the compositions have
a pH between 1.5, or 2.0, or 2.5 and a pH of 6.0, or 5.5, or 5.0,
or 4.5, or 4.0, or 3.5. The concentration of silicon is between
0 .1M and 1.5M. These compositions may be used for silicon
supplementation or for delivery of therapeutic silicate, i.e. to
treat a condition ameliorated by the delivery of therapeutic or
nutritional silicate. Preferably, the stabilised polymeric
silicate composition when formulated for oral delivery is a
liquid filled capsule. This may be to treat a condition in the
G tract or for the purpose of silicon supplementation. The
former may involve iron binding to ameliorate iron's toxic effect
in the colon. This and other embodiments of oral delivery may
require enteric or specialist coating for delayed release.
In a further application, the stabilised polymeric silicate
compositions may be administered to a subject intravenously by
dilution into a drip, typically a glucose or saline or sucrose
drip, with or without a buffer or agent to achieve a pH suitable
for administration. In this aspect of the present invention, the
determination of doses suitable for providing given levels of
silicate in circulation can be determined by doctors using
approaches well known in the art.
In an alternative application, the stabilised polymeric silicate
compositions may be formulated for topical administration for
example for application to the skin or surface of wounds.
Liquid pharmaceutical compositions generally include a liquid
carrier such as water, petroleum, animal or vegetable oils,
mineral oil or synthetic oil. Physiological saline solution,
dextrose or other saccharide solution or glycols such as ethylene
glycol, propylene glycol or polyethylene glycol may be included.
Where the silicate-containing supplement needs to be maintained in
a solid form, e.g. to control the delivery of a component of the
material, it may be necessary to select components of the
formulation accordingly, e.g. where a liquid formulation of the
material is made. Preservatives, stabilisers, buffers,
antioxidants and/or other additives may be included, as required,
for example in embodiments of the present invention in which the
polymeric silicate compositions are suitable for administration to
a subject via a drip.
In therapeutic applications, stabilised polymeric silicate
compositions of the present invention are preferably given to an
individual in a "prophylactically effective amount" or a
"therapeutically effective amount" (as the case may be, although
prophylaxis may be considered therapy) , this being sufficient to
show benefit to the individual (e.g. bioavailability) . The
actual amount administered, and rate and time-course of
administration, will depend on the nature and severity of what is
being treated. Prescription of treatment, e.g. decisions on
dosage etc. is within the responsibility of general practitioners
and other medical doctors, and typically takes account of the
disorder to be treated, the condition of the individual patient,
the site of delivery, the method of administration and other
factors known to practitioners. Examples of the techniques and
protocols mentioned above can be found in Remington' s
Pharmaceutical Sciences, 20th Edition, 2000, Lippincott, Williams
& Wilkins. A composition may be administered alone or in
combination with other treatments, either simultaneously or
sequentially, dependent upon the condition to be treated.
The compositions of the present invention may be used in
therapeutic applications in which delivery of silicate is
desirable, including a wide array of medical conditions that
include, but are not limited to, osteoporosis, osteopenia,
musculoskeletal and joint disorders, cancers of all types, skin
conditions, vascular diseases, cardiovascular diseases, coronary
heart diseases, inflammatory diseases, autoimmune diseases,
Alzheimer's disease, cognitive impairment, infections of all
types, wounds, ulcers, gastrointestinal disorders, liver disease,
kidney disease, immune related disorders or hormone related
disorders. Cosmetic aspects of the present invention include the
cosmetic improvement of hair, skin or nails, e.g. to provide them
with an improved appearance. The compositions of the present
invention may also be used in veterinary therapeutic applications
in which delivery of silicon is desirable, including, but not
limited to, treatment of wounds, ulcers and cancers. The
therapeutic uses of silicates are disclosed in WO 2009/052090, US
Patent Publication No: 2009/0130230 and US Patent Publication No:
2013/0149396, incorporated by reference in their entirety, and in
particular the compositions of the present invention may be used
in the treatment of conditions disclosed in these references with
the added advantage that the compositions of the present
invention are stabilised.
In a further aspect, the polymeric silicate compositions of the
present invention may be delivered orally not as bioavailable
silicon but, rather, to keep it in the distal gut lumen where
absorption is low, and to utilise the strong binding affinity of
polysilicic acid for certain cations, notably iron. Recent
evidence shows that iron in the colon is permissive for the
development of colonic cancers in susceptible individuals (13) .
Thus, in a further embodiment, the delivery of stabilised
polymeric silicate compositions targeted to the colon, at
concentrations that once in that environment favours only gradual
dissolution or even condensation of the polysilicic acid will
result in complexation/deactivation of luminal iron and, thereby,
prevention or reduction of tumorogenesis in the local
environment. This may be useful in the treatment or prevention
of cancer in the colon.
During infection and chronic disease, such as cancer, the body
induces an anaemic state, partly through the reduced mobilisation
of iron and the reduced shuttling of iron between different
cellular and extracellular compartments. The iron is locked up
into ferritin. This is because loose iron can enhance infection
and chronic disease states and so it needs to be sequestered.
Indeed, one commonly proposed strategy in disease treatment is
the sequestration of iron with chelators that lock it into an
immobile state. The finding that polymeric silicate compositions
of the present invention (e.g. SANS and uSANS) can mop up iron
means that when appropriately formulated for administration to a
subject, these may have a clinical role in sequestering iron and
helping combat disease. This may be through the binding of iron
in the gastrointestinal lumen thereby preventing iron's ingress
to the body and/or toxicity to intestinal cells, such as in the
colorectal region, or may be a systemic effect following
parenteral administration in cellular and/or extracellular
compartments .
Silicate nanoparticles that are transiently stable in vivo may
have suitable roles for re-activation of the immune system to
help treat infections and cancers, for example. Cancers may
include but are not restricted to: melanoma, skin cancers, lung
cancer, pancreatic cancer, colon rectal and other splanchnic
cancers, gastric cancer, breast cancer, lymphomas, leukaemias,
uterine cancers, prostate cancer, oesophageal cancer, bone
cancers, bladder cancers, cervical cancer, endometrial cancer,
brain cancer, eye cancers, ovarian cancer, testicular cancer,
liver cancer, renal cancer, head and neck cancers and includes
metastatic and primary cancers. Infection includes, but is not
limited to: infection with viruses, retroviruses and bacteria
such as Mycobacteria, Gram positive bacteria and Gram negative
bacteria, as well as helminths, parasites and other infectious
agents .
The transiently stable silicate nanoparticles may also act as a
reservoir for the release of silicic acid that itself is
effective in enhancing connective tissue health and may be useful
in osteoporosis, fracture healing, joint diseases, skin diseases,
blood vessel disorders, or for nutritional supplementation to
ensure adequate supply of silicate.
As such, administration may be by topical application, oral
administration or parenteral administration, the latter
especially by intravenous administration.
Other medical uses of the compositions of the present invention
include the treatment of hypertension, diabetes, bone diseases,
cardiovascular diseases, neurodegenerative pathologies, cancer of
all types not noted above, hyperacidity, osteoporosis, dental
calculus, Alzheimer disease, Creutzf eld- Jacob disease as well as
for wound healing.
Other medical uses of the compositions of the present invention
include the treatment of skin affected by burn, wounding or
action of pathogens or of caustic chemicals, including the
treatment of sun burn, or any skin disease including psoriasis,
eczema and dermatitis of other sorts.
Polyalkylene glycols such as PEG are especially well suited for
topical delivery of silicate as it forms an ointment and is
available in a range of different molecular weights, allowing the
tailoring of viscosity and other physical parameters that may
desirable in the final ointment.
It will be obvious to those in the art that topical application
delivery may be achieved using non- or only partially PEG based
ointments. In this case, upon initial stabilisation with PEG as
described herein, the silicates are incorporated in a non-PEG
based ointment, e.g. a PEG stabilised nanosilicate composition
incorporated in a further, different vehicle such as hydroxyethyl
cellulose .
An effective amount of one or more stabilised polymeric silicate
compositions herein may be formulated for topical application,
e.g. to the skin, teeth, nails or hair. These compositions can
be in the form of creams, lotions, gels, suspensions,
dispersions, microemulsions , nanodispersions , microspheres, hydro
gels, emulsions (oil-in-water and water-in-oil , as well as
multiple emulsions) and multilaminar gels and the like, (see,
for example, The Chemistry and Manufacture of Cosmetics,
Schlossman et al., 1998), and may be formulated as aqueous or
silicone compositions or may be formulated as emulsions of one or
more oil phases in an aqueous continuous phase (or an aqueous
phase in an oil phase) . The type of carrier utilized in the
present invention depends on the properties of the topical
composition. The carrier can be solid, semi-solid or liquid.
Suitable carriers are liquid or semi-solid, such as creams,
lotions, gels, sticks, ointments, pastes, sprays and mousses.
Specifically, the carrier is in the form of a cream, an ointment,
a lotion or a gel, more specifically one which has a sufficient
thickness or yield point to prevent the particles from
sedimenting. The carrier can itself be inert or it can possess
benefits of its own. The carrier should also be physically and
chemically compatible with the stabilised polymeric silicate
compositions or other ingredients formulated in the carrier.
Examples of carriers include water, hydroxyethyl cellulose,
propylene glycol, butylene glycol and polyethylene glycol, or a
combination thereof.
Materials and Methods
Preparation of small amorphous nano-silicates (SANS)
A 25±5mM solution of silicate was prepared from a concentrated
stock of sodium silicate. Next, an HC1 solution was used to
adjust pH to 6.810.2. The pH drop resulted in the formation of
amorphous colloidal silicates. The solution was left to
equilibrate for 16-24 hours during which it increased to pH
7.110.2. The process leading to the formation of the stabilised
polymeric silicate compositions of the present invention is shown
schematically in Figure 1 .
Methodology
The aliquots were diluted to ca, 1 mM in 10 mM HEPES buffer and
pH adjusted, if needed, to pH 6.7-7, 25 h after initial SANS
stock preparation. A molybdic acid assay was used to determine
concentration of soluble silicate over time.
Molybdic acid assay
100 of the test solution or standard (prepared from Sigma
Aldrich Si ICP standard, 1000 mg/L) and 200 molybdic acid
colouring solution (0.6105 g NH Mo7 4H20 , 15 mL 0.5 N H2SO4, 85 mL
H2O) were transferred to a 96 well plate and mixed for 10 min.
After the incubation, the absorbance (405 nm) was measured the
concentration of soluble silicic acid was determined using the
standard curve.
PEG stabilised ultra small amorphous nano-silicates (uSANS)
A suspension of nanoparticulate silicates (0.5M Si) was prepared
by first diluting a concentrated solution of sodium silicate
(resulting pH is greater than 10.5) and then dropping the pH to
approximately 1.0 in less than 5 sec by a bolus addition of
concentrated HC1 . The pH was then raised to 3.0 and 1M PEG 200
added. This suspension was then diluted to lmM Si (24 h later)
for the dissolution assay.
Non- stabilised ultra small amorphous nano-silicates (uSANS)
The same process was used as for the PEG-stabilised material (0.5
mM; pH 3 ) , but without addition of PEG.
Change in particle size upon raising a non-stabilised suspension
of ultra small amorphous nano-silicates (uSANS; 0.5 ) to pH 7.0
A non-stabilised suspension of uSANS (0.5M Si) was prepared by
first diluting a concentrated solution of sodium silicate
(resulting pH is greater than 10.5) and then dropping the pH to
approximately 1.0 in less than 5 sec by a bolus addition of
concentrated HC1 , and then raising it to pH 3.5. The suspension
was subsequently diluted to 40 mM and the pH raised to 7.0 to
induce controlled particle growth.
Change in particle size upon raising a non-stabilised suspension
of ultra small amorphous nano-silicates (uSANS) (0.5 ) to pH 4.0
Process: A non-stabilised suspension of uSANS (0.5M Si) was
prepared by first diluting a concentrated solution of sodium
silicate (resulting pH is greater than 10.5) and then dropping
the pH to approximately 1.0 in less than 5 sec by a bolus
addition of concentrated HC1 . The pH was then raised to 4.0 to
induce controlled particle growth.
Transient particle size stability at pH 4.0 of a suspension of
uSANS (0.5M) stabilised wit PEG
A non-stabilised suspension of nanoparticulate silicates (0.5M
Si) was prepared by first diluting a concentrated solution of
sodium silicate (resulting pH is greater than 10.5) and then
dropping the pH to approximately 1.0 in less than 5 sec by a
bolus addition of concentrated HC1 . The pH was then raised to
4.0 and 1M PEG added.
Particle size stability of sucrose stabilised ultra-small
amorphous nano- silicates (uSANS) at physiological pH
A sucrose-stabilised suspension of uSANS (0.5M Si) was produced
by diluting a concentrated solution of sodium silicate (resulting
pH is greater than 10.5) and adding sucrose (such that the final
composition contains 1 .5M sucrose) . Next the pH was dropped to
approximately 1.0 in less than 5 sec by a bolus addition of
concentrated HC1 . This was followed by sodium hydroxide addition
to obtain pH 3.5. Next, this suspension was diluted down to
40mM Si and the pH adjusted to 7 to simulate intravenous
administration.
Size of small amorphous nano- silicates (SANS) particles before
and after freezing
Fresh SANS suspension (30 mM, preparation as in Figure 3 ) was
kept at -20°C for 16 hours and thawed 1-3 hours prior to
incorporation into PEG cream.
Silicon release from disperse and agglomerated small amorphous
nano- silicates (SANS) particles (both at 5mM) in a PEG cream
Release assay: Si-containing PEG creams (lOg) were transferred to
and allowed to settle at the bottom of a Falcon tube for at least
12 hours. Next, 10ml of a 50mM bicarbonate buffer (pH 7 ) was
added on top of the cream layer and the release of silicon
determined overtime by ICP-OES. The assay was run at room
temperature ~20°C. Agglomerated materials were produced by
freezing and thawing as described above.
Incorporation into a PEG cream
PEG 3350 (5.25 g ) was melted. A SANS suspension (2.3 g of 30mM
suspension) is mixed with PEG 400 (6.15 g ) at 65-70 °C and added
to the PEG melt. The resulting mixture was homogenised and
allowed to cool to room temperature.
Silicon release from SANS particles (5mM Si) and uSANS (40 and
60mM Si) in a PEG cream
Methodology: Same as described in Figure 12.
Incorporation of uSANS into a PEG Cream
Method 1 : PEG 3350 (5.25 g ) was melted and sodium hydroxide was
added to ensure the pH of the cream, once formed, was above pH 6 .
PEG 200- stabilized silicate nanoparticles (2.3 g of 0 .5M
suspension) were mixed with PEG 400 (6.15 g ) at 65-70°C and added
to the PEG melt. The resulting mixture was homogenised and
allowed to cool to room temperature.
Method 2 : PEG 3350 (5.25 g ) was melted and sodium hydroxide was
added to ensure the pH of the cream, once formed, was above pH 6 .
PEG 200- stabilized silicate nanoparticles (2.3 g of 0 .5M
suspension) were mixed with PEG 400 (6.15 g ) at room temperature
and added to the PEG melt. The resulting mixture was homogenised
and allowed to cool to room temperature.
Silicon release from PEG 200-stabilised polymeric silicate in a
PEG ointment
Stabilised silicate at pH 3 in which adjustment to pH 7 was
carried out at different stages in the formation of the PEG
cream.
Methodology: The preparation of PEG ointments comprises the
incorporation of PEG200 -stabilised suspension (0.5 M Si; pH 3.0)
into PEG cream, which further stabilised the materials. The
cream was formed by first mixing the suspension PEG 400, followed
by heating to 60-70°C and then adding PEG 3350. The pH
neutralisation was carried out adding NaOH after 1 ) PEG 200
stabilisation (before PEG 400), or 2 ) after addition of PEG 400,
or 3 ) with the addition of PEG3350.
Experiments to produce and test features of the stabilised
polymeric silicate compositions of the present invention
Amorphous poorly condensed materials
Amorphous nanosilicates can exhibit different levels of
condensation, which are not easily distinguished by standard
techniques, such as XRD. The present inventors observed that
exposure to even moderate temperatures (e.g. 60°C) can lead, over
time, to an increase in condensation that results in lower
dissolution rates (Figure 3 ) . This unwanted change is
particularly pronounced at higher temperatures, such as those
employed in drying or sterilisation processes, where even short
exposures result in a dramatic reduction in lability (Figure 4 ) .
In contrast, the synthetic methods described herein produce
stable nanoparticles that are labile, i.e. non-persistent in vivo
(Figure 5 ) . For reference, Ludox S 30® is an example of a
condensed silicate nanoparticle . Figure 19 shows that while
Ludox SM30® and the nanosilicates of the present invention have
similar particle sizes, that their respective different rates of
dissolution mean that dissolution rates are not size dependent.
Preparation of Small Amorphous Nano- Silicates (SANS)
A 30±3mM solution of silicate was prepared from a concentrated
stock of sodium silicate. Next, an HC1 solution was used to
adjust pH to 6.8+0.2. The pH drop resulted in the formation of
amorphous polymeric silicates. The solution was left to
equilibrate for 16-24 hours during which it increased to pH
7.110.2.
Methodology: Upon preparation, a SANS suspension was prepared and
immediately heated to 60°C. At specific time points, aliquots
were collected and allowed to cool to room temperature. The
aliquots were diluted to c . 1 mM in 10 mM HEPES buffer and
adjusted to pH 6.7-7, 25 h after initial SANS stock preparation.
A molybdic acid assay was used to determine concentration of
soluble silicate over time.
Molybdic acid assay: 100 of the test solution or standard
(prepared from Sigma Aldrich Si ICP standard, 1000 mg/L) and 200
molybdic acid colouring solution (0.6105 g NH4M07 4¾0, 15 L
0.5 N H2SO4, 85 mL H2O) were transferred to a 96 well plate and
mixed for 10 min. After the incubation, the absorbance (405 nm)
was measured the concentration of soluble silicic acid was
determined using the standard curve.
PEG Stabilised ultra Small Amorphous Nano-Silicates (uSANS)
A suspension of nanoparticulate silicates (0.5M Si) was prepared
by first diluting a concentrated solution of sodium silicate
(resulting pH is greater than 10.5) and then dropping the pH to
approximately 1.0 in less than 5 sec by a bolus addition of
concentrated HC1 . The pH was then raised to 3.0 and 1 PEG
added. This suspension was then diluted to lmM Si (24 h later)
for the dissolution assay.
Non stabilised Ultra-Small Amorphous Nano-Silicates (uSANS)
The same process was employed as for the PEG-stabilised material
(0.5 m ; pH 3), but without addition of PEG.
Size Tailorability
Using the process described herein, upon dropping the pH, small
particles (<5nm; typically <3.5 nm) are formed. However, larger
particle sizes can be achieved by raising the pH. Figure 6 shows
particle size growth upon raising the pH of a suspension of
nanoparticles . Usefully, the rate of growth can be determined by
selecting the appropriate pH and concentrations. Figure 7 shows
how a slower growth rate can be achieved by only raising the pH
to . A s stated above, size growth can be arrested by adding a
stabiliser (e.g. PEG, Figure 8 ) or diluting the suspension.
Transient Size Stability-
Growth retardants increase meta-stability (Figure 9A) in a pH
dependent fashion (Figure 9B) . This increase in stability
enables the processing and formulation of concentrated amorphous
silicates (e.g. incorporation in gels or creams) .
Size stability under physiological conditions
The enhanced dispersibility and stability of the silicates
described herein allow the administration of high concentrations
of nanoparticles at physiological pHs without the risk of
aggregation. This enables a range of applications relying on
parenterals, such as intravenous (i.v.), or oral delivery, and is
illustrated by sucrose stabilised silicate NPs, which remain
disperse and small once exposed to physiological pHs (Figure 10) .
Incorporation into a solid or semi-solid matrix
Effective release of silicon from silicate particles in a cream
requires that particles remain non-agglomerated. To illustrate
this, the agglomeration of non-stabilised amorphous silicates was
induced by freezing (Figure 11) prior to incorporation in a PEG
cream. A s a result, the release of silicon to a simulated
physiological fluid in contact with the cream was considerably
lower for the agglomerated particles (Figure 12) .
A s stated above, to preserve silicates in a poorly condensed form
these should be kept as aqueous suspensions. However, the
incorporation of aqueous suspensions into creams results in a
considerable dilution factor (typically 5-7 fold) of the silicate
active. Also, given that suspensions of unstabilised amorphous
silicates at physiological pH (pH 6-8) are not stable above 40
m , their final concentration in the cream is limited to ~ 8 mM.
However, using the stabilisation strategies described herein,
high concentrations of amorphous poorly condensed nanoparticles
can be incorporated into PEG creams and result in greater
release of the active agent (Figure 13) . The inventors have
also discovered that to prevent agglomeration, the pH adjustment
of the material is advantageously carried out once particles are
fully stabilized by all PEG components (Figure 14) .
TEM Analysis
TEM analysis was carried out using an ointment made using the
synthesis in Figure 14 in which NaOH was added after PEG 3350. A
small amounts ointment was suspended in ethanol . After
sonication, a drop of this suspension was placed on a grid, and
after drying, was examined in a FEI Tecnai F20 field emission gun
(FEG) transmission electron microscope (TEM) . The Tecnai F20
FEG-TEM was operated at 200 kV and is equipped with a Gatan Orius
SC600A CCD camera and an Oxford Instruments X-Max energy
dispersive X-ray (EDX) spectrometer. The final concentration of
Si was 83 mmol/kg as determined using EDX spectroscopy which
indicated the presence of silica as well as Cu. The TEM image
showed the nanoparticles of stabilised polymeric silicates
surrounded by a matrix of stabilising PEG 3350.
Iron sequestration
Silicate stabilised materials remove Fe from Fe-DMHP complex; at
5mM Si (Ratio 1:625 Fe:Si), all DMHP was unbound from the Fe
complex (Figure 15) . However, over time DMHP seemed to complex
iron again as seen by a shift towards the Fe-DMHP wavelength
(289.2 nm) in both stabilised and non-stabilised silicates
(Figure 16) .
Antimicrobial action of copper loaded Si NPs
Copper loaded silicate polymers showed antimicrobial activity,
but Growth retardants did not impact negatively in the bacterial
activity of copper. There was not a remarkable difference
between stabilised and non-stabilised materials (Figure 17) . In
practice, stabilisation would allow greater copper-loaded
silicate concentrations and no impact of the stabiliser on
efficacy .
Stabilised polymeric silicate compositions for GI administration
Compositions of the present invention may be tested to determine
their behaviour in the gastrointestinal tract. After exposure to
simulated digestion, the compositions undergo a rapid change such
that some silicic acid is released, that would be absorbed and
utilised by the body, whilst the remaining silicate forms larger
particles that would be safely excreted in the faeces.
Persistent nanoparticles and the concurrent risk of toxicity are
therefore avoided.
Stabilised polymeric silicate compositions for i.v.
administration
The gradual dilution of stabilised polymeric silicate
compositions stabilised using sucrose at pH 4.0 nanoparticles was
tested in a tubing containing saline at pH 7.4 and in equilibrium
with a larger body of saline, thereby mimicking intravenous
injection. Silicic acid is very rapidly formed enabling safe
parenteral delivery. Particle formation is not observed under
these conditions, but even if it were to happen, the
reticuloendothelium system would scavenge them effectively.
Exposure to potentially toxic nanoparticulate silicate is
minimum.
Stabilised polymeric silicate compositions formulated in a solid
or semi-solid matrix for topical application
Stabilised polymeric silicate compositions, optionally doped with
copper, of the present invention were incorporated in a hydroxy
ethyl cellulose gel (2% w/w) and for covering wounds. Optionally
metal ions such as Cu2+ or Ag+ may be included in the cream for
increasing its antibacterial properties.
Lactose treatment by heating- and particle size stability
according to US 5,807,951
0.91g of sodium metasilicate pentahydrate was dissolved in 10ml
of UHP water. 9.6 g of lactose was dissolved in 30ml of UHP water
at 50°C for 30 minutes. Both solutions were mixed and adjusted
to pH 8 with 0 .5M HC1 . The final material containing
approximately 70mM Si was analysed for particle size by DLS (A) .
An aliquot was collected, diluted to 40mM and adjusted to pH 7 to
mimic physiological conditions. This diluted suspension was also
characterised for particle size (B) . The same protocol as in
Figure 2 and the final suspension, approximately 70mM Si at pH 8 ,
was dried at 200°C.
Control of dissolution using metal ions
A uSANS dispersion (500 mM Si, pH 1.5) was spiked with soluble
metal ions and incubated at room temperature for 1 h . Figure 20
shows that low levels of metals inhibit the dissolution of uSANS
as determined by the molybdate assay.
References :
All documents mentioned in this specification are incorporated
herein by reference in their entirety.
Jugdaohsingh et al ., Is there a biochemical role for silicon?, in
Metal Ions in Biology and Medicine, Volume 10, pages 45-55, 2008,
John Libbey Eurotext: Montrouge.
WO 2009/052090.
US Patent Publication No: 2009/0130230.
US Patent Publication No: 2013/0149396.
US 5,807,951 (Nippon Zoki Pharmaceutical Co., Ltd.)
US Patent Publication No: 2011/0229577 (Kerek) .
Kim et al. (Macromolecules , 45: 4225-4237, 2012) .
Gao et al (Colloids and Surfaces A : Physicochem. Eng. Aspects
350 : 33-37, 2009) .
Claims :
1 . A process for producing a stabilised polymeric silicate
composition comprising polymeric silicic acid and nanosilicate
particles having mean diameters of 20 nm or less, the process
comprising the steps of:
(a) providing an aqueous solution of a soluble silicate at a
pH greater than or equal to 9.5;
(b) reducing the pH of the silicate solution to cause
polymerisation of the silicate to form polymeric silicic acid and
nanosilicate particles; and
(c) simultaneously or sequentially with steps (a) and/or (b)
adding to the silicate solution a stabilising agent that
comprises a polyalkylene glycol and/or a sugar thereby producing
a stabilised silicate composition in which the stabilising agent
inhibits formation of condensed silicates;
wherein the stabilised polymeric silicate composition is
aquated and wherein the process does not involve drying the
composition or heating it above 100°C.
2 . The process of claim 1 , wherein the stabilised polymeric
silicate composition comprises at least 5% water.
3 . The process of claim 1 or claim 2 , wherein the process does
not involve drying the composition or heating it above 70°C.
4 . The process of any one of claims 1 to 3 , wherein the
stabilised polymeric silicate composition is resorbable as
determined in an in vitro dissolution assay in which at least
25%, and optionally at least 35%, of the composition dissolves in
24 hours in HEPES buffer.
5 . The process of claim 4 , wherein the in vitro dissolution
assay is a molybdic acid assay for determining the soluble
silicic acid fraction.
6 . The process of any one of the preceding claims, which
comprises the further step after steps (b) and (c) of raising the
pH of the composition to a final pH by adding a base, and
optionally waiting for average particle size grow to the desired
size and then optionally adding further stabiliser and or
dropping the pH.
7 . The process of claim 6 , wherein the final pH is in the range
pH 3.0 to 9.0.
8 . The process of claim 6 or claim 7 , wherein the base is
sodium hydroxide or sodium carbonate.
9 . The process of any one of the preceding claims, further
comprising formulating the stabilised polymeric silicate
composition as a cream or ointment for topical administration to
a subject.
10. The process of claim 8 or claim 9 , wherein sodium hydroxide
is added to adjust the pH of the composition prior to formulating
the stabilised polymeric silicate composition as a cream or
ointment by mixing with polyalkylene glycol.
11. The process of claim 9 or claim 10, wherein formulating the
stabilised polymeric silicate composition as a cream or ointment
comprises mixing it with a solid or semi-solid matrix.
12. The process of claim 11, wherein the solid or semi-solid
matrix comprises one or more polyalkylene glycol polymers or one
or more hydroxyethyl cellulose gels.
13. The process of any one of claims 1 to , further comprising
formulating the stabilised polymeric silicate composition so that
it is formulated for oral administration or parenteral
administration of silicic acid to a subject.
14. The process of any one of the preceding claims, wherein in
step (a) the aqueous alkaline silicate solution is a Group 1 or
Group 2 metal silicate such as sodium silicate or potassium
silicate .
15. The process of any one of the preceding claims, wherein in
step (b) the pH is reduced to less than or equal to pH 4.0 by
adding an acid.
16. The process of any one of the preceding claims, wherein in
step (c) the pH is reduced to less than or equal to pH 3.0.
17. The process of any one of the preceding claims, wherein the
concentration of the silicate solution is between 5mM and 3.0 M .
18. The process of any one of the preceding claims, wherein the
concentration of the silicate solution is between 0.1 M and 1.5
M .
19. The process of any one of the preceding claims, wherein
stabilised polymeric silicate composition is stable for 1 month
or more, 2 months or more, 3 months or more, 6 months or more.
20. The process of any one of the preceding claims, wherein the
nanosilicate particles have a mean diameter of 10 nm or less.
21. The process of any one of the preceding claims, wherein the
nanosilicate particles have a mean diameter of 5 nm or less.
22. The process of any one of the preceding claims, wherein the
concentration of the silicate solution is more than 30mM.
23. The process of any one of the preceding claims, wherein in
step (a) the pH of the alkaline silicate solution is above pH
11.5.
24. The process of any one of the preceding claims, wherein two,
three, four or five stabilising agents are added in step (c) .
25. The process of any one of the preceding claims, wherein the
stabilising agent is sucrose or polyethylene glycol (PEG) .
26. The process of any one of the preceding claims, wherein the
stabilising agent is not lactose or mannitol.
27. The process of any one of the preceding claims, wherein in
step (b) the pH of the composition is lowered to a pH less than
or equal to pH 1.5.
28. The process of any one of the preceding claims, further
comprising adding a metal cation to the composition.
29. The process of claim 28, wherein the metal cation is Cu2+ ,
A g+, Ca2+ , Mg + , Fe3+ and/or Zn2+ .
30. The process of claims 28 or claim 29, wherein the metal
cation inhibit dissolution of the composition.
31. The process of claims 28 or claim 29, wherein the metal
cation provides the composition with antibacterial properties.
32. The process of any one of claims 29 to 31, wherein the metal
cation is added to provide a Si to metal ratio of between 100:1
and 10:1, and optionally to provide a Si to metal ratio of 20:1.
33. The process of any one of the preceding claims, wherein in
step (b) the pH is lowered over a period of less than 60 seconds,
less than 30 seconds, less that 10 seconds, or less than 5
seconds
34. A stabilised polymeric silicate composition comprising
polymeric silicic acid and nanosilicate particles having mean
diameters of 20 n or less as obtainable by the process of any
one of claims 1 to 33.
35. A stabilised polymeric silicate composition comprising
polymeric silicic acid and nanosilicate particles having mean
diameters of 20 n or less as obtainable by the process of any
one of claims 1 to 33 for use in a method of treatment.
36. The stabilised polymeric silicate composition for use in a
method of treatment of claim 35, wherein the composition is
formulated for parenteral administration.
37. The stabilised polymeric silicate composition for use in a
method of treatment of claim 36, wherein the parenteral
administration is intravenous (IV), intraperitoneal (IP) or
intramuscular (I ) administration.
38. The stabilised polymeric silicate composition for use in a
method of treatment of claim 36 or claim 37, wherein the
parenteral administration is intravenous (IV) administration via
an intravenous drip.
39. The stabilised polymeric silicate composition for use in a
method of treatment of any one of claims 36 to 38, wherein the
stabilising agent is sucrose and/or polyethylene glycol (PEG) .
40. The stabilised polymeric silicate composition for use in a
method of treatment of any one of claims 36 to 39, wherein the
composition is diluted or neutralised to a physiologically
acceptable pH for administration.
41. A stabilised polymeric silicate composition for use in a
method of treatment, wherein the composition comprising polymeric
silicic acid and nanosilicate particles having mean diameters of
20 nm or less and a stabilising agent comprising sucrose and/or a
polyalkylene glycol, wherein composition is formulated for
intravenous (IV) administration via an intravenous drip.
42. A stabilised polymeric silicate composition comprising
polymeric silicic acid and nanosilicate particles having mean
diameters of 20 nm or less as obtainable by the process of any
one of claims 1 to 33 for use in a method of promoting wound
healing and/or treating or preventing bacterial infection,
wherein the composition is formulated for topical administration.
43. A stabilised polymeric silicate composition for use in a
method of treatment, wherein the composition comprising polymeric
silicic acid and nanosilicate particles having mean diameters of
20 n or less and a stabilising agent comprising a polyalkylene
glycol, wherein composition is formulated for topical
administration, the composition is for use in a method of
promoting wound healing and/or treating or preventing bacterial
infection .
44. The stabilised polymeric silicate composition for use in a
method of treatment of claim 42 or claim 43, wherein the
composition is formulated as a cream or an ointment, optionally
wherein the cream or ointment comprises a polyalkylene glycol.
45. The stabilised polymeric silicate composition for use in a
method of treatment of any one of claims 42 to 44, wherein the
polyalkylene glycol is polyethylene glycol (PEG) .
46. The stabilised polymeric silicate composition for use in a
method of treatment of any one of claims 34 to 45, wherein the pH
is between 3.0 and 9.0.
47. The stabilised polymeric silicate composition for use in
method of treatment of any one of claims 34 to 46, wherein the
composition has a concentration of silicon of 2 .5mM or more,
5 .OmM or more, 25mM or more,40mM or more.
48. The stabilised polymeric silicate composition for use in
method of treatment of any one of claims 34 to 47, wherein
composition is for treating a human subject.
49. The stabilised polymeric silicate composition for use in a
method of treatment of any one of claims 34 to 47 for veterinary
administration .
50. A silicate-containing supplement comprising a stabilised
polymeric silicate composition comprising polymeric silicic acid
and nanosilicate particles having mean diameters of 20 nm or less
as obtainable by the process of any one of claims 1 to 33 for use
in the delivery of silicic acid to a human or animal subject.
51. The silicate-containing supplement of claim 50, wherein the
composition is in the form of a liquid filled capsule.
52. The silicate-containing supplement of claim 50 or claim 51,
wherein the composition is for direct oral administration.
53. The silicate-containing supplement of any one of claims 50
to 52, wherein the composition is a silicate supplement for
improving the appearance of hair, skin or nails.
54. A composition comprising a stabilised polymeric silicate
composition comprising polymeric silicic acid and nanosilicate
particles having mean diameters of 20 nm or less as obtainable by
the process of any one of claims 1 to 33 for use therapy.
55. The stabilised polymeric silicate composition for use in
therapy of claim 54 for use in method of treating osteoporosis,
osteopenia, musculoskeletal and joint disorders, cancer, skin
conditions, vascular diseases, cardiovascular diseases, coronary
heart diseases, inflammatory diseases, autoimmune diseases,
Alzheimer's disease, cognitive impairment, infections, wounds ,
ulcers, gastrointestinal disorders, liver disease, kidney
disease, immune related disorders or hormone related disorders.
56. The stabilised polymeric silicate composition for use in
therapy of claim 54, wherein the composition is for oral delivery
to bind cations in the gut, such as iron.
57. The stabilised polymeric silicate composition for use in
therapy of claim 54, wherein the composition is for intravenous
delivery for the treatment of cancer
58. The stabilised polymeric silicate composition for use in
therapy of claim 54, wherein the composition is for intravenous
delivery for the treatment of infections
59. A silicate-containing supplement comprising a stabilised
polymeric silicate composition comprising polymeric silicic acid
and nanosilicate particles having mean diameters of 20 nm or less
as obtainable by the process of any one of claims 1 to 33 for use
in the delivery of transiently stable silicate polymers to a
human or animal subject.
60. The silicate-containing supplement comprising a stabilised
polymeric silicate composition of claim 59, wherein the
composition is for oral supplementation and comprises particles
have mean diameters of 5 nm or less (uSANS) .