SONODYNAMIC THERAPY

Abstract

The invention provides polymeric particles comprising a matrix of a biocompatible polymer and polyethylene imine, said matrix having incorporated therein an anionic or hydrophobic sonosensitiser and, optionally, an immunomodulatory agent and/or an imaging agent. Such particles find use in methods of sonodynamic therapy, in particular in methods of combined sonodynamic therapy and immunotherapy, for example in the treatment of cancer, metastasis or micrometastasis derived from cancer. The invention is particularly suitable for the treatment of deep-sited, hard to treat tumours such as pancreatic cancer.

Claims

1. A polymeric particle comprising a matrix of a biocompatible polymer and polyethylene imine, said matrix having incorporated therein an anionic or hydrophobic sonosensitiser and, optionally, an immunomodulatory agent and/or an imaging agent.

2. A polymeric particle as claimed in claim 1 which is a microparticle or a nanoparticle, preferably a nanoparticle.

3. A polymeric particle as claimed in claim 1, wherein said matrix forms the body of the particle or wherein said matrix forms the shell of the particle.

4. A polymeric particle as claimed in claim 1, wherein said biocompatible polymer is selected from the group consisting of poly(caprolactone) (PCL), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLLA), and any derivative and/or blend thereof.

5. A polymeric particle as claimed in claim 1, wherein said biocompatible polymer is poly(lactic acid-co-glycolic acid) (PLGA) or a derivative thereof, e.g. a PEG derivative thereof, preferably wherein said biocompatible polymer is poly(D,L-lactic-co-glycolic acid).

6. A polymeric particle as claimed in claim 5, wherein said biocompatible polymer has a weight average molecular weight ranging from 7,000 to 240,000 Da, preferably from 50,000 to 150,000 Da, e.g. from 60,000 to 120,000 Da.

7. A polymeric particle as claimed in claim 5, wherein said PLGA has a lactic acid:glycolic acid ratio of about 75:25.

8. A polymeric particle as claimed in claim 1, wherein the polyethylene imine (PEI) polymer is branched, and/or wherein the PEI polymer has a weight average molecular weight ranging from 500 to 100,000 Da, preferably from 20,000 to 30,000 Da, e.g. about 25,000 Da.

9. A polymeric particle as claimed in claim 1, wherein the content of PEI in the particle as a percentage of the biocompatible polymer (e.g. PLGA) is in the range from 0.05 to 10 wt. %, preferably from 1 to 5 wt. %, e.g. about 2.1 wt. %

10. A polymeric particle as claimed in claim 1, wherein the sonosensitiser is selected from the following compounds or is a derivative of any of the following compounds: phenothiazine dyes (e.g. methylene blue, toluidine blue), Rose Bengal, porphyrins (e.g. Photofrin®), ATX-70, chlorins, benzochlorins, phthalocyanines, napthalocyanines, porphycenes, cyanines (e.g. Merocyanine 540 and indocyanine green), azodipyromethines (e.g. BODIPY and halogenated derivatives thereof), acridine dyes, purpurins, pheophorbides, verdins, psoralens, hematoporphyrins, protoporphyrins, curcumins, and their pharmaceutically acceptable salts.

11. A polymeric particle as claimed in claim 10, wherein the sonosensitiser is Rose Bengal.

12. A polymeric particle as claimed in claim 1 which further comprises an immunomodulatory agent.

13. A polymeric particle as claimed in claim 12, wherein said immunomodulatory agent is selected from the following and any pharmaceutically acceptable salts thereof: TLR7 agonists (e.g. i.e. Imiquimod, Resiquimod, Gardiquimod, Poly I:C (Polyinosinic: polycytidylic acid), CpG oligodeoxynucleotides, and Anti-galactosylceramide; immunoadjuvants such as indole 2,3 dioxygenase 1 inhibitors (e.g. Indoximod, Epacadostat, and Navoximod); and low molecular weight immune checkpoint inhibitors (e.g. BMS-1001, BMS-1166, and CCX872).

14. A polymeric particle as claimed in claim 13, wherein said immunomodulatory agent is Imiquimod.

15. A polymeric particle as claimed in claim 1 which further comprises an imaging agent, for example a near-infra-red imaging agent (e.g. indocyanine green, or an analogue or derivative thereof), a radiocontrast agent (e.g. diatrizoate or metrizoate), or an MR imaging agent (e.g. Omniscan).

16. A particulate composition comprising a plurality of polymeric particles as claimed in claim 1.

17. A method of sonodynamic therapy, e.g. a method of combined sonodynamic therapy and immunotherapy, said method comprising the step of administering to cells or tissues of a subject in need thereof a pharmaceutically effective amount of a particulate composition as claimed in claim 16, and subjecting said cells or tissues to ultrasound irradiation.

18. (canceled)

19. A method as claimed in claim 17 for the treatment of cancer, metastasis or micrometastasis derived from said cancer, in the treatment of circulating tumour cells (CTCs), or in the treatment of multiple primary tumours, preferably for use in the treatment of a deep-sited tumour, metastasis or micrometastasis derived from said tumour.

20. A method as claimed in claim 19 for the treatment of pancreatic cancer or metastatic pancreatic cancer.

21. A method as claimed in claim 17 for the prevention of secondary lesions in a subject (e.g. a patient).

22. A pharmaceutical composition comprising a polymeric particle as claimed in claim 1, together with at least one pharmaceutical carrier or excipient.

23. (canceled)

24. A method of sonodynamic therapy which comprises at least the following steps: (a) administering a particulate composition as claimed in claim 16 to affected cells or tissues of a subject in need thereof (e.g. a patient) and subjecting said cells or tissues to ultrasound irradiation; and (b) simultaneously, separately or sequentially administering to said subject (e.g. said patient) a pharmaceutically effective amount of an immune checkpoint inhibitor.

25. (canceled)

Description

[0114] The invention will now be described further with reference to the following non-limiting Examples and the accompanying figures in which:

[0115] FIG. 1 shows ROS production by RB, RB+ICG or RB+ICG+IM containing nanoparticles in the presence and absence of ultrasound (US). Error bars represent±the SD and n=3.

[0116] FIG. 2 shows nIR fluorescence imaging based study demonstrating particle uptake by pancreatic cancer cells (BxPC3). The inset shows a typical fluorescence image from which the data were derived. Error bars represent±SD where n=3.

[0117] FIG. 3 shows the treatment of BxPC3 (A), T110299 (B) and hPSC (C) cells with RB+ICG nanoparticles in the presence (+US) and absence (−US) of ultrasound. Error bars represent t SEM where n=5.

[0118] FIG. 4 shows the treatment of BxPC3 (A) and T110299 (B) with RB+ICG+IM nanoparticles in the presence (+US) and absence (−US) of ultrasound. Error bars represent±SEM where n=6.

[0119] FIG. 5 shows whole body (A)(n=3) and excised organ (B) (n=2) nIR fluorescence imaging to demonstrate tumour sequestration of RB+ICG nanoparticles. For A the error bars represent±SD.

[0120] FIG. 6 shows the treatment of human pancreatic xenograft tumours in SCID mice using RB+ICG nanoparticles (NP) in the present and absence of ultrasound (US). Arrows indicate treatment times. Error bars represent±SD, where n=3 for NP, n=4 for untreated and n=4 for NP+US.

[0121] FIG. 7 demonstrates an SDT-mediated abscopal effect in animals bearing multiple tumours. Target tumours (A) were treated with nanoparticles alone (NP), ultrasound alone (US) and a combination of each. Growth of the off-target tumour (B) from each group of animals was also monitored. Arrows indicate treatment times for the targeted tumour. Error bars represent±SEM and n=5 for each group.

[0122] FIG. 8 shows the comparative effect of RB+ICG (RB NP)- and RB+ICG+IM (IM NP)-mediated SDT on target and off-target tumours in the same animal. Arrows indicate the treatment times for the target tumour. Error bars represent+SEM and for the untreated control n=17, IM NP alone n=3, IM NP+US n=5, RB NP alone n=4, RB NP+US n=4.

[0123] FIG. 9 shows the protective effect provided against tumour establishment by RB+ICG+IM (IM NP)-mediated SDT. For comparative purposes, tumours were also treated with RB+ICG (RB NP)-mediated SDT. Arrows indicate treatment times at the target tumours. Error bars represent±SEM and for the untreated control n=3, IM−US n=4, IM+US n=3, RB−US n=4, RB+US n=5.

[0124] FIG. 10 shows the staining of residual tumour tissues for stroma (intense red colour—shown as dark grey) using Sirius Red. Magnification=50.

[0125] FIG. 11 shows the percentage of red pixels (stroma) per section frame in FIG. 10 where error bars represent±SEM; n=4 and * p<0.05 and ** P<0.01.

[0126] FIG. 12 shows tumour infiltrating (A) leukocytes (CD45.sup.+) and (B) cytotoxic T cells (CD8a.sup.+) in target and off-target tumours receiving no treatment (UNT), treated with RB and ICG containing nanoparticles plus ultrasound (RB NP+US) and treated with RB, ICG and imiquimod-containing nanoparticles plus ultrasound (IM NP+US). Error bars represent±SEM where n=4 and * p<0.05, *** p<0.001.

EXAMPLES

Example 1—Nanoparticle Preparation and Characterisation

[0127] 1.1 Nanparticle Preparation:

[0128] The following procedure was used to prepare nanoparticles containing Rose Bengal (RB), containing Rose Bengal plus indocyanine green (RB+ICG) or containing Rose Bengal, indocyanine green and imiquimod (RB+ICG+IM). 60 mg of poly(DL-lactic-co-glycolic acid) (PLGA)(75:25, MW 66,000-107,000) was dissolved in 2 mL acetone. A 1.25 mg aliquot of polyethylenimine (PEI)(branched, MW 25,000) (1.25 mg/mL) was added to 4 mL of acetone. For RB nanoparticles, 10 mg RB was dissolved in 2 ml ethanol. For RB+ICG nanoparticles, the ICG was dissolved in ethanol at a concentration of 1 mg/mL and for RB+ICG+IM nanoparticles, the IM was dissolved at a concentration of 1 mg/mL in methanol. For generation of the relevant nanoparticles the appropriate payload solution or combination of solutions were added dropwise to 40 mL of 1% polyvinyl alcohol (PVA) (87-90% to hydrolysed, MW 30,000-70,000). During addition of the payload solutions to the PVA, efficient emulsification was ensured by sonication using a 6 mm ultrasound probe (Vibra-Cell), at a frequency of 20 kHz, operated at 70% amplitude for 6 min. after which the suspension was stirred overnight to ensure solvent removal. Nanoparticles were recovered by centrifugation at 10,500 rpm for 20 minutes and then washed, firstly, with distilled water and then phosphate buffered saline (PBS). The pellets were re-suspended in 3 ml distilled water and lyophilised for storage.

[0129] 1.2 Nanparticle Characterisation:

[0130] Nanoparticle size was determined using dynamic light scattering using a Malvern Zetasizer (Malvern Instruments, UK). Particles were suspended at a concentration of 1 mg/mL in distilled water and both the size and polydispersion index was determined for each type of particle. The results are summarised in Table 1 and demonstrate that the particles ranged in size from 226 to 263 nm, which is compatible with exploiting the tumour enhanced permeability and retention (EPR) effect. In addition, the particles had relatively low PDIs indicating a relatively monodisperse preparation.

TABLE-US-00001 TABLE 1 DLS analysis of nanoparticles Sample Diameter (nm) PDI RB 235-258 0.03-0.2 RB + ICG 231-263 0.03-0.2 RB + ICG + IM 226-259  0.09-0.16

[0131] To determine the loading efficiency (LE) of RB, ICG and IM, the relevant particles were dissolved in dimethyl sulfoxide (DMSO) and the concentrations of each payload were determined by measuring the absorbance at 565 nm and 790 nm, respectively. The loading efficiency of IM was determined using HPLC. The loading efficiency is expressed as the mass of the relevant payload as a % of the theoretical maximum possible. The data obtained are shown in Table 2.

TABLE-US-00002 TABLE 2 Loading efficiencies of payloads into nanoparticles. Sample LE (RB)% LE (ICG)% LE (IM)% RB 47.2 0 0 RB + ICG 36.1 26.8 0 RB + ICG + IM 38.8 26.4 20.5

Example 2—Preparation of RB Nanoparticles in the Presence and Absence of PEI

[0132] In order to demonstrate the value of incorporating PEI into the formulation, RB particles were prepared in the presence and absence of PEI. Following preparation, both formulations were visually compared. The preparation containing PEI was more intensely coloured and clearly demonstrated that these particles contained more RB.

Example 3—Ultrasound-Mediated Reactive Oxygen Species Generation

[0133] In order to demonstrate that particles were capable of generating reactive oxygen species (ROS) when exposed to ultrasound, thereby demonstrating their potential for use in SDT, particles (RB or RB+ICG or RB+ICG+IM) were suspended at a concentration of 1 mg/mL in phosphate buffered saline (PBS). 2 mL of this was added to 50 mL of 10 μM 1,3-diphenylisobenzofuran (DPBF) prepared in a water/ethanol mixture (50:50) which had been aerated for 10 min. During exposure to ultrasound at a frequency of 1 MHz, a power density of 3.5 W/cm.sup.2, a duty cycle of 50% (at a pulse repetition frequency of 100 Hz) for 30 min, 1 mL samples were harvested at 5 min intervals and the absorbance at 410 nm was determined to assess the oxidative bleaching of DBPF. The results are shown in FIG. 1 and they demonstrate that the particles are able to generate ROS in the presence of ultrasound. The results also confirmed the potential of these particles for use in SDT.

Example 4—nIR Fluorescence Imaging to Monitor Cellular Uptake of RB+ICG Nanoparticles

[0134] In order to demonstrate that incorporating ICG could provide a nIR imaging capability, the cellular uptake of RB+ICG particles using nIR imaging was assessed. 5×10.sup.3 human pancreatic cancer cells (BxPC3) were plated into each well of a 96-well plate in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum and cultured overnight at 37° C. in a humidified 5% CO.sub.2 atmosphere. 1 mg of particles was suspended in 0.1 mL of PBS and this was subsequently diluted 1:10 in serum free RPMI 1640 medium. Following removal of the original medium from each well, 100 μL of the particle suspension was placed in each well. Over a 3 h period this suspension was periodically removed and replaced with serum containing medium. A Xenogen IVIS® Lumina imaging system, equipped with an ICG filter set, was used to read the fluorescence signal and data were analysed using Living Image® software v.2.6. The data are presented in FIG. 2 and demonstrate that the ICG component of the nanoparticle affords an imaging capability that can be exploited to identify particle location.

Example 5—SDT Treatment Using RB+ICG Nanoparticles—In Vitro Studies

[0135] To demonstrate that the RB+ICG particles could elicit cytotoxic effects in vitro, three cell lines were employed as targets. BxPC3 cells are human pancreatic cancer cells, T110299 are mouse pancreatic cancer cells derived from a KPC genetically engineered mouse model carrying both Kras and p53 mutations, and hPSC cells are human pancreatic stellate cells. The latter serve as precursors of pancreatic fibroblasts and result in desmoplasia or the formation of fibrous connective tissues that form a pathological obstacle to effective treatment. While BxPC3 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, the T110299 cells were cultured in high glucose DMEM supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% L-glutamine and 1% penicillin/streptomycin, and the hPSC line was cultured in high glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. BxPC3 cells, T110299 cells and hPSC were plated into 96-well plates at concentrations of 5×10.sup.3, 1.5×10.sup.3 and 3×10.sup.3 cells per well, respectively and cultured overnight at 37° C. in a humidified 5% CO.sub.2 atmosphere. Spent medium was removed from the wells and replaced with fresh serum-containing medium and both the BxPC3 and T110299 were treated with concentrations of nanoparticles at 0, 0.1, 0.5, 1 and 5 μM with respect to RB. Cells were also treated with free RB at a concentration of 0.5 μM. The hPSC cell line was treated with nanoparticles at 0, 0.01, 0.05, 0.1 and 0.5 μM with respect to RB and these cells were also treated with free RB at a concentration of 0.5 μM for comparative purposes. Both BxPC3 and T110299 cells were incubated for 24 h after which the medium was replaced. The hPSC line was incubated for 4 h with particles and the medium was then replaced. The latter was done because if the cells were treated for 24 h with the nanoparticles, no cells remained viable following subsequent treatment with ultrasound. Cells were then treated with ultrasound by placing the individual wells in contact with the ultrasound transducer and contact was mediated by an ultrasound gel. Each well was treated with ultrasound for 30 s at a frequency of 1 MHz, a power density of 3 W/cm.sup.2 and a duty cycle of 50% at a pulse repetition of 100 Hz. Plates were incubated for a further 24 h and cell viability was determined using the MTT assay. Cell viability was expressed as a % of the untreated control for each cell line. The data are shown in FIGS. 3A, B and C and they demonstrate: (i) that all 3 cell lines exhibited reduced cell viability in the presence of ultrasound and nanoparticles; (ii) for all three cell lines little or no toxicity was exhibited by the particles in the absence of ultrasound; (iii) the ultrasound conditions employed in the treatments had little or no effect on cell viability; and (iv) for all three cell lines the reduction in viability obtained with ultrasound together with particles containing 0.5 μM RB exhibited a similar reduction in cell viability to cells treated with ultrasound and the free RB at that concentration. Surprisingly, it was found that the hPSC were more susceptible to SDT than the two cancer cell lines since a lower concentration of nanoparticles provided a greater reduction in cell viability. This could prove useful in the treatment of pancreatic cancer in particular, since specific eradication of such cells may have a negative impact on desmoplasia.

Example 6—SDT Treatment Using RB+ICG+IM Nanoparticles—In Vitro Studies

[0136] In order to confirm that the nanoparticles could still deliver an SDT effect following incorporation of IM into the particles, both BxPC3 and T110299 cells were treated with the RB+ICG+IM containing nanoparticles. Cells were prepared and treated as described in Example 5 and the results are shown in FIGS. 4A and 4B. The data confirm that inclusion of the IM does not have a negative impact on the ability of the nanoparticles to impart a cytotoxic effect in the presence of ultrasound.

Example 7—Distribution of RB+ICG Nanoparticles In Vivo

[0137] In order to confirm that the incorporation of ICG into the nanoparticles provides an imaging capability and to further exploit this aspect to assess both tumour sequestration of the particles and organ distribution, a human pancreatic tumour xenograft model (BxPC3) was employed in SCID mice. All animals were treated humanely and in accordance with licenced protocols under the UK Animal (Scientific procedures) Act 1986. Tumours were generated by injecting 1×10.sup.6 cells suspended in 100 μL of a 1:1 mixture of complete medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin) and Matrigel™ subcutaneously into the rear dorsum of 8 week old SCID mice. Once tumours had reached an average size of 110 mm.sup.3, animals were anaesthetised using Hypnorm/Hypnovel administered intraperitoneally and a 100 μL aliquot of nanoparticles suspended in PBS to ensure delivery of RB at a concentration of 250 μM, was then administered intravenously to each animal. Following administration of the nanoparticles, the anesthetised animals were imaged using nIR fluorescence imaging as described in Example 4. In analysing the data a reference point on an extremity of the animal was chosen as a surrogate of whole body fluorescence and the ratio of the signal obtained from the tumour region was expressed relative to that reference to semi-quantitatively determine selective uptake of nanoparticles by the tumour. The data are shown in FIG. 5A and they demonstrate that the signal in the tumour, relative to whole body fluorescence increased greater than 6-fold in the first 7 h and this subsequently decreased to 4-fold at 24 h. These data demonstrate that the nanoparticles were being sequestered in the tumour following administration. Preferential uptake by the tumour was confirmed by sacrificing the animals and surgically removing and imaging relevant organs. The data in FIG. 5B indicate that in relative terms a significant signal was obtained from both the tumour and the liver with both kidney and brain exhibiting the next highest signal. Although a similar signal was obtained for both the liver and the tumour using the nanoparticles, Yue et al. (May, 2019 Nat Commun. 2019 May 2; 10(1):2025) using a liposomal formulation bearing a sensitiser and immune modulator as payloads, demonstrated that the liver contained over twice the amount of liposomes than the tumour at 24 h. In comparative terms the invention is superior to the liposomal formulation described in Yue et al. since it clearly demonstrates a greater degree of tumour sequestration of the polymeric nanoparticles. In overall terms the data confirm that incorporation of ICG into the nanoparticles provides an imaging capability and this has been exploited to demonstrate a higher degree of preferential tumour sequestration of the nanoparticles than previously demonstrated.

Example 8—SDT Treatment of Human Pancreatic Xenograft Tumours Using RB+ICG Nanoparticles

[0138] To demonstrate that the RB+ICG nanoparticles could be employed for SDT, BxPC3 tumours were established in SCID mice as described for Example 7. When tumours had reached an average size of 110 mm.sup.3, nanoparticles were administered intravenously to mice as described for Example 6. 30 min. after administration of nanoparticles, the relevant animals were treated with ultrasound for 3.5 min at a frequency of 1 MHz, a power density of 3.5 W/cm.sup.2 and a duty cycle of 30%, using a pulse repetition frequency of 100 Hz. Animals were treated with nanoparticles and ultrasound as indicated by the arrows in FIG. 6. Control animals either received nanoparticles alone at the times indicated by the arrows in FIG. 6 or were untreated. Tumour volume was monitored at the times indicated in FIG. 6. The data demonstrate that for animals treated with nanoparticles followed by ultrasound, tumour growth was dramatically reduced. These results demonstrate that these nanoparticles can elicit an SDT effect in vivo.

Example 9—Demonstration of an Abscopal Effect Using RB+ICG Nanoparticle-Mediated SDT

[0139] In order to demonstrate that RB+ICG nanoparticle-induced SDT can elicit an abscopal effect, i.e. impart a therapeutic effect at an off-target tumour, two tumours were induced in each animal. For these studies the cell line, T110299 described above in Example 5, was used to generate tumours in 8 week old immunocompetent C57BL/63 mice. A tumour was established on each side of the rear dorsum of each mouse by subcutaneously injecting each site with a 100 μL aliquot containing 0.5×10.sup.6 T110299 cells suspended in a 1:1 mixture of complete medium (DMEM supplemented as described in Example 5) and PBS. When tumours reached an average size of 130 mm.sup.3 animals were treated. When treating with nanoparticles and with the exception of the time of treatment, these were administered intravenously as described for Example 8. When treating with ultrasound, with the exception of the days on which animals were treated, conditions were the same as those described in Example 8 and in all cases only the target tumour received ultrasound. Animal groups consisting of 5 animals each were (1) untreated, (2) treated with ultrasound alone, (3) treated with RB+ICG nanoparticles alone and (4) treated with RB+ICG nanoparticles together with ultrasound. The size of both the target and off-target tumours were monitored and the data obtained are shown in FIGS. 7A and B. As shown in FIG. 7A, the target tumours receiving the combined treatment with both nanoparticles and ultrasound exhibited a dramatic decrease in tumour growth, while nanoparticles or ultrasound had any impact on tumour growth. These data confirm those described in Example 8, demonstrating that the nanoparticles could be employed for SDT. In examining growth of the off-target tumour, i.e. the tumour that did not receive ultrasound treatment in each animal (FIG. 7B), again a dramatic decrease in tumour growth in animals receiving the combined treatment of nanoparticles plus ultrasound was observed and again, no difference in growth was observed in animals treated with nanoparticles or ultrasound alone. The data demonstrate that the RB+ICG nanoparticles can elicit a potent abscopal effect. In contrast, the liposomal formulation described by Yue et al., (2019) failed to elicit an abscopal effect in the absence of an immune checkpoint inhibitor using an ectopic model, even when their formulation contained the immune modulator imiquimod. Although they describe an abscopal effect in the absence of an immune checkpoint inhibitor using an orthotopic model, their effect is weaker than that observed at their primary target lesion. In overall terms the data obtained using the invention suggest that treatment of a single tumour with RB-ICG-mediated SDT could potentially be used to control disease at metastatic sites.

Example 10—Enhanced Abscopal Effect Using RB+ICG+IM Nanoparticle-Mediated SDT

[0140] Since an abscopal effect was detected using the RB+ICG nanoparticles, the immunomodulatory, imiquimod (IM), was then incorporated. This is a toll-like receptor agonist (TLR7) and it has been suggested that it and similar compounds (resiquimod, gardiquimod, 3M-011) can serve as immunomodulators capable of activating antigen presenting cells together with T and natural killer (NK) cells. Although TLR7 or TLR7/8 agonists have been used both in preclinical and clinical studies relating to the generation of protective immune responses in cancer, their utility in the treatment of pancreatic cancer is confusing with some reports suggesting a protective response and others even suggesting a potential cancer promoting response [Chi et al. (2017); Scholch et al. (2014); Zou et al. (2015); Ochi et al. (2012) and Grimmig et al. (2017)]. Therefore, in order to determine if IM would contribute any therapeutic advantage, this was incorporated into the nanoparticles as described in Example 1.

[0141] The target model employed in this study was the immunocompetent mouse model used in Example 9 and the comparative efficacy of particles containing RB+ICG and RB+ICG+IM was assessed, each in the presence and absence of ultrasound as indicated by arrows in FIG. 8 using the ultrasound treatment conditions described in Example 9. Particles were administered intravenously at the times indicated by arrows in FIG. 8 to relevant groups of tumour bearing animals at the concentration (with respect to RB) as described for Example 9 and tumour volume was monitored. Control animals were untreated, treated with particles alone or treated with ultrasound alone. The data obtained are shown in FIG. 8 where A shows the effects of treatment on the ultrasound targeted tumour in each animal and B shows the effects on the tumour that did not receive ultrasound treatment. From the data in FIG. 8A, it can be seen that both the RB+ICG and the RB+ICG+IM particles deliver a similar response in terms of delivering a decrease in tumour growth. It was interesting to note also that the IM-containing particles did not exhibit any stimulatory effect on tumour growth in the group of animals that were treated with nanoparticles alone. However, at the off-target tumour (FIG. 8B) it was noted that the particles containing the IM resulted in a greater reduction in tumour growth, demonstrating that the inclusion of IM in the particles potentiated the therapeutic effect at the off-target tumour. These results demonstrate that the RB+ICG+IM nanoparticles could be used as a means of potentiating the effects of SDT, particularly for treating metastatic disease.

Example 11—the Protective Effect of RB+ICG+IM-Mediated SDT

[0142] Since it was shown that the abscopal effect delivered at the off-target tumour using RB+ICG+IM-mediated SDT was potentiated by inclusion of the IM in the particles, it was of interest to determine if this effect could protect animals from developing new tumours. If this was proven to be the case, it would suggest that the RB+ICG+IM-mediated SDT was capable of generating immunological memory by way of eliciting an adaptive immune response. To this end, the immunocompetent mouse pancreatic tumour model was again used as the target. In these studies a single tumour was established in each animal as described in Example 9. Animals were treated with RB+ICG- and RB+ICG+IM-mediated SDT as described for Example 10 and 24 h later, cells were injected into an off-target site on each animal in an attempt to establish a second tumour. The target tumour continued to be treated (nanoparticles and ultrasound) as indicated in FIG. 9. Growth of both the treated target tumour and any tumour that developed at the off-target site was monitored. The results are shown in FIG. 9, where A shows growth of target tumours and B shows growth of any tumours that established at the off-target site. The data clearly demonstrate that a decrease in tumour growth was observed in target tumours treated with RB+ICG- and RB+ICG+IM-mediated SDT, confirming previous results in Example 10. However, at the off-target site the only group that did not exhibit establishment of tumours was the group of animals treated with RB+ICG+IM-mediated SDT. In the study by Yue et al. (2019) all animals treated with their formulation in the absence of an immune checkpoint inhibitor developed tumours when rechallenged. With the invention, the results presented herein demonstrate that RB+ICG+IM-mediated SDT can provide a protective effect against the development of secondary lesions and confirms that it could play a role in control of metastatic disease. This result is surprising since it has previously been suggested that TLR7/8 agonists could play a developmental role in pancreatic cancer [Ochi et al. (2012) and Grimmig et al. (2017)].

Example 12—Impact of RB+ICG Nanoparticle-Mediated SDT on Tumour Stroma

[0143] Since particle-mediated SDT exhibited a more potent effect with pancreatic stellate cells rather than cancer cells as demonstrated in Example 5 and FIG. 3, and since these cells serve as stromal precursors, it was decided to exploit histology together with specific stromal staining to examine residual tumour tissues in animals treated with particle-mediated SDT. Animals from the study described in Example 9 were sacrificed on day 11 and residual tumour tissues were surgically excised, fixed in 4% paraformaldehyde, embedded in paraffin and sectioned using a microtome into 5 μm sections prior to staining. The tumour sections were de-paraffinized in xylene and hydrated in graded ethanol prior to staining with haematoxylin for 3 minutes, followed by washing in warm tap water for 30 minutes and undergoing secondary staining with Picrosirius Red (Sirius Red stain for stroma) for 1 hour. After 2 quick washes in 0.5% acetic acid, ascending concentrations of ethanol were employed to dehydrate sections which were then cleared in two washes of xylene before mounting occurred with resinous mounting medium and glass coverslips. Images were captured using a Zeiss Microscope, and the percentage of Sirius Red positive pixels was quantified across all treatment groups using ImageJ (National Institutes of Health, USA).

[0144] The data obtained are shown in FIG. 10 and the stroma tissue is stained an intense red colour in all sections. However, direct observation of sections from tumours that were either untreated or treated with NPs alone demonstrated that the stroma is much more prevalent than in sections of residual tissue examined following treatment with NPs plus ultrasound. This was confirmed by quantitative comparison of red pixels (stroma) in all images using ImageJ as shown in FIG. 11. These results, taken together with the data from Example 5, demonstrate that NP-mediated SDT is capable of significantly modifying tumour stroma and this potentially offers a significant therapeutic benefit. It is well known that the existence of stroma in many solid tumours is a negative prognostic marker primarily because it promotes invasion and metastasis, and because it presents a barrier to entry of therapeutic drugs or cells of the immune system. It has been shown that drugs which disrupt the stromal barrier can be exploited to enable more effective drug- or immune-based therapies (Kianna et al., Cancer Research, 79: 372-386, 2019). This example therefore demonstrates a clear and hitherto unforeseen advantage associated with NP-mediated SDT, particularly in the context of assisting immunotherapy. A SDT-induced decrease in tumour stroma would permit cells of the immune system to penetrate tumour tissues in order to be activated by either endogenously-generated antigens resulting from the SDT treatment (e.g. damage associated molecular patterns, DAMPs) and/or benefit from an exogenously added stimulant of the immune system simultaneously provided by the nanoparticles (e.g. imiquimod in the RB+ICG+IM nanoparticles).

Example 13—SDT-Mediated Enhanced Infiltration of Leukocytes and Cytotoxic T Cells into Tumours Following Treatment with RB+ICG or RB+ICG+IM Nanoparticles

[0145] Since a dramatic abscopal effect was evident in particle-mediated SDT (as shown in FIGS. 7 and 8) it was decided to see if treatment of tumours resulted in infiltration of immune cells into those treated tumours. Animals bearing two tumours were established as described in Example 9 and were either untreated, treated with RB and ICG-containing nanoparticles plus ultrasound or treated with RB, ICG and imiquimod-containing nanoparticles plus ultrasound. As in Example 9, only one tumour in each animal (target) was exposed to ultrasound. Animals were sacrificed 48 h after treatment and both the target and off-target tumours were harvested from each animal. Tissues were homogenised in RPMI 1640 medium supplemented with 4% foetal bovine serum, 160 μL collagenase type II (30 mg/mL), 50 μL DNAse (2 μg/mL) and mixed gently for 15 min at room temperature. The suspension was filtered to remove clumps, washed with PBS by centrifugation at 150 g for 5 min and incubated with anti-CD45 (CD45.sup.+, leukocytes) and anti-CD8a (CD8a.sup.+, cytotoxic T cells) antibodies (Thermo Scientific, UK) for 30 min in the dark. Samples were washed twice with PBS by centrifugation and analysed using a Beckman Coulter Gallios flow cytometer (Beckman Coulter, UK) and post-acquisition analysis was completed using Kaluza analysis software (Beckman Coulter, UK).

[0146] The data obtained from these experiments are shown in FIGS. 12A and 12B. The data demonstrate no significant infiltration of leukocytes (CD45.sup.+) into the untreated and RB+ICG nanoparticle-mediated SDT treated tumours, whereas a very dramatic and statistically significant (P<0.001) degree of infiltration of leukocytes occurred in the target tumour treated with the RB, ICG and imiquimod-containing nanoparticle-mediated SDT (FIG. 12A, Target; IMQ NP+US). Based on the effects of particle-mediated SDT on stroma (as shown in Example 11), one might have expected to find enhanced infiltration into SDT-treated target tumours for both the RB+ICG and RB+ICG+IMQ nanoparticle-mediated treatments (although it should be noted that SDT treatment might also be expected to catastrophically impact negatively on tumour vascularisation and this could preclude infiltration). Although undetectable infiltration of CD45.sup.+ occurred in the target tumour treated with RB and ICG-containing nanoparticles plus ultrasound, it is apparent that a sufficient immune response is generated to provide a dramatic abscopal effect (FIGS. 7 and 8). In addition, although there is no statistically significant increase in infiltration of CD45.sup.+ following treatment with each nanoparticle at the off-target tumours, there does appear to be an upwards trend observable (FIG. 12A, Off-target). This results from the generation of a tumour-specific immune response in the form of tumour homing immune cells that accumulate via the intact vasculature of the off-target tumour.

[0147] Incorporating imiquimod into the particles, enabling simultaneous delivery of both the sonosensitiser and the immunoadjuvant, results in a significant enhancement of tumour infiltrating leukocytes and this effect may be responsible for the enhanced abscopal effect observed in FIG. 8 at the off-target tumour following treatment with the imiquimod-containing particle. These suggestions are further supported by the data presented in FIG. 12B. This shows cytotoxic T cell (CD8a.sup.+) infiltration and again similar patterns were observed at both the target and off-target tumours where a statistically significant (p<0.05) degree of infiltration was observed at the target tumours following treatment with the imiquimod-containing particles (IMQ NP+US) and an upward trend in cytotoxic T cell infiltration occurred in the off-target tumours when treated with each particle and subjected to ultrasound.

[0148] The data presented herein demonstrate a clear advantage associated with systemic co-delivery of the imiquimod together with the sonosensitiser in a single particle since (1) imiquimod is clinically incompatible with systemic administration; and (2) particle deposition at the target tumour enables co-placement of the sonosensitiser and immune stimulant at the target site, so that the latter is present during SDT-induced generation of DAMPs, thereby enabling a highly localised form of in situ vaccination.