U.S. patent application number 16/895743 was filed with the patent office on 2020-09-24 for particles.
The applicant listed for this patent is The University of Liverpool. Invention is credited to Pierre Chambon, Jane Ford, Marco Giardiello, Neil Kitteringham, Andrew Owen, Steven Rannard, Hannah Rogers.
Application Number | 20200297652 16/895743 |
Document ID | / |
Family ID | 1000004874066 |
Filed Date | 2020-09-24 |
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United States Patent
Application |
20200297652 |
Kind Code |
A1 |
Rannard; Steven ; et
al. |
September 24, 2020 |
PARTICLES
Abstract
Particles comprising a branched polymer and either a block
copolymer or a linear dendritic hybrid represent a category of
useful materials. They may be used in for example drug delivery
applications. They may be prepared by a method comprising the steps
of: dissolving the branched polymer and block copolymer or linear
dendritic hybrid, and optionally other component(s), in a solvent
to form a solution; adding said solution to a different liquid; and
removing said solvent to form a dispersion of co-precipitated
particles.
Inventors: |
Rannard; Steven;
(Merseyside, GB) ; Ford; Jane; (Merseyside,
GB) ; Rogers; Hannah; (Merseyside, GB) ;
Chambon; Pierre; (Liverpool, GB) ; Giardiello;
Marco; (Merseyside, GB) ; Owen; Andrew;
(Merseyside, GB) ; Kitteringham; Neil;
(Merseyside, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Liverpool |
Liverpool |
|
GB |
|
|
Family ID: |
1000004874066 |
Appl. No.: |
16/895743 |
Filed: |
June 8, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15326775 |
Jan 17, 2017 |
10675251 |
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|
PCT/GB2015/052089 |
Jul 17, 2015 |
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16895743 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/427 20130101;
C08G 81/025 20130101; A61K 9/5153 20130101; C08F 220/1804 20200201;
A61K 9/5192 20130101; C08F 2438/01 20130101; A61K 31/4745 20130101;
C08F 220/20 20130101; A61K 9/5146 20130101; A61K 31/192 20130101;
C08G 83/003 20130101; C08F 220/34 20130101; C08G 63/08 20130101;
A61K 9/5138 20130101; C08F 222/102 20200201; A61K 31/513 20130101;
C08L 33/10 20130101; C08F 2/38 20130101; A61K 31/536 20130101 |
International
Class: |
A61K 9/51 20060101
A61K009/51; A61K 31/4745 20060101 A61K031/4745; C08G 83/00 20060101
C08G083/00; A61K 31/192 20060101 A61K031/192; C08F 220/20 20060101
C08F220/20; C08G 63/08 20060101 C08G063/08; C08F 2/38 20060101
C08F002/38; C08L 33/10 20060101 C08L033/10; A61K 31/427 20060101
A61K031/427; A61K 31/513 20060101 A61K031/513; A61K 31/536 20060101
A61K031/536; C08G 81/02 20060101 C08G081/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 18, 2014 |
GB |
1412841.7 |
Claims
1. Particles comprising both a branched polymer and a block
copolymer.
2. Particles as claimed in claim 1 wherein the block copolymer is a
diblock copolymer.
3. Particles as claimed in claim 1 wherein the block copolymer
comprises a vinyl polymer block.
4. Particles as claimed in claim 1 wherein the branched polymer is
a branched vinyl polymer.
5. Particles as claimed in claim 1 wherein the branched polymer is
a branched vinyl polymer and wherein the block copolymer comprises
a vinyl polymer block.
6. Particles as claimed in claim 1 prepared by
co-precipitation.
7. Particles as claimed in claim 1 wherein the branched polymer is
a branched vinyl polymer, wherein the block copolymer comprises a
vinyl polymer block, and wherein the particles are prepared by
co-precipitation.
8. Particles as claimed in claim 3 wherein the vinyl polymer block
comprises HPMA, nBuMA, tBuMA, DEAEMA or styrene.
9. Particles as claimed in claim 1 wherein the block copolymer
comprises a PEG block.
10. Particles as claimed in claim 1 wherein the branched vinyl
polymer comprises one or more of the monomers HPMA, nBuMA, tBuMA,
styrene, and DEAEMA, and/or wherein the branched vinyl polymer
comprises a brancher selected from EGDMA and BDME.
11. Particles as claimed in claim 1 wherein the branched polymer is
a branched vinyl polymer comprising: HPMA and EGDMA; or HPMA, nBuMA
and EGDMA; or HPMA, tBuMA and EGDMA; or nBuMA and EGDMA; and the
block copolymer is a diblock copolymer comprising HPMA and PEO.
12. Particles as claimed in claim 1 further comprising a drug,
prodrug or other biologically active component.
13. Particles as claimed in claim 12 wherein the drug is an HIV
antiretroviral, anticancer drug, or ibuprofen.
14. Particles as claimed in claim 1 which are nanoparticles.
15. Particles as claimed in claim 1 in solid form.
16. A composition containing particles as claimed in claim 1
dispersed in water or an aqueous phase.
17. A method of drug delivery comprising administering a
therapeutically effective amount of the particles of claim 12 to a
patient in need thereof.
18. A method of treating HIV or cancer comprising administering a
therapeutically effective amount of the particles of claim 12 to a
patient in need thereof.
19. A method of preparing particles as defined in claim 1
comprising: dissolving the branched polymer and block copolymer,
and optionally other component(s), in a solvent to form a solution;
adding said solution to a different liquid; and removing said
solvent to form a dispersion of co-precipitated particles.
20. A method as claimed in claim 19 further comprising the
following subsequent steps, one or more times: adding further
solution (of the branched polymer and the block copolymer, and
optionally the other component(s), in the solvent) to the liquid;
and removing the solvent.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a Continuation of U.S.
Nonprovisional patent application Ser. No. 15/326,775, filed Jan.
17, 2017, which is the U.S. National Phase of International Patent
Application Serial No. PCT/GB2015/052089, filed Jul. 17, 2015,
which claims priority to GB Application No. 1412841.7, filed Jul.
18, 2014. The entire disclosures of the applications noted above
are incorporated herein by reference.
[0002] The present invention relates to particles comprising
organic polymers. These particles are useful in for example drug
delivery applications.
[0003] We have previously disclosed various polymeric materials and
technologies to produce molecular architectures which are useful in
various fields including biology and nanotechnology.
[0004] For example WO 2009/12220 discloses polymer-dendrimer
hybrids, also known as polydendrons, which comprise a branched
vinyl polymer scaffold carrying dendrons: these polydendrons
possess advantageous dendrimer-type properties in part due to their
multiply branched nature, without the disadvantages of complex
conventional dendrimer processes.
[0005] It is useful to be able to control the properties of such
materials including for example their size, size distribution,
functionality, hydrophobicity/hydrophilicity, and behaviour in
systems of use, e.g. in aqueous systems in the case of biological
applications.
[0006] We have now developed a new category of useful
materials.
[0007] From a first aspect the present invention provides particles
comprising a branched polymer and either a block copolymer or a
linear dendritic hybrid.
[0008] The branched polymer may be of various types. Two example
types are branched vinyl polymers and branched polyesters.
[0009] Branched vinyl polymers may be prepared by known methods,
from monofunctional vinyl monomers and difunctional vinyl monomers
(branching agents).
[0010] Branched polyesters may be prepared by for example ring
opening polymerization of monofunctional lactone monomers and
difunctional lactone monomers (branching agents).
[0011] Block copolymers comprise blocks or segments each of which
have repeating units of the same type within each block and which
are different between block segments. The present invention has
been found to be particularly effective with diblock copolymers,
i.e. copolymers which have two distinct blocks. Other block
copolymers may be used, e.g. triblock copolymers.
[0012] Linear dendritic hybrids, also known as linear dendritic
polymers, likewise comprise distinct segments, one of which is a
linear polymer chain and another of which is a dendron. We have
found that the present invention is particularly effective with a
linear dendritic hybrid which comprises a homopolymer terminating
in or initiated by a dendron. Nevertheless other types of linear
dendritic hybrid (e.g. wherein the linear polymer is itself a
copolymer, for example a statistical copolymer, block copolymer or
diblock copolymer) may be used in the present invention. Thus for
example a linear dendritic hybrid may comprise one block joined to
another block which is then joined to a dendron.
[0013] The particles may be nanoparticles, for example particles
wherein at least one dimension, or wherein the z-average diameter
(also referred to as the average hydrodynamic diameter), is no
greater than about 1000 nm. The particles may have z-average
diameters less than 800 nm, or less than 500 nm, or less than 300
nm, e.g. around 50 to 300 nm, or 50 to 250 nm, or 100 to 200
nm.
[0014] It is important to note that the particles of the present
invention contain two types of polymeric material, i.e. a branched
polymer and either a block copolymer or a linear dendritic hybrid
(and optionally further materials as discussed below) but that
these are not covalently bonded to each other. In contrast,
polydendrons, as disclosed in for example WO 2009/12220, contain
dendron units which are covalently attached to a branched vinyl
polymer scaffold. In the present invention, the different polymeric
materials are associated with each other within a particle
structure, but are not covalently bonded to each other. Preferably
most, or substantially all, or all, of the particles have the same
general structure. The regular way in which the parts associate,
intermingle or assemble brings about reliable and reproducible
properties.
[0015] The combination of polymeric structures in the present
invention brings about particular advantages.
[0016] The first essential component, the branched polymer
component, may be generally hydrophobic and therefore useful for
providing an environment for associating with, carrying, or
encapsulating, a hydrophobic compound, moiety or payload, for
example a drug. Alternatively it may have hydrophilic properties.
It may be responsive to pH or other conditions or stimuli such that
its properties, e.g. its hydrophobic/hydrophilic properties, its
structure, and/or its behaviour in liquid phases, may vary
depending on the environment.
[0017] The second essential component has at least two segments
(different homopolymer or copolymer blocks in the case of block
copolymers, or a linear polymer block plus a dendron in the case of
linear dendritic hybrids). This allows the resultant particles to
be suitable for or tailorable for use in a variety of media and
environments and for a variety of applications. It also allows one
of the segments to act as a stabilizer to stabilize the particles
within liquid media, for example in aqueous media, and optionally
to provide salt stability to stabilize the particles in a range of
salt conditions and/or in a range of physiological conditions which
might be encountered in living systems.
[0018] The block copolymer or linear dendritic hybrid may comprise
a hydrophobic segment. This enables it to associate with a
hydrophobic branched polymer (and optionally with hydrophobic
carried material). The hydrophobic segment may comprise a vinyl
polymer.
[0019] The block copolymer or linear dendritic hybrid may comprise
a hydrophilic segment. This is useful in for example aqueous
environments and enables the stabilisation of the nanoparticles.
The hydrophilic segment may comprise a polyether structure such as
polyethylene glycol (PEG) or PEO, for example.
[0020] Of course, the block copolymer or linear dendritic hybrid
may comprise both a hydrophobic segment and a hydrophilic segment
and thereby exhibit amphiphilic behaviour.
[0021] Thus we have developed particularly advantageous
combinations where the branched polymer is hydrophobic and forms a
hydrophobic core and where the block copolymer or linear dendritic
hybrid is amphiphilic such that one segment thereof can associate
with the hydrophobic core and another segment thereof can stabilise
the particles in aqueous media. These exhibit considerable
uniformity in terms of size, and stability in aqueous media and
under varied salt conditions.
[0022] Nevertheless other combinations of types of polymer and
consequent properties are also useful in other scenarios, and are
within the scope of the present invention.
[0023] The branched polymer, block copolymer or linear dendritic
hybrid may comprise chemistry which is responsive to pH or other
conditions or stimuli such that its properties, e.g. its
hydrophobic/hydrophilic properties, may vary depending on the
environment.
[0024] The linear dendritic hybrid comprises a linear polymer chain
and a dendron. Said linear polymer chain may itself be a copolymer,
e.g. a statistical copolymer or a block copolymer.
[0025] The particles may comprise a further material, for example a
chemical compound, organic compound, hydrophobic compound,
hydrophilic compound, drug, prodrug or therapeutically,
diagnostically or biologically useful material. Indeed, one of the
main uses of the present invention is in drug delivery.
[0026] The further materials, e.g. drugs or biologically active
materials, may be included as or within separate compounds within
the particles, or may be covalently bound to one of the polymeric
structures, e.g. to the branched polymer, block copolymer or linear
dendritic hybrid
[0027] From a further aspect the particles are prepared by
co-precipitation.
[0028] The method of preparation may comprise: [0029] dissolving
the branched polymer and either block copolymer or linear dendritic
hybrid, and optionally other component(s), in a solvent to form a
solution [0030] adding said solution to a different liquid [0031]
removing the solvent, to form a dispersion of co-precipitated
particles within the liquid.
[0032] The solvent may be removed by for example allowing said
solvent to evaporate or through dialysis.
[0033] The dispersion may be used for applications, e.g.
therapeutic applications, as it is, or alternatively the dispersion
may be concentrated or gelled, or the liquid may be removed to
result in solid particles.
[0034] The solvent in which the materials are dissolved may be an
organic solvent, suitably a solvent which is miscible with the
liquid in which precipitation occurs, e.g. water-miscible solvents.
For example the solvent in which the materials are dissolved may be
e.g. acetone, acetonitrile, dimethyl formamide, dimethyl sulfoxide,
dioxane, ethanol, isopropyl alcohol, methanol, tetrahydrofuran and
any combination and mixture of them or combinations comprising
water.
[0035] The solvent in which the materials are dissolved may be one
which is chosen because it evaporates relatively easily, e.g. one
which has a boiling point of 80 degrees C. or less, or 70 degrees
C. or less, or 60 degrees C. or less.
[0036] The liquid in which the precipitation occurs may for example
be water or other aqueous system, or other liquid in which the
materials do not dissolve, or dissolve less well in comparison to
the first solvent.
[0037] Various aqueous solutions can be used. Some examples
include: [0038] aqueous solutions of for example alkali
metal/halogen salts (e.g. NaCl, LiBr, KCl). at all concentrations
(up to saturation) [0039] aqueous solution within a pH range of 0
to 12, including acid and base (BrOnsted-Lowry mineral and organic
acids and bases) solutions (e.g. aqueous solutions of e.g. HCl,
H2SO4, acetic acid, NaOH, or KOH) [0040] surfactant (e.g. Sodium
dodecyl sulphate, amphiphilic diblock copolymers, TWEEN.RTM.,
BRIJ.RTM.) solutions in water at all concentrations (below and
above critical micellar concentration) [0041] biological media
[e.g. transport buffer, bovine serum albumin (BSA), dulbecco's
modified eagle's medium (DMEM), Roswell park memorial institute
1640 (RPMI 1640), foetal bovine serum (FBS)] in aqueous systems,
and mixtures of the same, at all suitable concentrations.
[0042] The aqueous systems listed above provide some indication of
the wide range of systems in which the present invention works,
consistent with applications in a variety of conditions including
therapeutic applications, where there is a need to endure varied
and sometimes extreme physiological environments.
[0043] The liquid in which precipitation occurs may be a mixture of
water and organic solvent e.g. acetone/water mixture, THF/water,
MeOH/water, EtOH/water, or IPA/water. The amount of water in such
mixtures may optionally be 2 parts water by volume to 1 part
organic solvent by volume, or greater than 2 parts water (e.g.
greater than 3 parts water, e.g. greater than 4 parts water) by
volume to 1 part organic solvent by volume.
[0044] One of the inventive points of distinction of the present
invention over the prior art is that the present invention involves
the co-precipitation of two specific types of polymer at the same
time from organic solvent into water. Whereas precipitation
(including nanoprecipitation) of organic polymers has previously
been disclosed in various documents and has been carried out with
varying degrees of success (see, for example, Slater et al, Soft
Matter 2012, 8, 9816-9827), this has not been in relation to a
combination of polymer types as defined in the present invention.
We are first to recognize, and demonstrate, the benefits of such
co-precipitation.
[0045] Optionally, the co-precipitation may be carried out multiple
times. Thus, after the formation of a dispersion of co-precipitated
particles within the liquid (and the subsequent removal of organic
solvent), the following method steps may additionally be carried
out, one or more times: [0046] adding further solution (of the
branched polymer and either the block copolymer or the linear
dendritic hybrid, and optionally the other component(s), in the
solvent) to the liquid [0047] removing the solvent.
[0048] We have found that, by carrying out multiple
co-precipitation, it is possible to increase the concentration of
particles within the liquid whilst substantially maintaining the
particle size, PdI and stability. In other words, multiple
nanoprecipitation can be used to provide more of the same type of
nanoparticles per unit volume, whereas it might have been expected
that multiple nanoprecipitation would result in significantly
larger particles, different dispersity characteristics, or
instability. This is particularly advantageous in facilitating drug
delivery where an enhanced payload can result in practical
therapeutic advantages and cost benefits.
The Branched Polymer Component
[0049] The branched polymer is an essential component of the
particles of the present invention.
[0050] The branched polymer is non-gelled and processable and
optionally of low viscosity. It is soluble in organic solvents, as
described above in relation to a suitable method of forming the
particles by precipitation from such solvents into a different
medium. It can be contrasted with polymer structures which are
insoluble and/or exhibit high viscosity, such as extensively
crosslinked insoluble polymer networks, high molecular weight
linear polymers, or microgels.
Branched Vinyl Polymers
[0051] The branched polymer may be a branched vinyl polymer. This
can be made by, but is not limited to being made by, living
polymerization, controlled polymerization or conventional
chain-growth polymerization techniques such as free radical
polymerisation. Several types of living and controlled
polymerization are known in the art and suitable for use in the
present invention. A preferred type of living polymerization is
Atom Transfer Radical Polymerization (ATRP); however other
techniques such as Reversible Addition-Fragmentation chain-Transfer
(RAFT) and Nitroxide Mediated Polymerisation (NMP) or conventional
free-radical polymerization controlled by the deliberate addition
of chain-transfer agents are also suitable syntheses.
[0052] The skilled person is aware of techniques to provide
branched but non-gelled vinyl polymers. For example, suitable
procedures are described in N. O'Brien, A. McKee, D. C.
Sherrington, A. T. Slark and A. Titterton, Polymer 2000, 41,
6027-6031; T. He, D. J. Adams, M. F. Butler, C. T. Yeoh, A. I.
Cooper and S. P. Rannard, Angew. Chem. Int. Ed. 2007, 46,
9243-9247; V. Butun, I. Bannister, N. C. Billingham, D. C.
Sherrington and S. P. Armes, Macromolecules 2005, 38, 4977-4982; I.
Bannister, N. C. Billingham, S. P. Armes, S. P. Rannard and P.
Findlay, Macromolecules 2006, 39, 7483-7492; and R. A. Slater, T. O
McDonald, D. J. Adams, E. R. Draper, J. V. M. Weaver and S P
Rannard, Soft Matter 2012, 8, 9816-9827. The non-gelled and soluble
products of the present invention are different to materials
disclosed in L. A. Connal, R. Vestberg, C J. Hawker and G. G. Qiao,
Macromolecules 2007, 40, 7855-7863 which are known to comprise
multiple cross-linking in a gelled network.
[0053] The polymerization of each vinyl polymer chain starts at an
initiator. Polymerization of monofunctional vinyl monomers leads to
linear polymer chains. Copolymerization with difunctional vinyl
monomers leads to branching between the chains. In order to control
branching and prevent gelation there should be less than one
effective brancher (difunctional vinyl monomer) per chain. Under
certain conditions, this can be achieved by using a molar ratio of
brancher to initiator of less than one: this assumes that the
monomer (i.e. the monofunctional vinyl monomer) and the brancher
(i.e. the difunctional vinyl monomer) have the same reactivity,
that there is no intramolecular reaction, that the two
functionalities of the brancher have the same or similar
reactivity, and that reactivity remains the same even after
part-reaction. Of course, the systems and conditions may be
different, but the skilled person understands how to control the
reaction and determine without undue experimentation how a
non-gelled structure may be achieved. For example, under dilute
conditions some branchers form intramolecular cycles which limit
the number of branchers that branch between chains even if the
molar ratio of brancher to initiator (i.e. polymer chain) is higher
than 1:1 in the reaction.
[0054] Initiators and other reagents used in the polymerisation
process are as known in the art. For example, in ATRP, convenient
and effective initiators include alkyl halides (e.g. alkyl
bromides) and in conventional free radical polymerisation,
effective initiators include azo compounds
Other Branched Polymers
[0055] Other suitable types of branched polymers include branched
polyesters. These may be prepared by for example ring opening
polymerization of monofunctional lactone monomers and difunctional
lactone monomers (branching agents). Ring opening polymerization
methods and materials are known in the art, for example from Nguyen
et al., Polym Chem 2014, 5, 2997-3008.
Amount of Branched Polymer in Relation to Linear Polymer and Other
Optional Components
[0056] The amount of branched polymer (e.g. branched vinyl polymer)
may optionally be no greater than 95 wt %, or no greater than 75 wt
%, or no greater than 50 wt %, or no greater than 10 wt %, or no
greater than 1 wt %. In this context wt % denotes the amount of
branched vinyl polymer as a percentage of the total mass of solid
material in the particles. The amount of branched polymer may
optionally be at least 0.1 wt %, or at least 0.5 wt %. For example
the amount of branched polymer may be between about 0.1 wt % and 95
wt %, e.g. between 1 wt % and 10 wt %.
[0057] Surprisingly we have found that relatively low amounts of
branched polymer (optionally around 1-10 wt % in some cases) are
effective in being able to direct the nanoprecipitation, and in
allowing the formation of regular particles of narrow dispersity.
Thus, in some applications this can bring a benefit in allowing the
amount of other component(s) (i.e. the block copolymer component or
the linear dendritic hybrid, plus any other components e.g. carried
materials) to be maximised.
The Block Copolymer Component
[0058] The block copolymer component can comprise various types of
polymer including for example vinyl polymers, polyethers (e.g. PEO
or PPO), polyesters (e.g. polycaprolactone or polylactic acid),
polyurethanes, polyamides, or polycarbonates.
[0059] There are (at least) two distinct blocks. The type of
polymer forming each block may be the same or different, though of
course if the type of polymer is the same then for the blocks to
differ the monomers, or mixture of monomers, must differ.
[0060] For example, the block copolymer may comprise two blocks
each of which is a vinyl polymer chain, wherein the vinyl monomer
making up one block differs from the vinyl monomer making up the
other block.
[0061] Alternatively the type of polymer forming each block may be
different. For example the block copolymer may comprise one block
which is a vinyl polymer chain and a second block which is a
polyether chain or a polyester chain.
[0062] One convenient known way of preparing a block copolymer
wherein one block is a vinyl chain is to use a macroinitiator in a
vinyl polymerisation process wherein the macroinitiator comprises
the other or another block. For example, the macroinitiator may be
a bromide initiator comprising a PEO or other polymer chain. In a
further variation, said initiator may be bifunctional and may
therefore allow the preparation of a triblock copolymer (for
example being an A-B-A block copolymer where A is a vinyl, often
hydrophobic, block, and B is a PEO, hydrophilic, block).
[0063] The molecular mass of the PEO may for example be greater
than 1,000 g/mol, for example between 1,000 and 10,000 g/mol; for
example PEG 2K or PEG 5K.
[0064] Another example is to use a polymer (e.g. a hydrophilic
polymer) bearing a hydroxyl group as a macroinitiator for ring
opening polymerisation (e.g. of cyclic esters, e.g.
.quadrature.-caprolactone or other cyclic structures). Known
ring-opening polymerisation techniques may be used.
[0065] Other ways to synthesise diblock copolymers include for
example one-pot methods in which one monomer (e.g. a vinyl monomer)
is polymerised first (generally a hydrophilic block first, but not
necessarily) and then, when this first reaction has reached the
desired monomer conversion, and without any purification of the
first block, the second monomer (which may for example lead to a
hydrophobic block if the first monomer leads to a hydrophilic
block) may be added in situ. In each case, the monomers added may
be a mixture of monomers leading to a statistical copolymerisation
within each block. Other known methods of preparing block
copolymers may be used.
The Linear Dendritic Hybrid
[0066] The linear dendritic hybrid comprises a polymer chain and a
dendron. The dendron is covalently attached to the polymer chain.
The polymer chain can comprise various types of polymer including
vinyl polymers, polyesters, polyamides, polycarbonates,
polyurethanes, or polyethers (e.g. PEO or PPO).
[0067] Linear dendritic hybrids are known from various documents,
e.g. F. Wurm, H. Frey, Prog. Polym. Sci. 2011, 36, 1-52.
[0068] The dendron may be incorporated in various ways. One
possible method uses dendrons as macromolecular initiators in for
example vinyl polymerisation. This allows the formation of for
example linear dendritic hybrids having a vinyl polymer chain with
a dendron at the end of the chain. Dendrons may also be used as
chain transfer agents to regulate and initiate a chain growth
polymerisation.
[0069] In order to be able to initiate polymerization, the dendrons
must bear suitable reactive functionality. For example, in ATRP,
dendrons which carry halides (e.g. bromides) at their focal points
can act as initiators. In this scenario, propagation starts at the
apex, or focal point, of the dendron "wedge". The skilled person is
well aware of the types of components and reagents which are used
in polymerisations including ATRP and other living or controlled
polymerizations and conventional free radical polymerisation, and
hence the type of functionality which must be present on or
introduced to dendrons for them to act as initiators or chain
transfer agents.
[0070] For example, one possible way of introducing bromo groups to
dendrons is to functionalize dendron alcohols with
alpha-bromoisobutyryl bromide. There are however many other ways of
functionalizing dendrons so that they can act as initiators and
chain transfer agents, and other types of functionality which will
initiate polymerization. The concept of a dendron initiator is
applicable to all suitable types of polymerization and the
functionality can be varied as necessary.
[0071] Another method of incorporating dendron comprises ring
opening polymerisation e.g. of cyclic esters, using dendrons with
suitable functional groups (e.g. --OH groups).
[0072] A further method uses dendrons as chain transfer agents for
polymerisation with reversible addition-fragmentation chain
transfer (RAFT) or conventional free radical polymerisation.
[0073] Other possible methods include post-functionalisation of
polymers via chain-end/dendron coupling, for example through the
use of a thiol group at the focal point.
[0074] Other methods known in the art for the incorporation of
dendrons into linear dendritic hybrids may be used.
[0075] There is no particular limitation regarding the type of
dendron that can be used, or the chemistry used to prepare the
dendrons. In some scenarios it is desirable to have particular
groups present at the surface (i.e. at the tips of the "branches"
of the dendron), and these may be incorporated during the synthesis
of the dendron. The dendrons are preferably non-vinyl.
[0076] Any suitable coupling chemistry may be used to build up the
dendrons. In one example, amines and alcohols may be coupled
together, for example using carbonyldiimidazole. This is, however,
merely one example and numerous other coupling methods, such as
Michael addition chemistry or well known esterification techniques,
are possible.
[0077] The dendrons may comprise various moieties including for
example amines (e.g. dendrons which are branched at tertiary amine
centres, or dendrons which terminate at NMe.sub.2 groups), hydroxyl
groups, acids, carboxyl groups, or PEG groups.
[0078] The branched polymer may optionally contain alcohol
groups.
[0079] The branched polymer may optionally contain amine
groups.
[0080] The branched polymer may optionally contain carboxyl
groups.
[0081] The block copolymer may optionally contain alcohol
groups.
[0082] The block copolymer may optionally contain amine groups.
[0083] The block copolymer may optionally contain carboxyl
groups.
[0084] The polymer chain of the linear dendritic hybrid may
optionally contain alcohol groups.
[0085] The polymer chain of the linear dendritic hybrid may
optionally contain amine groups.
[0086] The polymer chain of the linear dendritic hybrid may
optionally contain carboxyl groups.
EXAMPLES, FIGURES AND EXPERIMENTAL DETAILS
[0087] The present invention will now be described in further
non-limiting detail, by way of example, with reference to the
figures in which:
[0088] FIG. 1 shows a schematic representation of a branched vinyl
polymer, diblock copolymers, and a particle of the present
invention containing the branched vinyl polymer and diblock
copolymers, and also some monomers which may be used in the
preparation of the polymers;
[0089] FIG. 2 shows a schematic representation of one possible
co-precipitation procedure in accordance with the present
invention;
[0090] FIG. 3 shows SEM images of some nanoparticles in accordance
with the present invention;
[0091] FIGS. 4 and 5 shows the stability of particles of the
present invention to salt solution and over time;
[0092] FIG. 6 shows an SEM image of nanoparticles in accordance
with the present invention;
[0093] FIGS. 7 to 12 show particle size distributions of
drug-loaded particles;
[0094] FIG. 13 shows a schematic representation of a branched
polymer, linear dendritic hybrid, and a nanoparticle of the present
invention containing both, in two different architectures;
[0095] FIGS. 14 to 18 show some combinations of branched polymers
and linear dendritic hybrids and their properties;
[0096] FIG. 19 shows an SEM image of some nanoparticles; and
[0097] FIG. 20 shows the effect on cell viability of some
nanoparticles of the present invention.
PARTICLES CONTAINING BRANCHED POLYMERS AND BLOCK COPOLYMERS
[0098] One group of particles in accordance with the present
invention are those which contain branched polymers and block
copolymers.
[0099] FIG. 1 shows an overview of some components used. A branched
vinyl polymer (top left) contains linear chains formed from vinyl
monomers which are branched by use of a brancher. A wide variety of
vinyl monomers may be used, some examples of which are shown
(bottom left).
[0100] The vinyl monomers shown in the bottom left of FIG. 1, and
their abbreviations as used herein, are as follows: [0101] HPMA:
hydroxypropyl methacrylate. 2-hydroxypropyl methacrylate is shown,
though the invention can utilize not only this isomer alone but
optionally a commercially available mixture of this isomer and
2-hydroxyisopropyl methacrylate or the latter alone. [0102] nBuMA:
n-butyl methacrylate [0103] Styrene [0104] tBuMA: t-butyl
methacrylate [0105] DEAEMA: N,N-diethylaminoethyl methacrylate, a
monomer which is hydrophilic in comparison to the above-mentioned
monomers, and pH-responsive.
[0106] As indicated in FIG. 1, combinations of monomers may be used
e.g. HPMA and nBuMA, or HPMA and tBuMA.
[0107] Other materials used include the following, also shown in
FIG. 1: [0108] EBIB: ethyl alpha-bromoisobutyrate, an initiator
commonly used to initiate polymerisation [0109] EGDMA: ethylene
glycol dimethacrylate, a suitable brancher [0110] PEG 2K or PEG 5K:
polyethylene oxide of size 2K or 5K (although of course other sizes
are possible), which is bromo-functionalised so that it can act as
a macroinitiator for vinyl polymerisation. Because of the number of
ethylene oxide groups present in the polyethylene oxide (PEO), PEG
2K is also referred to as PEO.sub.45 and PEG 5K is also referred to
as PEO.sub.114.
[0111] Other monomers, branchers, initiators and other materials
can also be used including: [0112] BDME, 1,4-butanediol
di(methacryloyloxy)-ethyl ether, a pH responsive brancher
[0113] Diblock copolymers of varying lengths are shown (top
middle): these can for example have one block formed from a vinyl
monomer selected from those shown, e.g. HPMA, and a second block of
different chemistry e.g. polyethylene oxide. One possible
preparative procedure uses bromo-functionalised polyethylene oxide
(e.g. PEG 2K or 5K) (FIG. 1, bottom right) as a macroinitiator for
vinyl polymerisation. The resultant amphiphilic diblock copolymer
can be co-precipitated with the branched vinyl polymer to form a
particle which is schematically illustrated top right. In this
example the hydrophilic polyethylene oxide chains of the block
copolymer are shown on the outside of the particle, as would be the
case in aqueous systems.
[0114] As shown in FIG. 2, the co-precipitation may for example be
carried out by adding both polymers, in suitable organic solvent,
to water, and then allowing the organic solvent to evaporate.
[0115] Some examples of particle size (dynamic light scattering
size) and polydisersity (PDI) values for co-precipitates of
branched vinyl polymer (poly-HPMA-EGDMA) with amphiphilic diblock
copolymer (one block being PEG 5K or PEG 2K, and the other being
HPMA.sub.40, HPMA.sub.80, or HPMA.sub.120) in various ratios are
shown in the following table:
TABLE-US-00001 Branched polymer:diblock PEG2K PEG2K PEG2K PEG5K
PEG5K PEG5K polymer HPMA40 HPMA80 HPMA120 HPMA40 HPMA80 HPMA120
90/10 Z-ave (d nm) 113 114 120 111 112 132 PDI 0.067 0.045 0.066
0.055 0.045 0.059 80/20 Z-ave (d nm) 94 127 111 103 106 127 PDI
0.123 0.061 0.033 0.152 0.118 0.043 70/30 Z-ave (d nm) 106 115 108
121 106 113 PDI 0.029 0.053 0.037 0.061 0.124 0.069 60/40 Z-ave (d
nm) 115 111 103 107 103 117 PDI 0.047 0.037 0.069 0.028 0.065 0.059
50/50 Z-ave (d nm) 167 124 226 154 141 135 PDI 0.024 0.055 0.035
0.048 0.063 0.051
[0116] It can be seen that, in these examples, the sizes (z-average
diameters) of the co-nanoprecipitated particles range from 90 to
230 nm, and that there are narrow PDI values throughout showing
their consistency and uniformity.
[0117] SEM shows the sizes and spherical nature of the
nanoparticles. FIG. 3 shows some examples: co-precipitates of
branched vinyl polymer (poly-HPMA-EGDMA) with PEG 5K-HPMA.sub.120
in the ratios 80:20 (top) and 90:10 (middle) and with PEG
2K-HPMA.sub.120 in the ratio 50:50 (bottom).
[0118] FIG. 4 shows the stability of various particles to salt
solution (0.5M NaCl). "Blank" denotes before salt is added,
"Instant" immediately after, and 1, 7 and 21 the number of days
afterwards. The particles exhibit considerable salt stability,
which is important for use in physiological environments. Previous
examples of particles formed solely from branched vinyl polymers
alone have been shown to be unstable in the presence of salts
(Slater et al, Soft Matter 2012, 8, 9816-9827).
[0119] The following example provides more details of some
nanoparticles containing hydrophobic cores and amphiphilic diblock
stabilisers:
Example 1: Nanoparticles Containing Branched Hydrophobic Core
Polymer and Linear Amphiphilic Diblock Copolymer
Preparation of Polymers
[0120] ATRP was used for the synthesis of linear
PEO.sub.45-p(HPMA.sub.120) and the branched statistical copolymer
of EGDMA and HPMA p(HPMA.sub.50-EGDMA.sub.0.9). Polymers were
analysed by triple detection gel permeation chromatography (GPC)
and .sup.1H NMR. Monomer to polymer conversion was monitored by
.sup.1H NMR using anisole as an internal reference.
[0121] The linear amphiphilic block copolymer was synthesised
during a one step ATRP reaction in methanol at 30.degree. C. For
introduction of the hydrophilic block a PEO.sub.45-Br
macroinitiator (2 kDa) was added accordingly to HPMA along with the
catalytic system Cu(I)Cl/2,2'-Bipyridine (Bpy) in the following
ratio [Macroinitiator]:[Monomer]:[Cu(I)Cl]:[Bpy]=1:120:1:2. The
targeted number average degree of polymerisation (DP.sub.HPMA) was
120 HPMA monomer units and GPC confirmed a number average
DP.sub.HPMA=127 monomer units (M.sub.n=20300 Da) with a narrow
dispersity ( =1.23).
[0122] The branched statistical copolymerisation of HPMA and EGDMA
was initiated with ethyl 2-bromo isobutyrate (EBIB) using the
catalytic system previously described in the following ratio
[Macroinitiator]:[EGDMA]:[Monomer]:[Cu(I)Cl]:[Bpy]=1:0.9:50:1:2.
The molar ratio of [EGDMA]/[EBIB]<1 is a crucial parameter in
order to have control over the branching reaction and avoid
gelation. As expected, the presence of EGDMA dramatically increases
the polymer molecular weight (for example, M.sub.w=295,000 Da) and
dispersity ( =7.56) in contrast to the linear amphiphilic polymer
which displayed a monomodal narrow molecular weight distribution.
In addition, .sup.1H NMR analysis of both linear
PEO.sub.45-p(HPMA.sub.120) and branched
p(HPMA.sub.50-EGDMA.sub.0.9) showed high monomer conversion
(>98%).
Nanoprecipitation and Co-Nanoprecipitation
[0123] Polymeric nanoparticles were prepared by nanoprecipitation
which corresponds to a solvent switch through a rapid precipitation
into water (ambient temperature). It is hypothesised that during
the association of the p(HPMA.sub.50-EGDMA.sub.0.9) hydrophobic
branched core, the linear HPMA chains from the diblock copolymer
also become incorporated into the hydrophobic core allowing the
PEO.sub.45 (2 kDa) chains to be present at the surface of the
resulting particles and prevent aggregation by steric
stabilisation. Varying weight fractions of
p(HPMA.sub.50-EGDMA.sub.0.9).sub.x:PEO.sub.45-p(HPMA.sub.120).sub.y
(x:y) were dissolved in acetone at a total concentration of 5 mg
mL.sup.-1 for six hours to ensure complete solubilisation. Rapid
precipitation of 1 mL of the polymer solution into 5 mL of water
gave a final nanoparticle concentration of 1 mg mL.sup.-1 after
complete acetone evaporation. The nanoparticle dispersions were
analysed by dynamic light scattering (DLS) and scanning electron
microscopy (SEM). The resulting z-average diameters (nm),
polydispersity indexes (PDI), zeta potentials (mV) and number
average diameters (nm) are collected in the following table.
TABLE-US-00002 TABLE DLS and SEM characterisation of nanoparticles
obtained by co-nanoprecipitation of p(HPMA.sub.50-EGDMA.sub.0.9),
PEO.sub.45-p(HPMA.sub.120) and
p(HPMA.sub.50-EGDMA.sub.0.9).sub.x:PEO.sub.45-
p(HPMA.sub.120).sub.y(x:y) acetone solutions into water. DLS DLS
SEM Z- Number Number average Diameter Diameter Zeta Ratio Diameter
Average Average Potential Entry Sample (%) (d nm) PDI (d nm) (d nm)
(mV) 1 p(HPMA.sub.50-EGDMA) 100 148 0.08 112 .+-. 6 117 -40.5 2
PEO.sub.45-p(HPMA.sub.120) 100 296 0.135 188 .+-. 80 190 -17.5 3
p(HPMA.sub.50-EGDMA):PEO.sub.45- 90:10 107 0.075 84 .+-. 2 81 -19.3
p(HPMA.sub.120) 4 p(HPMA.sub.50-EGDMA):PEO.sub.45- 80:20 126 0.046
99 .+-. 4 105 -24.4 p(HPMA.sub.120) 5
p(HPMA.sub.50-EGDMA):PEO.sub.45- 70:30 116 0.067 93 .+-. 3 117
-20.6 p(HPMA.sub.120) 6 p(HPMA.sub.50-EGDMA):PEO.sub.45- 60:40 119
0.107 91 .+-. 1 88 -21.5 p(HPMA.sub.120) 7
p(HPMA.sub.50-EGDMA):PEO.sub.45- 50:50 174 0.093 137 .+-. 3 110
-19.7 p(HPMA.sub.120)
[0124] Self assembly of the branched p(HPMA.sub.50-EGDMA.sub.0.9)
and amphiphilic PEO.sub.45-p(HPMA.sub.120) polymers during
co-nanoprecipitation from acetone into H.sub.2O produced well
defined particles indicated by the low PDI values (0.046-0.107) and
size homogeneity respectively obtained from DLS measurements and
SEM observations.
[0125] The co-nanoprecipitated particles exhibited zeta potential
values ranging between -19 mV and -25 mV (Table 1, entries
3-7).
Salt Stability
[0126] The salt stability of the co-precipitated particles was
demonstrated by adding aliquots (20 .mu.L) of an aqueous 0.5M NaCl
salt solution to 1 mL of the nanoprecipitated dispersions.
Z-average diameters and PDI were measured during a period of 0-21
days.
[0127] Co-nanoprecipitated particles demonstrated excellent salt
stability over 21 days after 20 .mu.L addition, and maintainance of
narrow polydispersity. Nanoparticles containing only branched vinyl
polymer crashed out of solution immediately on NaCl addition
whereas nanoparticles containing both branched vinyl polymer and
diblock amphiphiic copolymer remained as a stable nanoparticle
dispersion. SEM analysis of the nanoparticles (0.1 mg mL.sup.-1)
showed the spherical nature of the particles and
co-nanoprecipitates made up of groups of particles.
[0128] FIG. 5 shows how the z-average diameter and PDI vary on
addition of NaCl (20 .mu.L 0.5M NaCl) to a 1 mg mL.sup.-1 aqueous
dispersion of
p(HPMA.sub.50-EGDMA.sub.0.9):PEO.sub.45-p(HPMA.sub.120) 60:40. The
vertical axis denotes intensity (percent) and the horizontal axis
shows z-average diameter (d, nm). Solid line: before addition.
Dashed line: immediately after NaCl addition. Dotted line: 21 days
after NaCl addition.
[0129] FIG. 6 shows a scanning electron microscopy image of
p(HPMA.sub.50-EGDMA.sub.0.9):PEO.sub.45-p(HPMA.sub.120) 60:40; 1 mg
mL.sup.-1.
Conclusions from Example 1
[0130] Example 1 shows that the addition of an amphiphilic diblock
`stabiliser` (PEO 2000 Da) to a branched hydrophobic polymer at low
concentrations followed by co-nanoprecipitation can form particles
with desirable z-average diameters and very narrow PDI values. A
range of co-nanoprecipitated particles can be prepared. The
co-nanoprecipitated particles offer enhanced stability due to the
introduction of a steric stabiliser. This relies upon the formation
of an outer layer of material that prevents particles coming into
close proximity.
Loading of HIV Antivirals
[0131] The use of the nanoparticles to load various drugs including
the following HIV antiretrovirals was investigated:
##STR00001##
[0132] Drugs could be encapsulated inside the polymer particles in
a reliable and reproducible manner, allowing particles to be
produced with very narrow particle size distributions.
[0133] The use of branched vinyl polymers with a blend of two
monomers [HPMA plus either tert-butyl methacrylate (tBMA) or
n-butyl methacrylate (nBMA)] in the hydrophobic core was found to
be particularly effective in allowing high drug loadings to be
achieved. Good results were also obtained when nBMA was used as the
only monofunctional monomer in the branched vinyl polymer.
[0134] The results are shown in FIGS. 7 to 12 as follows:
TABLE-US-00003 Branched polymer (EGDMA used as FIG. Drug and
loading brancher in each case) Z-ave PdI 7 Ritonavir 20 wt %
tBMA-HPMA 123 nm 0.062 8 Ritonavir 20 wt % nBMA-HPMA 141 nm 0.072 9
Lopinavir 15 wt % tBMA-HPMA 166 nm 0.060 10 Lopinavir 15 wt %
nBMA-HPMA 137 nm 0.063 11 Efavirenz 15 wt % tBMA-HPMA 131 nm 0.037
12 Efavirenz 15 wt % nBMA-HPMA 117 nm 0.131
[0135] The block copolymer utilised to form particles shown in
FIGS. 7-12 was PEG 5K-HPMA.sub.120 and the ratio of branched
polymer to linear polymer was 50:50.
[0136] The drug loading could be increased by further tailoring the
polymer chemistry to allow drug loadings of 25 wt % to be achieved
for Efavirenz. The drug-loaded particles were stable extended
periods of time. Further experiments investigated the loading of
different drug types and details of these (and also of polymer
synthesis and co-nanoprecipitation in this context) are as
follows.
Typical Nanoparticle Preparation Including 10 wt % Loading for
Efavirenz, Ritonavir and Lopinavir
[0137] During a typical nanoparticle preparation, 5.5 mL of a 1
mg/mL acetone solution of efavirenz, ritonavir or lopinavir is
added to a vial and left to evaporate overnight. To this vial 25 mg
of the branched polymer and 25 mg of the polymer diblock were added
and dissolved in 10 mL of acetone during 6-8 hours to ensure
complete solubilisation. 1 mL of the 5 mg/mL solution of polymers
and dissolved drug was added to 5 mL of stirring distilled water
(500 rpm) and left for 24 hours for complete acetone evaporation
(final concentration of polymer 1 mg/mL).
Anti-Cancer Drugs
[0138] Other examples of drugs which can be incorporated are
anti-cancer drugs.
[0139] Irinotecan is a hydrophobic anticancer drug and SN-38 is a
hydrophobic anticancer active metabolite of irinotecan.
[0140] Irinotecan in particular was effectively encapsulated at 10
wt % or 15 wt % into co-nanoprecipitated particles with low PdI
values, for example where the branched polymer comprised nBMA or
tBMA-HPMA and the amphiphilic polymer comprised PEG5K-HPMA120.
Irinotecan (Typical 10 wt % Loading)
[0141] During a typical nanoparticle preparation 5.5 mL of a 1
mg/mL acetone solution of irinotecan was added to a vial and left
to evaporate overnight. To this vial, 25 mg of the branched polymer
and 25 mg of the polymer diblock were added and dissolved in 10 mL
of acetone during 6-8 hours to ensure complete solubilisation. 1 mL
of the 5 mg/mL solution of polymers and dissolved drug was added to
5 mL of stirring water (500 rpm) and left for 24 hours for complete
acetone evaporation.
SN-38 (Typical 2 wt % Loading)
[0142] During a typical nanoparticle preparation 1.03 mL of a 1
mg/mL THF/Acetonitrile (50:50) solution of SN-38 was added to a
vial and left to evaporate overnight. To this vial 25 mg of the
branched polymer core and 25 mg of the polymer diblock were added
and dissolved in 10 mL of acetone during 6-8 hours to ensure
complete solubilisation. 1 mL of the 5 mg/mL solution of polymers
and dissolved drug was added to 5 mL of stirring water (500 rpm)
and left for 24 hours for complete acetone evaporation.
Preparation of SN-38 Nanoparticles by DMSO Dialysis
[0143] During a typical nanoparticle preparation by dialysis, 2.65
mL of a 1 mg/mL DMSO solution of SN-38, 25 mg of the branched
polymer core and 25 mg of the polymer diblock were added and
dissolved in 7.35 mL of DMSO during 6-8 hours to ensure complete
solubilisation. 1 mL of the 5 mg/mL solution of polymers was added
to a dialysis bag with a molecular weight cut off (MWCO) of 2000
g/mol and left to dialyse in distilled water over 4 days (changing
the water every 4 hours)
ATRP Polymerisation--Formation of the Branched Polymer Core
p(HPMA.sub.50-EGDMA.sub.0.9)
[0144] The targeted number average degree of polymerisation
(DP.sub.n) was 50 repeat units. During a typical ATRP synthesis,
EBIB initiator (0.14 g, 0.69 mmol 1 eq.) and HPMA (5 g, 34.68 mmol
50 eq.) were added to a round-bottomed flask equipped with a
nitrogen inlet/outlet and a stirrer bar. Methanol was added (50
wt/wt %, based on HPMA) and the solution was stirred vigorously
under nitrogen for 10-15 minutes. The branching agent EGDMA (0.12
g, 0.62 mmol 0.9 eq. to EBIB initiator), copper catalyst Cu(I)Cl
(0.069 g, 0.69 mmol 1 eq.) and bpy (0.22 g, 1.39 mmol 2 eq.) were
added to the flask and the temperature was fixed at 30.degree. C.
The reaction was monitored by .sup.1H NMR spectroscopy and
terminated with methanol when the HPMA monomer had reached >99%
conversion. The polymer was purified using Dowex Marathon exchange
beads (.about.12 g) to remove excess copper catalyst followed by
passing the sample through a basic alumina column. Excess THF was
removed under vacuum to concentrate the sample before precipitation
into cold hexane. The resulting polymer was confirmed by .sup.1H
NMR in MeOD, triple detection GPC with an eluent of THF.
[0145] The polymerisation was carried out for all other
monomers:
nBMA p(nBMA.sub.50-EGDMA.sub.0.9), tBMA
p(tBMA.sub.50-EGDMA.sub.0.9), HPMA-nBMA
p(HPMA.sub.25-nBMA.sub.25-EGDMA.sub.0.9) and HPMA-tBMA
p(HPMA.sub.25-tBMA.sub.25-EGDMA.sub.0.9) Example Synthesis of
Poly(Ethylene Glycol) Mono-Functional ATRP Macro-Initiator
(PEO.sub.x-Br) (when x=45, the Macroinitiator is Referred to Herein
as PEG 2K)
[0146] During a typical synthesis, PEO.sub.45-OH (30 g, 15 mmol, 1
eq.) was dissolved in 100 mL of toluene in the presence of
triethylamine (2.275 g, 22.5 mmol, 1.5 eq.) and
4-dimethylaminopyridine (0.092 g, 0.75 mmol 0.05 eq.) in a two
necked round-bottomed flask fitted with an addition funnel, a
nitrogen inlet/outlet and a stirrer bar. 2-bromo-2-methylpropionyl
bromide (5.175 g, 22.5 mmol, 1.5 eq.) diluted with 25 mL of toluene
was placed in the addition funnel. The reactor was put under
stirring, cooled at about 0.degree. C. in an ice bath and the
2-bromo-2-methylpropionyl bromide solution was added slowly over a
period of 20-30 min After the addition was completed, the reactor
was allowed to reach room temperature and was left to stir for 24
hours. The formation of a white precipitate (triethylamine salt)
indicated the progress of the reaction. Then, the reaction medium
was warmed up in a water bath at about 50.degree. C., filtered and
concentrated on the rotary evaporator. The resulting product was
diluted in acetone and purified by precipitation in petroleum
ether. The last step was repeated once and the product was finally
dried under vacuum at 40.degree. C. for 24 hours. The resulting
macro-initiator was recovered with 70% yield and its structure was
confirmed by .sup.1H NMR in D.sub.2O, triple detection GPC with an
eluent of DMF and MALDI-TOF mass spectrometry.
PEG 5K
[0147] Same synthesis for PEO-5K initiator--PEO.sub.114-OH was used
rather than PEO.sub.45-OH.
ATRP Polymerisation--Synthesis of Linear
PEO.sub.45-P(HPMA.sub.120)
[0148] The targeted number average degree of polymerisation
(DP.sub.n) was 120 repeat units. In a typical ATRP synthesis,
PEO.sub.45-Br macroinitiator (0.62 g, 0.29 mmol 1 eq.) and HPMA (5
g, 34.68 mmol 120 eq.) were added to a round-bottomed flask
equipped with a nitrogen inlet/outlet and a stirrer bar. Methanol
was added (33.5 w/v %, based on HPMA) and the solution was stirred
vigorously under nitrogen for 10-15 minutes. The copper catalyst
Cu(I)Cl (0.029 g, 0.29 mmol 1 eq.) and bpy (0.09 g, 0.58 mmol 2
eq.) were added to the flask and the temperature was fixed at
30.degree. C. The reaction was monitored by .sup.1H NMR
spectroscopy and terminated with methanol when the HPMA monomer had
reached 99% conversion. The polymer was purified using Dowex
Marathon exchange beads (.about.12 g) to remove excess copper
catalyst followed by passing the sample through a basic alumina
column. Excess THF was removed under vacuum to concentrate the
sample before precipitation into petroleum ether 30/40. The
resulting polymer was confirmed by .sup.1H NMR in d.sub.6-DMSO,
triple detection GPC with an eluent of DMF.
[0149] The procedure described above was used for all other number
average degree of polymerisations for HPMA and the ATRP of
PEOSK-nBMA120.
Other Drugs and Other Drug Incorporation Methods
[0150] The present invention is also compatible with numerous other
drugs and also with other methods of incorporating drugs including
not just encapsulation as described previously but also chemical
bonding, sometimes referred to as conjugation, either to the
branched polymer or the linear polymer or both components of the
particle.
[0151] In this context Ibuprofen was used as a model drug. Free
ibuprofen was encapsulated, and it was also bonded via its acid
functionality to produce a prodrug model.
Ibuprofen Work--Prodrug Model
Synthesis of the Ibuprofen (IBU) Modified HPMA (IbuPMA)
[0152] During a typical synthesis HPMA (1.5 g, 10.40 mmol 1 eq.),
Ibuprofen (2.79 g, 13.53 mmol 1.3 eq), DMAP (0.64 g, 5.5 mmol, 0.5
eq) and DCC (2.79 g, 13.53 mmol, 1.3 eq) were dissolved in 40 mL of
THF in a round bottom flask and stirred at ambient temperature for
24 hours. The DCU salt was filtered and washed with THF followed by
rotary evaporation. DCM (100 mL) was added and washed with 1M
sodium bisulfate solution to remove excess DCU, then dried over
MgSO4, concentrated in vacuo and stored at 0.degree. C.
ATRP Polymerisation--Incorporated IBU Monomer: Targeted Total
DP80
[0153] Composition PEO.sub.114-p(HPMA.sub.60-IbuPMA.sub.20)
[0154] The targeted number average degree of polymerisation
(DP.sub.n) was HPMA.sub.60-IbuPMA.sub.20. PEO.sub.114-Br
macroinitiator (0.59 g, 0.12 mmol 1 eq.) and HPMA (1 g, 6.94 mmol
60 eq.) and IbuPMA (0.77 g 2.3 mmol, 20 eq.) were added to a
round-bottomed flask equipped with a nitrogen inlet/outlet and a
stirrer bar. Methanol was added (37 w/w %, based on HPMA+IbuPMA)
and the solution was stirred vigorously under nitrogen for 10-15
minutes. The copper catalyst Cu(I)Cl (0.0114 g, 0.12 mmol 1 eq.)
and bpy (0.036 g, 0.23 mmol 2 eq.) were added to the flask and the
temperature was fixed at 30.degree. C. The reaction was monitored
by .sup.1H NMR spectroscopy and terminated with methanol when the
monomers had reached 99% conversion. The polymer was purified using
a neutral alumina column flushed with THF to remove excess copper
catalyst. Excess THF was removed under vacuum to concentrate the
sample before precipitation into cold petroleum ether 30/40. The
resulting polymer was confirmed by .sup.1H NMR in MeOD, triple
detection GPC with an eluent of THF.
Post Modification of PEO.sub.114-HPMA.sub.120--Targeted 40 HPMA
Monomer Units
[0155] PEO.sub.114-HPMA.sub.120 (1 g, 0.033 mmol, 1 eq.), Ibuprofen
(0.27 g, 1.33 mmol, 40 eq.), DCC (0.274 g, 1.33 mmol, 40 eq.) and
DMAP (1.times.10.sup.-3 g) were dissolved in THF (12 mL) and left
over 24 hours. The DCU was filtered off and washed with THF and
concentrated in vacuo. The excess DMAP and DCU was dissolved in DCM
and washed with 1M sodium bisulfate and dried with MgSO4 and dried
under vacuum.
ATRP Polymerisation--Branched IBU Modified Copolymer
p(HPMA.sub.60-IBU.sub.20-EGDMA.sub.0.9)
[0156] The targeted degree of polymerisation (DP.sub.n) was 50
repeat units. During a typical ATRP synthesis, EBIB initiator
(0.024 g, 0.12 mmol 1 eq.) and HPMA (1.04 g, 7.23 mmol 60 eq.) and
IbuPMA (0.8 g, 2.41 mmol, 20 eq.) were added to a round-bottomed
flask equipped with a nitrogen inlet/outlet and a stirrer bar.
Methanol was added (50 wt/wt %, based on HPMA) and the solution was
stirred vigorously under nitrogen for 10-15 minutes. The branching
agent EGDMA (0.021 g, 0.11 mmol 0.9 eq. to EBIB initiator), copper
catalyst Cu(I)Cl (0.012 g, 0.12 mmol 1 eq.) and bpy (0.038 g, 0.24
mmol 2 eq.) were added to the flask and the temperature was fixed
at 30.degree. C. The reaction was monitored by .sup.1H NMR
spectroscopy and terminated with methanol when the HPMA monomer had
reached >99% conversion. The polymer was purified using a
neutral alumina column flushed with THF. Excess THF was removed
under vacuum to concentrate the sample before precipitation. The
polymer was precipitated from MeOH into cold petroleum ether 30/40.
The resulting polymer was confirmed by .sup.1H NMR in MeOD, triple
detection GPC with an eluent of THF.
Nanoparticle Preparation
[0157] IBU Modified
Diblock--p(EO).sub.114-p(HPMA.sub.60-HPMA:IBU.sub.20):p(HPMA.sub.50-EGDMA-
.sub.0.9)
[0158] During a typical nanoparticle preparation, 25 mg of
PEO.sub.114-p(HPMA.sub.60-HPMA:IBU.sub.20) and 25 mg of
p(HPMA.sub.50-EGDMA.sub.0.9) were added to 10 mL of MeOH during 6-8
hours to ensure complete solubilisation over 6-8 hours. 1 mL of the
5 mg/mL solution of polymers was added to 5 mL of stirring
distilled water (500 rpm) and left for 24 hours for complete
evaporation.
IBU Modified Branched Polymer Core
p(HPMA.sub.60-IBU.sub.20-EGDMA.sub.0.9)
[0159] During a typical nanoparticle preparation, 25 mg of
PEO.sub.114-p(HPMA.sub.120) and 25 mg of
p(HPMA.sub.60-IbuPMA.sub.20-EGDMA.sub.0.9) were added to 10 mL of
MeOH during 6-8 hours to ensure complete solubilisation over 6-8
hours. 1 mL of the 5 mg/mL solution of polymers was added to 5 mL
of stirring distilled water (500 rpm) and left for 24 hours for
complete evaporation.
[0160] The same procedure can be repeated for other variations of
the nanoparticles using the experimental above. For free IBU
addition, this can simply be added into the methanol before
solubilisation at the desired ratio to allow mixtures of
encapsulated IBU and IBU-prodrugs within the particles.
Further Example Showing that a Different Type of Block Copolymer--a
Tri-Block A-B-A (Hydrophobic-Hydrophilic-Hydrophobic) Polymer--can
be Prepared, and Used in Combination with a Branched Polymer and
Drug Encapsulation
[0161] To illustrate a variation of the chemistry which may be used
within the scope of the present invention, a bifunctional initiator
was prepared and used as the "B" block in the preparation of an
"A-B-A" tri-block copolymer. This tri-block copolymer was then used
in combination with a branched vinyl polymer and drug molecules to
form suitable co-nanoprecipitated particles.
Synthesis of PEG Bifunctional Macroinitiator
[0162] During a typical synthesis, OH-PEG.sub.104-OH (M.sub.n
.about.4600 g, 1 equiv. 20 g, 4.3 mmol), TEA (3 equiv. 0.013 mol,
1.83 mL) and DMAP (0.1 equiv, 4.0 mmol, 0.053 g) were added to
toluene (80 mL) in a two necked round bottom flask fitted with a
nitrogen inlet/outlet and stirrer bar. .alpha.-Bromo isobutyryl
bromide (3 equiv. 13 mmol, 1.61 mL) was diluted in toluene (20 mL)
and added drop-wise over 15 minutes via a dropping funnel and left
stirring over 24 hours. The reaction mixture was then filtered and
the excess solvent removed in vacuo. The product was dissolved in a
minimal amount of acetone, and precipitated into cold petroleum
ether 40-60.degree. C. (1:10 product:solvent) and left to dry under
vacuum at 40.degree. C. for 48 hours.
Synthesis of p(HPMA.sub.x-b-PEG.sub.104-b-HPMA.sub.x) Block
Polymer
[0163] During a typical synthesis, Br-PEG.sub.104-Br (1 equiv. 2.17
g, 0.43 mmol), HPMA (80 equiv. 5.0 g, 34.6 mmol), anisole (0.1 ml)
and anhydrous methanol (50 wt %) were added to a round bottomed
flask fitted with an nitrogen inlet/outlet and stirrer bar. To this
solution, anhydrous methanol (50 wt %, based on HPMA), CuCl (1
equiv. 0.042 g, 0.43 mmol) and bpy (2 equiv. 0.135 g, 0.867 mmol)
were added and the reaction mixture was degassed under nitrogen for
15-20 minutes. The reaction was left stirring for 24 hrs and high
conversion was confirmed .sup.1H NMR spectra. The reaction was
terminated by addition of methanol (.about.50 ml) and the remaining
solution was then passed through a neutral alumina column to remove
the catalytic system. The product was purified by concentrating the
solution in vacuo and precipitation of the polymer into cold hexane
(1:10 product: solvent). The resulting block copolymer was analysed
by .sup.1H NMR and GPC.
Branched p(DEAEMA.sub.50) polymer was synthesized as previously
described. Encapsulation of Drug Molecules into Co-Nanoprecipitated
Particles Consisting of an A-B-A Triblock and a Branched Polymer
Core
[0164] A stock solution of solubilised drug molecules (Ritonavir
and Lopinavir) was added to a sample vial (up to 5 wt % of each)
and the acetone was left to evaporate. To this vial, the branched
polymer, A-B-A triblock polymer (varying ratios of each) and
acetone were added to make up a solution of 5 mg/mL. To ensure
complete solubilisation this solution was left rolling over 24
hours. During a typical co-nanoprecipitation, 1 mL of the
polymer/drug solution was rapidly added to 5 mL of stirring water.
The solution was left stirring overnight to ensure complete solvent
removal and subsequent nanoparticles dispersions were measured by
dynamic light scattering.
Stable nanoparticles resulted, whether encapsulating Ritonavir
alone, Lopinavir alone, or a 1:1 by weight mixture of both.
Particles Containing Branched Polymers and Linear Dendritic
Hybrids
[0165] A second group of particles in accordance with the present
invention are those which contain branched polymers and linear
dendritic hybrids.
Examples of Polymers Prepared by ATRP and of Combinations of Such
Polymers in Particles
[0166] Some non-limiting examples of components used in branched
vinyl polymers and block copolymers have been described and defined
above.
Linear dendritic hybrids may comprise monomers as referred to
above, and additionally, of course, comprise dendrons. In that
context, some non-limiting examples of components used in polymers
of the present invention include the following. [0167] G2-M: a
generation 2 dendron initiator having the structure:
[0167] ##STR00002## [0168] G2-Bz: a generation 2 dendron initiator
with benzyl-functionalised ends having the structure:
[0168] ##STR00003## [0169] G1-M: a generation 1 dendron initiator
having the structure:
[0169] ##STR00004## [0170] G0-M: a generation 0 initiator having
the structure:
##STR00005##
[0171] Schematic representations of co-nanoprecipitates of branched
polymers and linear dendritic hybrids are shown in FIG. 13. The
dendrons are shown as wedges. In some examples (top right) the
dendrons are hypothesised to be at least partially within the
particles whereas in other examples (bottom right) the dendrons are
hypothesised to be mainly exposed at the surface. This can be
controlled by varying pH, for example when using pH responsive
materials such as DEAEMA.
[0172] Some examples of combinations of polymers, and the
properties of the resultant particles under different pH
conditions, are shown in FIGS. 14 to 18. FIGS. 14 to 18 use the
abbreviation CNP which denotes co-nanoprecipitates. An SEM image is
shown in FIG. 19 for the pH 7.8 particles containing EBIB
tBuMA-EGDMA and G2-M DEAEMA (FIG. 14).
[0173] .sup.a All diameters are given as z-average values as
measured by dynamic light scattering.
[0174] .sup.b All zeta potentials are given as surface charge
values as measured by dynamic light scattering
Further Examples of Combinations of Branched Vinyl Polymers and
Linear Dendritic Hybrids
[0175] Further examples of combinations of branched vinyl polymers
and linear dendritic hybrids, prepared by ATRP, include those
listed in the following table. This exemplifies a range of
chemistries (including hydrophilic, hydrophobic, and pH responsive)
and different copolymer approaches (including block copolymers and
statistical copolymers). It will also be seen that the block
copolymer approach may be combined with the linear dendritic hybrid
approach, i.e. a linear dendritic hybrid may comprise a dendron
connected to one block which is then connected to another block.
Furthermore, in place of G2-M or G2-Bz, G1-M, G0-M or EBIB may be
used instead, i.e. the linear chains may be initiated by smaller
dendrons or by initiators which are not dendrons (in which case the
materials co-precipitated with the branched vinyl polymer are not
linear dendritic hybrids but merely linear polymers, of which the
present invention is concerned with a subset, namely block
copolymers).
TABLE-US-00004 branched polymer linear dendritic hybrid
EBIB-HPMA.sub.50-EGDMA.sub.0.95 G2-M-HPMA.sub.50 as above
G2-M-DEAEMA.sub.50 EBIB-tBuMA.sub.50-EGDMA.sub.0.95
G2-M-DEAEMA.sub.50 as above G2-M-HPMA.sub.50 as above
G2-M-PtBuMA.sub.50 as above Block copolymers:
G2-M-PDEAEMA.sub.25-tBuMA.sub.25 or
G2-M-PDEAEMA.sub.17-tBuMA.sub.33 or
G2-M-PDEAEMA.sub.33-tBuMA.sub.17 as above as above, except that the
copolymers are random copolymers rather than block copolymers as
above G2-Bz-DEAEMA.sub.50 EBIB-DEAEMA.sub.50-EGDMA.sub.0.95
G2-M-DEAEMA.sub.50 and EBIB-DEAEMA.sub.50-BDME.sub.2.0 as above
EBIB-DEAEMA.sub.50-EGDMA.sub.0.95 G2-M-HPMA.sub.50
EBIB-HPMA.sub.x-DEAEMA.sub.y-EGDMA.sub.0.9 G2-M-HPMA.sub.50
(copolymer wherein x, y = 25, 25 or 17, 33 or 33, 17) as above
G2-M-DEAEMA.sub.50 as above G2-M-HPMA.sub.x-DEAEMA.sub.y (random
copolymer wherein x, y = 17, 33 or 25, 25 or 33, 17) as above (as
above, except that the copolymer is a diblock copolymer rather than
a random copolymer) EBIB-HPMA.sub.50-EGDMA.sub.0.95
G2-M-HPMA.sub.x-DEAEMA.sub.y (random copolymer wherein x, y = 17,
33 or 25, 25 or 33, 17) as above (as above, except that the
copolymer is a diblock copolymer rather than a random copolymer)
EBIB-DEAEMA.sub.50-EGDMA.sub.0.95 G2-M-HPMA.sub.x-DEAEMA.sub.y
(random copolymer wherein x, y = 17, 33 or 25, 25 or 33, 17) as
above (as above, except that the copolymer is a diblock copolymer
rather than a random copolymer)
[0176] The materials in the above table may for example be
co-nanoprecipitated in a ratio of 90:10 branched:linear. One
suitable method involves: dropping 0.2 ml (5 mg ml.sup.-1 in
acetone, THF or IPA) of linear polymer and 1.8 ml (5 mg ml.sup.-1
in acetone, THF or IPA) of branched polymer into 10 ml water; and
allowing the organic solvent to evaporate overnight to form a 1 mg
ml.sup.-1 nanoparticle dispersion in water.
[0177] A range of pH conditions may be used and optionally
conditions and monomers may be chosen to provide particular
structures. Merely by way of non-limiting example, at low pH
protonation of amine moieties (e.g. in DEAEMA or in dendrons) means
that amine moieties are more likely to be exposed towards the
outside of the particles
Examples of Polymers Prepared by Ring Opening Polymerization and of
Such Polymers in Particles
[0178] Another method of polymerization which can be used in the
present invention is ring opening polymerisation (ROP). For
example, lactone monomers may be ring opened by reaction with
alcohols under suitable conditions as known in the art.
[0179] For example, the polymerization of caprolactone monomer may
be initiated by benzyl alcohol to produce benzyl-polycaprolactone
(abbreviated herein as Bz-PCL).
[0180] The ring opening of single lactone rings such as
polycaprolactone results in linear polymers.
[0181] Branched polymers may be obtained by using branchers, e.g.
monomers which have two lactone rings connected together, e.g. BOD
(4,4'-bioxepanyl-7,7'-dione). The following scheme shows a method
of preparing BOD:
##STR00006##
A branched polyester may be prepared by copolymerizing a
monofunctional lactone (e.g. caprolactone) and a difunctional
lactone (e.g. BOD) using an initiator (e.g. Bz-OH):
[0182] Such branched polycaprolactones may be used instead of the
branched vinyl polymers, in combination with linear polymers (i.e.
block copolymers or linear dendritic hybrids).
[0183] Examples of combinations of branched polycaprolactone
(prepared by ROP) and linear dendritic hybrids (prepared by ATRP),
which we have co-nanoprecipitated, include those listed in the
following table. As before, in place of G2 it is possible to
instead use G1, G0 or EBIB.
TABLE-US-00005 branched polycaprolactone linear dendritic hybrid
Bz-PCL.sub.50-BOD.sub.0.6 G2-M-DEAEMA.sub.50 as above
G2-M-HPMA.sub.50 as above block copolymers:
G2-M-DEAEMA.sub.25-tBuMA.sub.25 or G2-M-DEAEMA.sub.17-tBuMA.sub.33
or G2-M-DEAEMA.sub.33-tBuMA.sub.17 as above as above except random
copolymers instead of block copolymers
[0184] ROP may also be used to prepare the linear dendritic hybrid
(or other linear polymer component), and ROP and ATRP may be
combined.
[0185] Polyesters may also be used in the linear dendritic hybrid,
and non-limiting examples of suitable initiators for ROP in that
context include the following. [0186] G2-pOH: a generation 2
dendron bearing a hydroxyl group to initiate ROP, of the following
structure:
[0186] ##STR00007## [0187] G1-pOH: a generation 1 dendron bearing a
hydroxyl group to initiate ROP, of the following structure:
[0187] ##STR00008## [0188] G0-pOH: a generation 0 initiator of the
following structure:
##STR00009##
[0189] Examples of linear dendritic hybrids which may be combined
with branched polymers, e.g. Bz-PCL.sub.50-BOD.sub.0.6, include:
[0190] G1-p-PCL.sub.50-DEAEMA.sub.20 [0191] G2-p-PCL.sub.50 [0192]
G2-p-PCL.sub.30 [0193] G2-p-PCL.sub.20
Example Experimental Procedures for ATRP and ROP Polymerisations
and for the Preparation of Materials Used in these
Polymerisations
1. Polymerisation by ATRP
1.1 ATRP Dendron Initiator Synthesis
1.1.1 Synthesis of G1-M-OH
[0194] 2-(Dimethylamino)ethyl acrylate (6.0 g, 42 mmol, 6 eq.) was
added to a 50 mL round 2 necked round-bottomed flask containing
isopropanol (IPA) (12 mL). The flask was deoxygenated under a
positive N.sub.2 purge for 10 minutes. 1-amino-2-propanol (0.5246
g, 7.0 mmol, 1 eq.) dissolved in IPA (12 mL) was added drop wise
while the solution was stirring in an ice bath under a positive
flow of N.sub.2. The final mixture was stirred for a further 10
minutes at 0.degree. C. before being allowed to warm to room
temperature and left stirring for 48 hrs. The solvent was removed
and the product left to dry in vacuo overnight. Found C, 57.45; H,
9.77; N, 11.12%. C.sub.17H.sub.35N.sub.3O.sub.5 requires, C, 56.43;
H, 9.68; N, 11.62%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.08
(d, 3H), 2.18-2.62 (m, 22H), 2.69 (m, 2H), 2.89 (m, 2H), 3.77 (m,
1H), 4.16 (m, 4H). .sup.13C NMR (100 MHz, CDCl3) .delta. 19.8,
32.6, 45.6, 49.7, 57.8, 62.0, 63.7, 76.9, 128.4, 130.9, 172.5. m/z
(ES MS) 362.3 [M+H]+, 384.3 [M+Na]+.
1.1.2 Synthesis of G2-M-OH
[0195] 2-(Dimethylamino)ethyl acrylate (6.0 g, 42 mmol, 6 eq.) was
added to a 50 mL round 2 necked round-bottomed flask containing IPA
(12 mL). The flask was deoxygenated under a positive N.sub.2 purge
for 10 minutes. Bis(3-aminopropyl)amino)propan-2-ol (1.3221 g,
6.984 mmol, 1 eq.) dissolved in IPA (12 mL) was added drop wise
while the solution was stirring in an ice bath under a positive
flow of N.sub.2. The final mixture was stirred for a further 10
minutes at 0.degree. C., allowed to warm to room temperature and
left stirring for 48 hrs. The solvent was removed and the product
left to dry in vacuo overnight. Found C, 58.32; H, 9.92; N, 12.87%.
C.sub.37H.sub.75N.sub.7O.sub.9 requires, C, 58.27; H, 9.84; N,
12.86%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.13 (d, 3H),
1.67 (m, 4H), 2.26-2.65 (m, 50H), 2.77 (m, 8H), 3.87 (m, 1H), 4.17
(m, 8H). m/z (ES MS) 762.6 [M+H]+, 784.6 [M+Na]+.
1.1.3 Synthesis of G0-M
[0196] 1-dimethylamino-2-propanol (1.1207 g, 10.86 mmol, 1 eq.),
triethanolamine (TEA) (1.5390 g, 15.2 mmol, 1.4 eq.) and dimethyl
amino pyridine (DMAP) (132.7 mg, 1.086 mmol, 0.1 eq.) were added to
a 250 mL 2 necked round-bottomed flask containing dichloromethane
(DCM) (160 mL). The flask was deoxygenated under a positive N.sub.2
purge for 10 minutes. .alpha.-bromoisobutyryl bromide (2.622 g, 1.4
mL, 11.4 mmol, 1.05 eq.) was added drop wise while the solution was
stirring in an ice bath under a positive flow of N.sub.2. The
reaction mixture was allowed to warm to room temperature and left
stirring overnight. The organic phase was washed with saturated
sodium hydrogen carbonate (NaHCO.sub.3) solution (3.times.30 mL).
The solution was dried with anhydrous Na.sub.2SO.sub.4. Found C,
42.87; H, 7.20; N, 5.55%. C.sub.9H.sub.18NO.sub.2Br requires, C,
42.86; H, 7.14; N, 5.55%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
1.27 (d, 3H), 1.89 (s, 6H), 2.17-2.55 (m, 8H), 5.07 (m, 1H).
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 17.6, 30.9, 46.1, 56.1,
63.5, 70.6, 76.9, 170.8. m/z (ES MS) 252 [M+H]+.
1.1.4 Synthesis of G1-M
[0197] G1-OH (1.1207 g, 10.86 mmol, 1 eq.), TEA (1.5390 g, 15.2
mmol, 1.4 eq.) and DMAP (132.7 mg, 1.086 mmol, 0.1 eq.) were added
to a 250 mL 2 necked round-bottomed flask containing DCM (160 mL).
The flask was deoxygenated under a positive N.sub.2 purge for 10
minutes. .alpha.-bromoisobutyryl bromide (2.622 g, 1.4 mL, 11.4
mmol, 1.05 eq.) was added drop wise while the solution was stirring
in an ice bath under a positive flow of N.sub.2. The reaction
mixture was allowed to warm to room temperature and left stirring
overnight. The organic phase was washed with saturated sodium
hydrogen carbonate (NaHCO.sub.3) solution (3.times.160 mL). The
solution was dried with anhydrous Na.sub.2SO.sub.4 and the product
left to dry in vacuo overnight. Found C, 49.41; H, 7.90; N, 8.23%.
C.sub.21H.sub.40N.sub.3O.sub.6Br requires, C, 49.41; H, 7.84; N,
8.24%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.22 (d, 3H), 1.89
(s, 6H), 2.24-2.69 (m, 22H), 2.83 (m, 4H), 4.20 (m, 4H), 5.0 (m,
1H). .sup.13C NMR (100 MHz, CDCl3) .delta. 18.6, 30.9, 32.8, 50.0,
56.4, 58.8, 60.3, 69.6, 77.2, 125.7, 144.3, 172.3. m/z (ES MS)
510.2 [M+H]+, 534.2 [M+Na]+.
1.1.5 Synthesis of G2-M
[0198] G2-OH (5.1431 g, 6.749 mmol, 1 eq.), TEA (0.9561 g, 9.449
mmol, 1.4 eq.) and DMAP (82.5 mg, 0.6749 mmol, 0.1 eq.) were added
to a 250 mL 2 necked round-bottomed flask containing DCM (160 mL).
The flask was deoxygenated under a positive N.sub.2 purge for 10
minutes. .alpha.-bromoisobutyryl bromide (1.629 g, 0.88 mL, 7.087
mmol, 1.05 eq.) was added drop wise while the solution was stirring
in an ice bath under a positive flow of N.sub.2. The reaction
mixture was allowed to warm to room temperature and left stirring
overnight. The organic phase was washed with saturated sodium
hydrogen carbonate (NaHCO.sub.3) solution (3.times.160 mL). The
solution was dried with anhydrous Na.sub.2SO.sub.4 and the product
left to dry in vacuo overnight. Found C, 54.05; H, 8.85; N, 10.76%.
C.sub.41H.sub.80N.sub.7O.sub.10Br requires, C, 54.01; H, 8.78; N,
10.76%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.26 (d, 3H),
1.56 (m, 4H), 1.91 (s, 6H), 2.22-2.67 (m, 50H), 2.76 (m, 8H), 4.19
(m, 8H), 5.04 (m, 1H). .sup.13C NMR (100 MHz, CDCl3) .delta. 24.5,
25.5, 28.4, 45.6, 62.2, 64.2, 77.2, 173.5. m/z (ES MS) 912.5
[M+H]+, 934.5 [M+Na]+, 950.5 [M+K]+.
1.1.6 Synthesis of G2-Bz-OH
[0199] Benzyl acrylate (6.7966 g, 42 mmol, 6 eq.) was added to a 50
mL round 2 necked round-bottomed flask containing IPA (12 mL). The
flask was deoxygenated under a positive N.sub.2 purge for 10
minutes. Bis(3-aminopropyl)amino)propan-2-ol (1.3221 g, 6.984 mmol,
1 eq.) dissolved in IPA (12 mL) was added drop wise while the
solution was stirring in an ice bath under a positive flow of
N.sub.2. The final mixture was stirred for a further 10 minutes at
0.degree. C., allowed to warm to room temperature and left stirring
for 48 hrs. The solvent was removed and the product left to dry in
vacuo overnight.
1.1.7 Synthesis of G2-Bz
[0200] G2-Bz (1.664 g, 1.86 mmol, 1 eq.), TEA (0.2639 g, 2.6 mmol,
1.4 eq.) and DMAP (22.8 mg, 0.1866 mmol, 0.1 eq.) were added to a
250 mL 2 necked round-bottomed flask containing DCM (110 mL). The
flask was deoxygenated under a positive N.sub.2 purge for 10
minutes. .alpha.-bromoisobutyryl bromide (0.5354 g, 0.29 mL, 2.329
mmol, 1.25 eq.) was added drop wise while the solution was stirring
in an ice bath under a positive flow of N.sub.2. The reaction
mixture was allowed to warm to room temperature and left stirring
overnight. The organic phase was washed with saturated sodium
hydrogen carbonate (NaHCO.sub.3) solution (3.times.110 mL). The
solution was dried with anhydrous Na.sub.2SO.sub.4 and the product
left to dry in vacuo overnight.
[0201] Found C, 63.40; H, 6.96; N, 4.18%.
C.sub.53H.sub.68N.sub.3O.sub.10Br requires, C, 64.44; H, 6.89; N,
4.26%. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.22 (d, 3H), 1.54
(m, 4H), 1.90 (s, 6H), 2.24-2.65 (m, 18H), 2.77 (m, 8H), 5.00 (m,
1H), 5.09 (s, 8H), 7.33 (m, 20H). .sup.13C NMR (100 MHz, CDCl3)
.delta. 18.1, 30.6, 32.3, 44.1, 48.8, 51.7, 52.7, 66.7, 76.7,
128.6, 135.8, 144.1, 172.3. m/z (ES MS) 988.4 [M+H]+.
1.2 pH Responsive Brancher Synthesis
1.2.1 Synthesis of 1,4-Butanediol di(methacryoyloxy)-ethyl ether
(BDME)
[0202] 1,4-butanediol divinyl ether (BDVE) (5.6 ml, 35.21 mmol) was
added to a two-necked 250 ml round bottomed flask equipped with a
condenser, a magnetic stirrer and a positive flow of nitrogen. A
small amount of radical inhibitor 4-tert-butylcatechol (end of a
spatula) was added and the mixture deoxygenated using a nitrogen
purge for 15 minutes. Once dissolved, the temperature was raised to
70.degree. C. Methacrylic acid (MAA) (14.9 ml, 175.8 mmol) was
added dropwise over 10 minutes through a septa. The reaction was
allowed to proceed at 70.degree. C. for a further 6 hours with
stirring. After this time, the reaction was stopped by cooling and
exposing to the air. The crude product was dissolved in chloroform
(100 ml) and washed with basic H.sub.2O (.about.pH12, 3.times.100
ml). The combined washings were collected and dried over NaSO.sub.4
and the solvent removed by rotary evaporation.
[0203] (Found: C 61.45; H 8.28%. C.sub.16H.sub.26O.sub.6 requires C
61.15; H 8.28%); .sup.1H NMR (400 MHz; CDCl.sub.3; Me.sub.4Si)
.delta. 1.44 (6H, d, CH.sub.3CH), 1.65 (4H, m,
CH.sub.2CH.sub.2CH.sub.2), 1.95 (6H, s, CH.sub.3C.dbd.CH2),
3.50-3.69 (4H, m, OCH.sub.2CH.sub.2), 5.60 and 6.15 (4H, 2s,
CH.sub.2.dbd.CCH.sub.3) and 5.95-5.99 (2H, q, CHCH.sub.3). .sup.13C
NMR (400 MHz; CDCl.sub.3; Me.sub.4Si) .delta. 18.27 (s), 20.83 (s),
26.29 (s) 68.85 (s), 96.93 (s), 125.90 (s), 136.37 (s) and 167.01
(s). m/z (EI) 314.2 (M.sup.+-C.sub.16H.sub.26O.sub.6 requires
314).
1.3 Polymerisation of HPMA
1.3.1 Polymerisation of HPMA.sub.50
[0204] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units P(HPMA).sub.50;
n.sub.HPMA/n.sub.Initiator: 50), bpy (173.3 mg, 1.1096 mmol, 2
eq.), HPMA (4 g, 27.7 mmol, 50 eq.) and methanol (MeOH) (56% v/v
based on HPMA) were placed into a 25 mL round-bottomed flask. The
solution was stirred and deoxygenated using a nitrogen (N.sub.2)
purge for 15 minutes. Cu(I)Cl (54.9 mg, 0.5548 mmol, 1 eq.) was
added to the flask and left to purge for a further 5 minutes. G2-M
(0.5054 g, 0.5548 mmol, 1 eq.) was added to the flask under a
positive flow of N.sub.2, and the solution was left to polymerise
at 30.degree. C. Reactions were terminated when >99% conversion
was reached, as judged by .sup.1H NMR (cf. FIG. 8), by exposure to
oxygen and addition of THF. The catalyst residues were removed by
passing the mixture over a basic alumina column. THF was removed
under vacuum to concentrate the sample before precipitation into
hexane and drying in the vacuum oven overnight.
1.3.2 Polymerisation of HPMA.sub.50-EGDMA.sub.x
[0205] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units P(HPMA).sub.50;
n.sub.HPMA/n.sub.Initiator: 50), bpy (173.3 mg, 1.1096 mmol, 2
eq.), HPMA (4 g, 27.7 mmol, 50 eq.), EGDMA (99.0 mg, 0.4993 mmol,
0.9 eq) and methanol (MeOH) (38.9% v/v based on HPMA) were placed
into a 25 mL round-bottomed flask. The solution was stirred and
deoxygenated using a nitrogen (N.sub.2) purge for 15 minutes.
Cu(I)Cl (54.9 mg, 0.5548 mmol, 1 eq.) was added to the flask and
left to purge for a further 5 minutes. EBIB (0.1082 g, 0.5548 mmol,
1 eq.) was added to the flask under a positive flow of N.sub.2, and
the solution was left to polymerise at 30.degree. C. Reactions were
terminated when >99% conversion was reached, as judged by
.sup.1H NMR, by exposure to oxygen and addition of THF. The
catalyst residues were removed by passing the mixture over a basic
alumina column. THF was removed under vacuum to concentrate the
sample before precipitation into hexane.
1.4 Polymerisation of DEAEMA
1.4.1 Polymerisation of DEAEMA.sub.50
[0206] In a typical synthesis, targeting a DP.sub.n=50 monomer
units PDEAEMA.sub.50; n.sub.DEAEMA/n.sub.Initiator: 50), bpy (134.9
mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.) and IPA
(56% v/v based on DEAEMA) were placed into a 25 mL round-bottomed
flask. The solution was stirred and deoxygenated using a nitrogen
(N.sub.2) purge for 15 minutes. Cu(I)Cl (42.8 mg, 0.4318 mmol, 1
eq.) was added to the flask and left to purge for a further 5
minutes. G2-M (0.3934 g, 0.4318 mmol, 1 eq.) was added to the flask
under a positive flow of N.sub.2, and the solution was left to
polymerise at 40.degree. C. Reactions were terminated when >99%
conversion was reached, as judged by .sup.1H NMR, by exposure to
oxygen and addition of acetone. The catalyst residues were removed
by passing the mixture over a basic alumina column. Acetone was
removed under vacuum to concentrate the sample before precipitation
into cold petroleum ether (40.degree. C.-60.degree. C.) and drying
in the vacuum oven overnight.
1.4.2 Polymerisation of DEAEMA.sub.50-EGDMA.sub.x
[0207] In a typical synthesis, targeting a DP.sub.n=50 monomer
units PDEAEMA.sub.50; n.sub.DEAEMA/n.sub.Initiator: 50), bpy (134.9
mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), EGDMA
(77.0 mg, 0.3886 mmol, 0.9 eq) and IPA (38.9% v/v based on DEAEMA)
were placed into a 25 mL round-bottomed flask. The solution was
stirred and deoxygenated using a N.sub.2 purge for 15 minutes.
Cu(I)Cl (42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask and
left to purge for a further 5 minutes. EBIB (84.2 mg, 0.4318 mmol,
1 eq.) was added to the flask under a positive flow of N.sub.2, and
the solution was left to polymerise at 40.degree. C. Reactions were
terminated when >99% conversion was reached, as judged by
.sup.1H NMR, by exposure to oxygen and addition of acetone. The
catalyst residues were removed by passing the mixture over a basic
alumina column. Acetone was removed under vacuum to concentrate the
sample before precipitation into cold petroleum ether (40.degree.
C.-60.degree. C.) and drying in the vacuum oven overnight. The
polymerisation conditions and procedure is identical to those
described for linear polymers above.
1.4.3 Polymerisation of DEAEMA.sub.50-BDME.sub.2.0
[0208] In a typical synthesis, targeting a DP.sub.n)=50 monomer
units PDEAEMA.sub.50; n.sub.DEAEMA/n.sub.Initiator: 50), bpy (134.9
mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol, 50 eq.), BDME
(291.2 mg, 0.8637 mmol, 0.9 eq) and IPA (38.9% v/v based on DEAEMA)
were placed into a 25 mL round-bottomed flask. The solution was
stirred and deoxygenated using a N.sub.2 purge for 15 minutes.
Cu.sub.(I)Cl (42.8 mg, 0.4318 mmol, 1 eq.) was added to the flask
and left to purge for a further 5 minutes. EBIB (84.2 mg, 0.4318
mmol, 1 eq.) was added to the flask under a positive flow of
N.sub.2, and the solution was left to polymerise at 40.degree. C.
Reactions were terminated when >99% conversion was reached, as
judged by .sup.1H NMR, by exposure to oxygen and addition of
acetone. The catalyst residues were removed by passing the mixture
over a basic alumina column. Acetone was removed under vacuum to
concentrate the sample before precipitation into cold petroleum
ether (40.degree. C.-60.degree. C.) and drying in the vacuum oven
overnight. The polymerisation conditions and procedure is identical
to those described for linear polymers above.
1.5 Polymerisation of tBuMA
1.5.1 Polymerisation of tBuMA.sub.50
[0209] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units (tBuMA.sub.50);
n.sub.tBumA/n.sub.Initiator: 50), bpy (175.7 mg, 1.125 mmol, 2
eq.), tBuMA (4 g, 28.129 mmol, 50 eq.) and aqueous isopropanol
(IPA/H.sub.2O) (92.5/7.5%) (50.4% v/v based on tBuMA) were placed
into a 50 mL round-bottomed flask. The solution was stirred and
deoxygenated using a nitrogen (N.sub.2) purge for 15 minutes.
Cu(I)Cl (55.7 mg, 0.5626 mmol, 1 eq.) was added to the flask and
left to purge for a further 5 minutes. G2-M (0.3934 g, 0.5626 mmol,
1 eq.) was added to the flask under a positive flow of N.sub.2, and
the solution was left to polymerise at 20.degree. C. and samples
were taken periodically from the reaction mixture for .sup.1H NMR
analysis. Reactions were terminated when >99% conversion was
reached, as judged by .sup.1H NMR, by exposure to oxygen and
addition of THF. The catalyst residues were removed by passing the
mixture over a basic alumina column. THF was removed under vacuum
to concentrate the sample before precipitation into hexane and
drying in the vacuum oven overnight.
1.5.2 Polymerisation of tBuMA.sub.50-EGDMA.sub.0.95
[0210] In a typical synthesis, targeting a number average degree of
polymerisation (DP.sub.n)=50 monomer units (tBuMA.sub.50);
n.sub.tBumA/n.sub.Initiator: 50), bpy (175.7 mg, 1.125 mmol, 2
eq.), tBuMA (4 g, 28.13 mmol, 50 eq.), EGDMA (105.9 mg, 0.5345
mmol) and aqueous isopropanol (IPA/H.sub.2O) (92.5/7.5%) (38.4% v/v
based on tBuMA) were placed into a 50 mL round-bottomed flask. The
solution was stirred and deoxygenated using a nitrogen (N.sub.2)
purge for 15 minutes. Cu(I)Cl (55.7 mg, 0.5626 mmol, 1 eq.) was
added to the flask and left to purge for a further 5 minutes. EBIB
(0.1097 g, 0.5626 mmol, 1 eq.) was added to the flask under a
positive flow of N.sub.2, and the solution was left to polymerise
at 20.degree. C. and samples were taken periodically from the
reaction mixture for .sup.1H NMR analysis. Reactions were
terminated when >99% conversion was reached, as judged by
.sup.1H NMR, by exposure to oxygen and addition of THF. The
catalyst residues were removed by passing the mixture over a basic
alumina column. THF was removed under vacuum to concentrate the
sample before precipitation into hexane and drying in the vacuum
oven overnight.
1.6 Polymerisation of DEAEMA-tBuMA
1.6.1 Polymerisation of G2-DEAEMA.sub.x-Stat-tBuMA.sub.y
[0211] In a typical synthesis, targeting a DP.sub.n=50 monomer
units PDEAEMA.sub.25-tBuMA.sub.25; n.sub.DEAEMA/n.sub.Initiator:
50), bpy (134.9 mg, 0.8637 mmol, 2 eq.), DEAEMA (2 g, 10.80 mmol,
25 eq.), tBuMA (1.5352, 10.80 mmol, 25 eq.) and IPA/H.sub.2O
(92.5/7.5%) (30.6% v/v based on DEAEMA/tBuMA) were placed into a 50
mL round-bottomed flask. The solution was stirred and deoxygenated
using a nitrogen (N.sub.2) purge for 15 minutes. Cu(I)Cl (42.8 mg,
0.4318 mmol, 1 eq.) was added to the flask and left to purge for a
further 5 minutes. G2-M (0.3934 g, 0.4318 mmol, 1 eq.) was added to
the flask under a positive flow of N.sub.2, and the solution was
left to polymerise at 40.degree. C. Reactions were terminated when
>99% conversion was reached, as judged by .sup.1H NMR, by
exposure to oxygen and addition of acetone. The catalyst residues
were removed by passing the mixture over a basic alumina column.
Acetone was removed under vacuum to concentrate the sample before
precipitation into cold petroleum ether (40.degree. C.-60.degree.
C.) and drying in the vacuum oven overnight.
1.6.2 Polymerisation of
EBIB-DEAEMA.sub.x-Stat-tBuMA.sub.yEGDMA.sub.0.9
[0212] In a typical synthesis, targeting a DP.sub.n=50 monomer
units PDEAEMA.sub.25-tBuMA.sub.25; n.sub.DEAEMA/n.sub.Initiator:
50), bpy (134.9 mg, 0.8637 mmol, 2 eq.), DEAEMA (2 g, 10.80 mmol,
25 eq.), tBuMA (1.5352, 10.80 mmol, 25 eq.), EGDMA (77 mg, 0.9
mmol, 0.9 eq.) and IPA/H.sub.2O (92.5/7.5%) (30.6% v/v based on
DEAEMA/tBuMA) were placed into a 50 mL round-bottomed flask. The
solution was stirred and deoxygenated using a nitrogen (N.sub.2)
purge for 15 minutes. Cu(I)Cl (42.8 mg, 0.4318 mmol, 1 eq.) was
added to the flask and left to purge for a further 5 minutes. EBIB
(84.2 mg, 0.4318 mmol, 1 eq.) was added to the flask under a
positive flow of N.sub.2, and the solution was left to polymerise
at 40.degree. C. Reactions were terminated when >99% conversion
was reached, as judged by .sup.1H NMR, by exposure to oxygen and
addition of acetone. The catalyst residues were removed by passing
the mixture over a basic alumina column. Acetone was removed under
vacuum to concentrate the sample before precipitation into cold
petroleum ether (40.degree. C.-60.degree. C.) and drying in the
vacuum oven overnight.
1.6.3 Polymerisation of G2-DEAEMA.sub.x-Block-tBuMA.sub.y
[0213] In a typical synthesis, targeting a DP.sub.n=50 monomer
units PDEAEMA.sub.25-tBuMA.sub.25; n.sub.DEAEMA/n.sub.Initiator:
50), bpy (134.9 mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol,
50 eq.) and IPA (38.9% v/v based on DEAEMA) were placed into a 50
mL round-bottomed flask. The solution was stirred and deoxygenated
using a nitrogen (N.sub.2) purge for 15 minutes. Cu(I)Cl (42.8 mg,
0.4318 mmol, 1 eq.) was added to the flask and left to purge for a
further 5 minutes. G2-M (0.3934 mg, 0.4318 mmol, 1 eq.) was added
to the flask under a positive flow of N.sub.2, and the solution was
left to polymerise at 40.degree. C. The reaction was allowed to
reach 80-90% conversion before tBuMA (1.5352, 10.80 mmol, 25 eq.),
bpy (134.9 mg, 0.8637 mmol, 2 eq.) and Cu(I)Cl (42.8 mg, 0.4318
mmol, 1 eq.) dissolved in H.sub.2O/IPA (92.5/7.5%) (27.3% v/v based
on tBuMA) was added to the reaction mixture and left to polymerise
overnight. Reactions were terminated when >99% conversion was
reached, as judged by .sup.1H NMR, by exposure to oxygen and
addition of acetone. The catalyst residues were removed by passing
the mixture over a basic alumina column. Acetone was removed under
vacuum to concentrate the sample before precipitation into cold
petroleum ether (40.degree. C.-60.degree. C.) and drying in the
vacuum oven overnight.
1.6.4 Polymerisation of
EBIB-DEAEMA.sub.x-Block-tBuMA.sub.yEGDMA.sub.0.9
[0214] In a typical synthesis, targeting a DP.sub.n=50 monomer
units PDEAEMA.sub.25-tBuMA.sub.25; n.sub.DEAEMA/n.sub.Initiator:
50), bpy (134.9 mg, 0.8637 mmol, 2 eq.), DEAEMA (4 g, 21.59 mmol,
50 eq.) and IPA (38.9% v/v based on DEAEMA) were placed into a 50
mL round-bottomed flask. The solution was stirred and deoxygenated
using a nitrogen (N.sub.2) purge for 15 minutes. Cu(I)Cl (42.8 mg,
0.4318 mmol, 1 eq.) was added to the flask and left to purge for a
further 5 minutes. EBIB (84.2 mg, 0.4318 mmol, 1 eq.) was added to
the flask under a positive flow of N.sub.2, and the solution was
left to polymerise at 40.degree. C. The reaction was allowed to
reach 80-90% conversion before tBuMA (1.5352, 10.80 mmol, 25 eq.),
bpy (134.9 mg, 0.8637 mmol, 2 eq.), EGDMA (77 mg, 0.9 mmol, 0.9
eq.) and Cu(I)Cl (42.8 mg, 0.4318 mmol, 1 eq.) dissolved in
H.sub.2O/IPA (92.5/7.5%) (27.3% v/v based on tBuMA) was added to
the reaction mixture and left to polymerise overnight. Reactions
were terminated when >99% conversion was reached, as judged by
.sup.1H NMR, by exposure to oxygen and addition of acetone. The
catalyst residues were removed by passing the mixture over a basic
alumina column. Acetone was removed under vacuum to concentrate the
sample before precipitation into cold petroleum ether (40.degree.
C.-60.degree. C.) and drying in the vacuum oven overnight.
2. Polymerisation by ROP
2.1 ROP Dendron Initiator Synthesis
2.1.1 Synthesis of G1-pOH
[0215] 2-(Dimethylamino)ethyl acrylate (6.0 g, 42 mmol, 6 eq.) was
added to a 50 mL round 2 necked round-bottomed flask containing IPA
(12 mL). The flask was deoxygenated under a positive N.sub.2 purge
for 10 minutes. Ethanolamine (0.4266 g, 6.9843 mmol, 1 eq.)
dissolved in IPA (12 mL) was added drop wise while the solution was
stirring in an ice bath under a positive flow of N.sub.2. The final
mixture was stirred for a further 10 minutes at 0.degree. C. before
being allowed to warm to room temperature and left stirring for 48
hrs. The solvent was removed and the product left to dry in vacuo
overnight.
2.1.2 Synthesis of G2-pOH
[0216] CDI (39.137 g, 0.241 mol) was added to a 500 mL 2-neck RBF
fitted with a reflux condenser, magnetic stirrer and a dry N.sub.2
inlet. Dry toluene (350 mL) was added and the flask was purged with
N.sub.2 for 10 minutes. The solution was stirred at 60.degree. C.
and t-Butanol (35.7 g, 46 mL, 0.483 mol) was added via a warm
syringe (Note: t-Butanol is a low melting solid, hence warm it in a
water bath at 35.degree. C. to allow it to melt to a liquid to
easily get it out of the bottle by syringe). The mixture was left
stirring at 60.degree. C. for 6 hours under a positive flow of
nitrogen. Following this, BAPA (16.077 g, 17.14 mL, 0.121 mol) was
added dropwise whilst stirring and maintaining the temperature at
60.degree. C. Upon addition, a white solid precipitate began to
form in the flask (imidazole). The reaction was left stirring for a
further 18 hours at 60.degree. C. under a positive flow of
nitrogen, and then allowed to cool to room temperature. The pale
yellow solution was filtered to remove any solid imidazole, and
concentrated in vacuo. The remaining oil was dissolved in
dichloromethane (250 mL) washed with distilled water (3.times.250
mL) and finally a saturated brine solution (150 mL). The organic
layer was dried with anhydrous Na.sub.2SO.sub.4, filtered and
concentrated in vacuo to give G1-BAPA as a white solid powder. To
remove any remaining residual solvents, the compound was placed
under high vacuum overnight. (38 g, 95%) Found C, 57.84; H, 10.45;
N, 12.91%. C.sub.16H.sub.33N.sub.3O.sub.4 requires, C, 57.98; H,
10.04; N, 12.68%. .sup.1H NMR (400 MHz, CDCl.sub.3) 5.19 (s, br,
NH--disappears on addition of D.sub.2O), 3.21 (t, 4H), 2.65 (t,
4H), 1.65 (q, 4H), 1.44 (s, 18H).sup.13C NMR (100 MHz, CDCl.sub.3)
156.48, 79.34, 47.77, 39.29, 30.11, 28.79. m/z (ES MS) 332.3
[M+H].sup.+
[0217] A mixture of G1-BAPA (20 g, 0.06 mol) 16 mmol) in
1,4-dioxane (200 mL), bromoethanol (7.54 g, 0.6 mol), 30 mg of
sodium iodide, and potassium carbonate (25.0 g, 1.8 mol) was
refluxed overnight. After concentration of the reaction mixture, it
was extracted with ethyl acetate (200 mL), washed with water (100
mL), dried over sodium sulfate, and filtered, and the solvent was
removed under reduced pressure. Purification of the crude product
by flash chromatography (2:1, ethyl acetate-hexane) produced
G1-OH.
[0218] In a 1 L RBF, G1-OH (33.70 g) was dissolved in ethyl acetate
(330 mL) and concentrated HCl (35.03 g, 30 mL, d=1.18 36% active)
was added very slowly. CO.sub.2 began to evolve. The reaction
vessel was left open and stirring for 6 hours. The ethyl acetate
was then removed in vacuo, and a crude .sup.1H NMR (D.sub.2O) of
the remaining oil taken. The crude .sup.1H NMR showed signs of
incomplete decarboxylation (see page 7 for spectra), so the oil was
re-dissolved in 250 mL ethyl acetate and heated to 55.degree. C.
for 5 hours. After removal of ethyl acetate, the crude oil was
dissolved in 4M NaOH (300 mL), and then reduced down by half
(approx.) on the rotary evaporator (60.degree. C.). Following this,
the oily mixture was extracted twice with CHCl.sub.3 (300 mL). The
organic layers were then combined, dried with anhydrous
Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give
bis(3-aminopropyl)amino)propanol as a pale yellow oil.
[0219] 2-(Dimethylamino)ethyl acrylate (6.0 g, 42 mmol, 6 eq.) was
added to a 50 mL round 2 necked round-bottomed flask containing IPA
(12 mL). The flask was deoxygenated under a positive N.sub.2 purge
for 10 minutes. Bis(3-aminopropyl)amino)propanol (1.2271 g, 6.984
mmol, 1 eq.) dissolved in IPA (12 mL) was added drop wise while the
solution was stirring in an ice bath under a positive flow of
N.sub.2. The final mixture was stirred for a further 10 minutes at
0.degree. C., allowed to warm to room temperature and left stirring
for 48 hrs. The solvent was removed and the product left to dry in
vacuo overnight.
2.2 Synthesis of Bifunctional Caprolactone
2.2.2 Synthesis of 4,4'-bioxepanyl-7,7'-dione (BOD)
[0220] Urea hydrogen peroxide (CO(NH.sub.2).H.sub.2O.sub.2) (20 g,
0.21 mol) was added to a 500 mL round-bottom flask containing
formic acid (100 mL). The solution was stirred for 2 h at room
temperature. The flask was then immersed in an ice bath to control
the exotherm resulting from the following stage of the procedure.
Bicyclohexanone (10 g, 0.05 mol) was slowly added to the solution
over a period of 5-10 min. The reaction mixture was stirred for 4 h
whilst the ice bath was changed periodically. 200 mL of water was
then added to the mixture followed by extraction with chloroform
(200 mL.times.4). The organic fraction was collected and washed
with a saturated aqueous sodium bicarbonate solution then dried
overnight with Na.sub.2SO.sub.4. After removing the solvent under
reduced pressure, a white powder was isolated and analysed by NMR
and compared to the literature reported values.
2.3 Polymerisation by ROP
2.3.1 Ring Opening Polymerisation of .epsilon.-Caprolactone
[0221] The typical protocol for the homopolymerisation of CL for a
target number average degree of polymerization DP=50 was as
follows. A 50 mL single necked round bottomed flask was purged with
nitrogen for 15 minutes. SnOct.sub.2 catalyst (0.0018 g, 0.0044
mmol) was added by syringe and needle and the flask purged further.
Distilled CL (3.773 g, 3.5 mL, 33.06 mmol) was introduced into a 50
mL flask and the flask purged for a further 10 minutes. Anhydrous
G1pOH (0.2316 g, 0.6666 mmol) was added via syringe. The flask was
then immersed in a preheated oil bath at 110.degree. C. and
vigorously stirred for the required reaction time of .about.20
hours. The reaction was killed by submerging the reaction in an ice
bath and the polymer purified by dissolving in THF and
precipitating into hexane.
2.3.2 Ring Opening Polymerisation of .epsilon.-Caprolactone and
4,4'-Bioxepanyl-7,7'-Dione (PCL-BOD)
[0222] The typical protocol for the homopolymerisation of CL for a
target number average degree of polymerization DP=50 was as
follows. A 50 mL single necked round bottomed flask containing BOD
(0.9183 g 4.059 mmol) was purged with nitrogen for 15 minutes.
SnOct.sub.2 catalyst (0.0079 g, 0.0195 mmol) was added by syringe
and needle and the flask purged further. Distilled CL (23.28 g,
21.6 mL, 204 mmol) was introduced into a 50 mL flask and the flask
purged for a further 10 minutes. Anhydrous BzOH (0.7315 g, 0.7 mL,
6.764 mmol) was added via syringe. The flask was then immersed in a
preheated oil bath at 110.degree. C. and vigorously stirred for the
required reaction time of .about.20 hours. The reaction was killed
by submerging the reaction in an ice bath and the polymer purified
by dissolving in THF and precipitating into hexane.
In Vitro Cell Viability Experiments
[0223] The effect of nanoparticles carrying SN38, in accordance
with the present invention, on the viability of murine CT-26 cells,
was investigated. It was found that, whilst SN38 is responsible for
a reduction in cell viability, the nanoparticle carrier itself does
not affect cell viability.
[0224] The following polymer compositions (mixtures of branched
polymers and block copolymers) were used:
JF1: p(HPMA.sub.50-co-EGDMA.sub.0.9):p(PEG.sub.114-b-HPMA.sub.120)
50:50 wt % JF2:
p(nBuMA.sub.50-co-EGDMA.sub.0.8):p(PEG.sub.114-b-HPMA.sub.120)
50:50 wt % JF3:
p(tBuMA.sub.25-co-HPMA.sub.25-co-EGDMA.sub.0.9):p(PEG.sub.114-b-HPMA.sub.-
120) 50:50 wt % The following protocol was followed.
Nanoparticle Samples--5 wt % Encapsulation of SN-38
[0225] 1. Add 30 uL of sample (JF1-3) to 970 uL media (final
concentration is 4 uM) [0226] 2. Add 15 uL of sample (JF1-3) to 970
uL media and 15 uL water (final concentration is 2 uM) [0227] 3.
Add 7.5 uL of sample (JF1-3) to 970 uL media and 22.5 uL water
(final concentration is 1 uM)
For SN38 in DMSO
[0227] [0228] 1. Make 2.5 mM stock SN38 solution (2 mg in 2000 uL
DMSO) [0229] 2. Add 1.6 uL of stock to 968.4 uL media and then add
30 uL sterile water (final concentration is 4 uM) [0230] 3. Add 2
uL of stock to 2 ul of DMSO (serial dilution of 1:2), add 1.6 uL of
this to 968.4 uL media and 30 uL sterile water (final concentration
is 2 uM) [0231] 4. Add 2 uL of previous intermediate stock to 2 uL
DMSO (serial dilution of 1:2), add 1.6 uL of this to 968.4 uL media
and 30 uL sterile water (final concentration is 1 uM)
For Controls
[0231] [0232] 1. Add 1.6 uL DMSO to 968.4 uL media and then add 30
uL sterile water [0233] 2. Add 30 uL sterile water to 970 uL
media
For Blanks
[0233] [0234] 1. Add 30 uL of sample (B1-3) to 970 uL media (final
concentration is 4 uM) [0235] 2. Add 15 uL of sample (B1-3) to 970
uL media and 15 uL water (final concentration is 2 uM) [0236] 3.
Add 7.5 uL of sample (B1-3) to 970 uL media and 22.5 uL water
(final concentration is 1 uM)
[0237] Aspirate media on 96 well plate and dose with 100 uL of each
solution in triplicate followed by detection with a 96 well plate
reader, absorbance 490 nm.
[0238] As shown in FIG. 20, after a 48 hour incubation period, the
5 wt % SN38 encapsulated nanoparticles samples, and the SN38 in
DMSO/water sample, resulted in reduced cell viability
(approximately 25% to 45%, compared to 100% for the control). In
contrast, the blank samples (i.e. those containing the polymeric
constituents of the nanoparticles but not the SN38) resulted in
100% cell viability, within error margins.
* * * * *