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.

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

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 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 .Math. nm) PDI (d .Math. nm) (d .Math. 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 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 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 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 PolymerisationFormation 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 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 (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 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 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 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 initiatorPEO.sub.114-OH was used rather than PEO.sub.45-OH.

ATRP PolymerisationSynthesis 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 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 (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 WorkProdrug 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 C.

ATRP PolymerisationIncorporated 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 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.120Targeted 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 (110.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 PolymerisationBranched 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 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 Diblockp(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 Copolymera Tri-Block A-B-A (Hydrophobic-Hydrophilic-Hydrophobic) Polymercan 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 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. -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 C. (1:10 product:solvent) and left to dry under vacuum at 40 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 (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:

##STR00002## [0168] G2-Bz: a generation 2 dendron initiator with benzyl-functionalised ends having the structure:

##STR00003## [0169] G1-M: a generation 1 dendron initiator having the structure:

##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:

##STR00007## [0187] G1-pOH: a generation 1 dendron bearing a hydroxyl group to initiate ROP, of the following structure:

##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 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) 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) 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 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) 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. -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 (330 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) 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) 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. -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 (3160 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) 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) 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. -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 (3160 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) 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) 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 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. -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 (3110 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) 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) 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 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 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 (pH12, 3100 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) 1.44 (6H, d, CH.sub.3CH), 1.65 (4H, m, CH.sub.2CH.sub.2CH.sub.2), 1.95 (6H, s, CH.sub.3CCH2), 3.50-3.69 (4H, m, OCH.sub.2CH.sub.2), 5.60 and 6.15 (4H, 2s, CH.sub.2CCH.sub.3) and 5.95-5.99 (2H, q, CHCH.sub.3). .sup.13C NMR (400 MHz; CDCl.sub.3; Me.sub.4Si) 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 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 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 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 C.-60 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 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 C.-60 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 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 C.-60 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 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 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 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 C.-60 C.) and drying in the vacuum oven overnight.

1.6.2 Polymerisation of EBIB-DEAEMA.SUB.x.-Stat-tBuMA.SUB.y.EGDMA.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 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 C.-60 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 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 C.-60 C.) and drying in the vacuum oven overnight.

1.6.4 Polymerisation of EBIB-DEAEMA.SUB.x.-Block-tBuMA.SUB.y.EGDMA.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 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 C.-60 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 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 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 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 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 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 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 (3250 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, NHdisappears 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 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 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 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 mL4). 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 -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 C. and vigorously stirred for the required reaction time of 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 -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 C. and vigorously stirred for the required reaction time of 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 Samples5 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

[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

[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

[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.