Nanoparticles for cancer therapy and diagnosis

Abstract

The present invention relates generally to methods and materials for use in photothermal or sonodynamic therapy. The invention novel nanoparticles for use in delivering sensitizers to solid tumour target, wherein the nanoparticles are composed of a polymers or co-polymer of monomers linked by peptide bonds, wherein the polymer or co-polymer comprises one or both of glutamate or derivatised glutatamic acid, and optionally a further, different, monomer which is a naturally occurring amino acid or synthetic monomer having a side chain group, wherein the polymer or co-polymer is not composed only of glutamate. The pendant groups and/or side chains of the polymer or co-polymer interact non-covalently with the sensitizer.

Claims

1. A nanoparticle for use in delivering an amphiphilic or hydrophobic sensitizer to a solid tumour target, wherein exposure of said sensitizer to either NIR or ultrasound causes ablation of said tumor, wherein the nanoparticle contains, and binds non-covalently to, the sensitizer, wherein the nanoparticle is composed of a polymer or co-polymer of at least 30 monomers linked by peptide bonds, wherein the polymer or co-polymer comprises one or both of monomers M1 and M2, wherein M1 is glutamic acid, wherein M2 is glutamic acid wherein a carboxyl group is derivatised with a pendant group, wherein the pendant group is selected from: a lipid; a group comprising an aromatic ring; a positively-charged group; an aliphatic group; wherein the polymer or co-polymer optionally further comprises monomer M3 which is different to M1 and M2, wherein M3 is either a naturally occurring amino acid having a side-chain group, or M3 is a synthetic monomer having a side chain group wherein the side chain group is selected from: a positively charged group; a polar uncharged group; a hydrophobic group, wherein the polymer or co-polymer comprises at least M2 or M3, and whereby the pendant groups and/or side chains of the polymer of co-polymer interact non-covalently with the sensitizer.

2. A nanoparticle as claimed in claim 1 further encapsulating said sensitizer, which is non-covalently bound to the pendant groups and/or side chains of the polymer or co-polymer.

3. A nanoparticle as claimed in claim 1, which is a co-polymer of M1 and M3.

4. A nanoparticle as claimed in claim 1, wherein M3 is selected from lysine, arginine, histidine, asparagine and glutamine, tyrosine, tryptophan, phenylalanine, methionine, leucine, and isoleucine, more preferably selected from, tyrosine and lysine.

5. A nanoparticle as claimed in claim 4 wherein the co-polymer is Glu/Tyr or Glu/Lys.

6. A nanoparticle as claimed in claim 1, which is a polymer of M1 and M2.

7. A nanoparticle as claimed in claim 1, wherein the carboxyl group is derivatised with a pendant group which is lipid.

8. A nanoparticle as claimed in claim 1, wherein each of the monomers M1 and M2 is of formula (I) or (II), ##STR00005## wherein in M1, Z is OH, wherein in M2, Z is Q.sup.A; wherein Q.sup.A is a pendant group selected from: a lipid; a group comprising an aromatic ring; a positively-charged group; an aliphatic group and wherein M3 is of formula (III); ##STR00006## wherein q and r are 0 or 1; wherein X.sup.A, X.sup.B, X.sup.C, X.sup.D, X.sup.E and X.sup.F are selected from H or Q.sup.B; wherein at least one of X.sup.A, X.sup.B, X.sup.C, X.sup.D, X.sup.E and X.sup.F is Q.sup.B; wherein Q.sup.B is a side chain group selected from: a positively charged group; a polar uncharged group; a hydrophobic group.

9. A nanoparticle as claimed in claim 8 wherein q and r are 0 and/or only one of X.sup.A, X.sup.B, X.sup.C, X.sup.D, X.sup.E and X.sup.F is Q.sup.B.

10. A nanoparticle is claimed in claim 8 wherein Q.sup.A is a pendant group which is a lipid and/or Q.sup.B is the sidechain of a naturally occurring amino acid.

11. A nanoparticle as claimed in claim 1 having a mass selected from the list consisting of: 5-500 kDa; 10-200 kDa, 20-150 kDa or 20-100 kDa; 20-50 kDa and 50-100 kDa.

12. A nanoparticle as claimed in claim 1, wherein the sensitizer is selected from the list consisting of: porphyrins, chlorins, phthallocyanines and cyanine dyes.

13. A process for preparing a nanoparticle as claimed in claim 1, which process comprises: (a) providing the polymer or co-polymer; (b) optionally derivatising the free carboxyl group of the glutamic acid with the pendant group; (c) preparing nanoparticles by sonication of the polymer or co-polymer in a solvent in the presence of the sensitizer; (d) optionally lyophylising the nanoparticles.

14. A method of treating a solid tumour, the method comprising administering to a subject a nanoparticle encapsulating a sensitizer according to claim 1 such as to deliver said sensitizer to the tumour, and exposing said sensitizer to either NIR or ultrasound to cause ablation of said tumor.

15. A method as claimed in claim 14, wherein the nanoparticle is administered by intravenous injection or infusion into the tumour or vicinity of the tumour and wherein ultrasound is locally applied to the vicinity of the tumour.

16. A method as claimed in claim 14, wherein the solid tumor is selected from the group consisting of adenocarcinomas, carcinomas, hemangiomas, liposarcomas, lymphomas, melanomas, and sarcomas.

17. A method as claimed in claim 14 which is a tumour theranostic method wherein the method of treating is performed in combination with a method of imaging the solid tumour in a subject, wherein the sensitizer fluoresces upon irradiation with NIR, and wherein in the presence of the tumour the nanoparticle is digested by a tumour associated protease, thereby enhancing the fluorescence on application of MR.

18. A method as claimed in claim 14 wherein the imaging is for the accurate intraoperative delineation of tumour or the detection of residual undetected disease, post-surgical resection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing (s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1: A schematic illustration of the mechanism and effect of SDT.

(3) FIG. 2: Examples of potential interactions between the PGA-based co-polymers and sensitizing agents

(4) FIG. 3: SDS-PAGE for PGATyr digested by cathepsin B at 370 C over 3 days. Digestion by cathepsin B at acidic pH is characteristic of the tumour environment.

(5) FIG. 4: Time-dependent evolution of the fluorescence spectra of free ICG (ICG), ICG-containing nanoparticles (ICGNP) and nanoparticles in the presence of cathepsin B (d.ICGNP), incubated at 37° C. in the time course of 2 days. The red arrow indicates the decrease or increase of ICG fluorescence after 24 h incubation.

(6) FIG. 5: Average signal obtained from NIR imaging of 3D matrices containing BxPC3 pancreatic cancer cell spheroids. Systems were incubated with free ICG (ICG), ICG-containing nanoparticles (ICGNP), ICGNP in the presence of cathepsin B and E-64, which is an inhibitor of cathepsin B, (ICGNP+CathB+E64) and ICGNP in the presence of cathepsin B (ICGNP+CathB). (n=3)

(7) FIG. 6: a. Illustration of nanoparticle digestion in the tumour extracellular environment, b. Mean radius of the undigested (Day 0) and cathepsin B-digested hematoporphyrin-carrying PGATyr-nanoparticles, c. Cellular uptake of hematoporphyrin (HP) in systems incubated with free HP (HP), HP-containing PGATyr nanoparticles (HPNPs), HP-containing PGATyr nanoparticles with cathepsin-B (HPNPs+CB) and HP-containing PGATyr nanoparticles with cathepsin-B inhibitor (HPNPs+E64) (n=3).

(8) FIG. 7: Cell viability of LNCaP cells treated in the absence (No HPNPs) and the presence of nanoparticles (HPNPs), at 10 μg/mL HP concentration, without (No US) and with ultrasound exposure (30 sec) at different parameters and pH 7.4 (a.) and at 5 μg/mL HP, 3 W/cm.sup.2, 50% DC at pH 6.4 (b.) (*p<0.05, ***p<0.001, n=4).

(9) FIG. 8: Left hand side: Plot of % change in subcutaneous tumour volume for SCID mice treated with (i) no treatment (black) (ii) ultrasound only (blue) (iii) PGATyr-based nanoparticles carrying hematoporphyrin (green) and (iv) PGATyr-based nanoparticles carrying hematoporphyrin with ultrasound (red). Animals were administered with the formulation on Day 0 and tumours were exposed to ultrasound (3 min) on Days 1 and 2. Right hand side: The corresponding body weight of animals. Error bars represent ± the standard error where n=4.

DETAILED DESCRIPTION OF THE INVENTION

(10) As explained above, PGA based polymers have been used previously in nanoparticles, although not in the context of the present invention. For example Zhu et al. (2014) describe nanoparticulate formulations that is based on polyglutamate-based a co-polymers that carry proteins, as distinct from sensitizing agents (Zhu Y, Akagi T, Akashi M. Self-assembling stereocomplex nanoparticles by enantiomeric poly(γ-glutamic acid)-poly(lactide) graft copolymers as a protein delivery carrier. Macromol Biosci. 2014 Apr. 14(4):576-87).

(11) Furthermore PGA salts, and derivatives and co-polymers thereof, can be obtained commercially in various molecular weights (for example from Sigma-Aldrich/Merck as “POLY-L-GLUTAMIC ACID”, or “Poly-L-γ-glutamic acid sodium salt”), or prepared as described in the Examples herein. Monomer Glu residues (or derivatives thereof) within PGA can be polymerised via the gamma- or alpha-carboxyl groups.

(12) Typically, to prepare the nanoparticles of the invention, PGA polymer or co-polymer is provided. This can be derivatised (optionally after blocking the terminal amine group) using methods known in the art. Thus in addition to the synthetic methodologies discussed herein, alternative reactions and strategies useful for the preparation of the derivatised polymers and co-polymers disclosed herein are known in the art. See, for example, March, “Advanced Organic Chemistry,” 5.sup.th Edition, 2001, Wiley-Interscience Publication, New York).

(13) Nanoparticles comprising the PGA polymer of co-polymer (optionally derivatised) and encompassing sensitizer can also be prepared using conventional methods for forming nanoparticles. For example by use of poly-vinyl alcohol (PVA) and sonication, followed by dialysis using a suitable molecular weight cut off dialysis material, and ultrafiltration (again using a suitable molecular weight cut off). Other methods of preparing nanoparticles are known in the art—see e.g. Nagavarma, B. V. N., et al. “Different techniques for preparation of polymeric nanoparticles—a review.” Asian J. Pharm. Clin. Res 5.3 (2012): 16-23.

(14) Preferred polymers are in the mass range 5-500 kDa e.g. 10-200 kDa, e.g. 20-150 kDa, e.g. 20-100 kDa, e.g. 50-100 KDa or 20-50 kDa. Preferred polymers include will include 30 to 6000 monomer units.

(15) Thus in one aspect there is provided a process for preparing a nanoparticle of the invention, which process comprises:

(16) (a) providing the glutamate containing polymer or copolymer;

(17) (b) optionally derivatising the free carboxyl group of the glutamate with the pendant group;

(18) (c) preparing nanoparticles by sonication of the polymer or copolymer in a solvent in the presence of the sensitizer;

(19) Optionally the sensitizer containing nanoparticles are freeze dried for storage before use.

(20) In one aspect the invention provides such a process substantially as described herein, with reference to the Description and Figures.

Sensitizers

(21) The sensitizer or sensitizing agent employed in the present invention will be appropriate to the therapeutic or diagnostic modality.

(22) For example for SDT the sensitizing agent will generate cytotoxic free radicals when exposed to ultrasound.

(23) For NIR (including other photothermal) the sensitizing agent will absorb in the NIR region of the electromagnetic spectrum leading to highly localised increased temperature (hyperthermic effect) and the production of free radicals.

(24) Suitable sensitizers for both these interventions are known to those skilled in the art.

(25) For SDT the sensitizer is used for the production of cytotoxic free radicals and the subsequent ablation of tumours.

(26) Examples include porphyrins, which are macrocyclic compounds with bridges of one carbon atom joining pyrroles to form a characteristic tetrapyrrole ring structure. There are many different classes of porphyrin derivatives (such as hematoporphyrin, photofrin, tetraphenylporphyrins, etc.). Other example SDT compounds include chlorins (such as HPPH, foscan, verteporfin, chlorin(e6), etc.), phthallocyanines (such as silicon phthalocyanine (PC4), RLP068, etc.)

(27) A preferred sensitizer for the present invention is a hematoporphyrin.

(28) Near-infrared (NIR) fluorescence imaging agents typically have high extinction coefficients, large Stokes' shifts, and the ability to generate strong fluorescence emission at the range of 700 to 1000 nm (see e.g. Shi, Changhong, Jason B. Wu, and Dongfeng Pan. “Review on near-infrared heptamethine cyanine dyes as theranostic agents for tumour imaging, targeting, and photodynamic therapy.” Journal of Biomedical Optics 21.5 (2016): 050901). Examples include cynanine dyes and other commercially available NIRF dyes e.g. Cy5.5, IRDye800-CW, Indocyanine green (ICG). Examples of heterocyclic polymethine cyanines with dual imaging and targeting properties include IR-780, IR-783, IR-808, and MHI-148.

(29) A preferred sensitizer for the present invention is a ICG.

(30) Sensitizing agents that operate in the NIR region of the electromagnetic spectrum have particular utility for the confined ablation of tumours based on both the hyperthermic effect at the microscopic level and the production of free radicals

(31) The loading of number of sensitizers in the nanoparticle can be varied in view of a range of factors, including the identity of the sensitizer, the tumour to be treated, and the overall design of the therapeutic regimen. However, as explained above, a higher loading is generally advantageous.

(32) In some of these embodiments, the loading of the nanoparticle is about 20% to about 100% by weight, more preferably at least 70%, 80%, 90% or 95% by weight. Loading may be assessed using standard methods in the art e.g. spectrophotometry (absorbance and fluorescence), based on a standard curve for each sensitizer

Therapeutic and Diagnostic Utilities

(33) Extensive preclinical experimentation has demonstrated the advantages of SDT and NIR/photothermal therapy are targeted, minimally-invasive treatment modalities, particularly over photodynamic therapy and systemic chemotherapy. They do not require the administration of highly toxic agents; thus, accumulation of these agents in off-target organs, up to reasonable levels, is not an issue. Therefore, they are not associated with adverse side effects and they can be applied repeatedly. Further highlighting their benefits, no cancer cell population has shown resistance to therapy-triggered ROS production with simultaneous inhibition of ROS “neutralization”, thus far. This is particularly important, considering the issue of multidrug resistance development using even the most advanced anticancer agents. It is also of high importance because it has a vast potential to address the issue of tumour heterogeneity that has rendered current treatments for advanced tumours inadequate. Moreover, one of the major advantages of non-hyperthermic ablation therapies, including SDT, is their immunogenicity, i.e. they leave tumour-associated antigens intact, subsequently inducing anti-tumour immune response against the primary or locally recurrent and disseminated disease.

(34) Also provided by the present invention are methods of treatment, diagnosis, and theragnosis utilising the nanoparticles of the invention. In particular, there are methods provided of treating patients by NIR/photothermal therapy, or SDT for the ablation of a tumour in patient using said nanoparticles.

(35) Typically, the nanoparticle is used in an effective amount. An “effective amount”, as used herein, is an amount being fit for the purpose intended. Thus for diagnostics this will be sufficient to generate a signal. For therapy and theranostics this will be sufficient to show benefit to the individual, optionally in combination with other therapies as described herein. The actual amount administered, and rate and time-course of administration, will depend on the subject and the intervention in hand. Decisions on dosage etc. are within the responsibility of general practitioners and other medical doctors, and will typically take account of the purpose of the imaging and\or disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.

(36) Thus the invention provides a method of treating a solid tumour comprising administering to a subject a nanoparticle encapsulating a sensitizer as described herein to deliver said sensitizer to the tumour, and exposing said sensitizer to either NIR or ultrasound to cause ablation of said tumour.

(37) The term “treatment,” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress (prolonged survival), a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition.

(38) In the present invention treatment may be e.g. for the purpose of slowing tumour growth, halting tumour growth, or decreasing tumour size.

(39) Typically, the nanoparticle is administered by intravenous injection or infusion, systemically or, optionally, into the tumour or vicinity of the tumour, and the NIR or ultrasound is locally applied to the vicinity of the tumour. In the case of ultrasound, an appropriate “acoustic window” through which ultrasound can be transmitted is used to irradiate the target tumour. These treatments can also be applied intraoperatively (during surgical procedure), to eliminate residual disease after surgical resection or to irradiate the tumour bed and eliminate undetected residual microscopic disease post-surgical resection. Clinical-grade lasers for NIR treatment are commercially available e.g. from BIOMED 25 (Biomed Ltd, UK).

(40) Commercially available ultrasound systems for clinical SDT include those available from Intelect Transport Ultrasound by Chattanooga Medical Supply Inc., USA. Typically commercial systems capable of producing ultrasound at 0.5-5 MHz frequency and 0.5-10 W/cm.sup.2 power density range are suitable for use in the present invention.

(41) The treatment may be used in combination with chemotherapy and/or radiotherapy

(42) As explained above, for SDT the sensitizer is used for the production of cytotoxic free radicals. When the system is used with SDT, the site-specific application, minimally invasive, of low-intensity ultrasound may be used to further induce agent dispersion through dense and poorly-diffused tumours (Nomikou et al (2010) “Ultrasound-enhanced drug dispersion through solid tumours and its possible role in aiding ultrasound-targeted cancer chemotherapy.” Cancer Lett 288(1): 94-98).

(43) Thus, the nanoparticles described herein may thus be used to produce reactive oxygen species to selected cells (such as tumour cells) in vivo.

(44) Sensitizing agents that absorb in the NIR region are used to provide a localised hyperthermic effect and for the production of free radicals upon irradiation with NIR light.

(45) Thus, the nanoparticles described herein may thus be used to produce a localised hyperthermic effect to selected cells (such as tumour cells) in vivo.

(46) The nanoparticles described herein may be used as adjuvant material in relation to an effective anti-tumour immune response, since they are not immunosuppressive, can preserve tumour antigens, and may have antitumour immunoadjuvant properties(Seth A, et al. Infection-mimicking poly(γ-glutamic acid) as adjuvant material for effective anti-tumor immune response. Int J Biol Macromol. 2015 April; 75:495-504).

(47) The tumour may be a solid tumour, by which is meant an abnormal mass of tissue that results from the uncontrolled proliferation of cells. Typically, solid tumours do not contain cysts or liquid areas within the tissue mass. Solid tumours can arise in any part of the body, and may be benign (not cancerous) or malignant (cancerous). Most types of cancer other than leukemias can form solid tumours. Solid tumours include, for example, adenocarcinomas, carcinomas, hemangiomas, liposarcomas, lymphomas, melanomas and sarcomas.

(48) As explained herein the tumour microenvironment has certain characteristics such as a “leaky” vasculature and poor lymphatic drainage, high collagen concentration and Interstitial Fluid Pressure, increased concentration of proteolytic enzymes such as cathepsin B, acidic pH, and hypoxia.

(49) The tumour microenvironment also provides an enhanced permeability and retention (EPR) effect, and the overexpression of particular cell membrane receptors. Among these, the EPR effect involves atypical and leaky vasculature, as well as poor lymphatic drainage that promote nanoparticle accumulation and retention in tumours (*Nomikou N, Curtis K, McEwan C, Callan B, Callan J F, McHale A P. A versatile, stimulus-responsive nanoparticle-based platform for use in both sonodynamic and photodynamic cancer therapy. Acta Biomaterialia 2017; 49: 414-421)

(50) Optionally the nanoparticles may have a mean average diameter larger than the average diameter of the vasculature of the normal tissue and smaller than the diameter of the tumour vasculature.

(51) The size of nanoparticles of the invention decreases in the presence of cathepsin B thereby facilitating uptake of the nanoparticles and sensitizer, as well as adequate diffusion throughout a dense tumour mass. Thus cellular uptake of the nanoparticles is proportional to the intracellular and secreted levels of cathepsin B.

(52) Prostate cancer is the second most common cancer and the sixth leading cause of cancer death among men worldwide, with an estimated recorded amount of 1.1 million cases and 377,000 deaths in 2017, which accounts for 15% of all new cases of cancer in men

(53) Its incidence is set to increase with advancing age demographics in developing countries. Once the cancer has spread beyond the prostate, it becomes relatively unmanageable. The only available strategy to reduce progression-related toxicity and premature death is early detection combined with well-tolerated interventions. Current treatments leave much to be desired. In the recent quality of life report from the PROTECT study, half the men exhibited urinary incontinence at one year and two thirds reported significant sexual function impairment following surgical intervention. Focal therapy has resulted in a much better toxicity profile than standard-of-care treatments, but the interventions have proved resistant to widespread diffusion as they are difficult to perform.

(54) The nanoparticles of the invention are degraded by prostate-specific membrane antigen (PSMA) which has glutamate carboxypeptidase activity (see Chang, Sam S. “Overview of prostate-specific membrane antigen.” Reviews in urology 6.Suppl 10 (2004): S13).

(55) The nanoparticles can offer improved sensitizer (e.g. hematoporphyrin) diffusion and distribution throughout the dense and poorly-vascularised mass of prostate tumours, with a subsequent potential to improve the efficacy of SDT. They can optionally be used in conjunction with transrectal ultrasound.

(56) As explained above in the case of formulations containing an NIR-absorbing sensitizer (such as ICG or other dye) the fluorescence of the sensitizer is quenched while it is in the form of intact nanoparticles. However, as shown in the Examples below NIR fluorescence is recovered upon digestion with cathepsin-B.

(57) This recovery of fluorescence under the conditions mimicking those of the tumour microenvironment has significant potential in NIR imaging for the accurate intraoperative delineation of tumours or the detection of residual undetected microscopic disease, post-surgical resection.

(58) Thus there is also provided a method of imaging a solid tumour in a subject, the method comprising administering to the subject a nanoparticle encapsulating a sensitizer according to the invention wherein the sensitizer fluoresces in the presence of NIR, wherein in the presence of the tumour the nanoparticle is hydrolysed by a tumour associated protease thereby enhancing the fluorescence on application of NIR.

(59) As explained above, the protease may (for example) be cathepsin-B or prostate-specific membrane antigen.

Kits

(60) In another aspect the present invention provides kits comprising a nanoparticle as described herein, optionally with instructions for use for performing a method of treatment, diagnosis, or a theranostic method as described herein. Optionally the kit may also include a source of NIR or ultrasound.

(61) Wherever a composition (e.g. of nanoparticles) is described herein, it will be appreciated that the same composition for use in the therapeutic methods (including theranostic and diagnostic methods) described herein is also envisaged, as is the composition for use in the manufacture of a medicament for treating or imaging the relevant disease.

(62) Likewise where a method utilising nanoparticles is described, it will be appreciated that use of a nanoparticle encapsulating a sensitizer in that method, a nanoparticle encapsulating a sensitizer for use in that method, and use of a nanoparticle encapsulating a sensitizer in the preparation of medicament for that method is also envisaged.

(63) Thus the invention further provides: Use of a nanoparticle encapsulating a sensitizer of the invention for NIR or ultrasound therapy, imaging, or theranostics; A nanoparticle encapsulating a sensitizer of the invention for use in NIR or ultrasound therapy, imaging, or theranostics; Use of a nanoparticle encapsulating a sensitizer of the invention in the preparation of medicament for NIR or ultrasound therapy, imaging, or theranostics.

(64) A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

(65) Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

(66) It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

(67) Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

(68) Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

(69) The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

(70) The disclosure of all references cited herein, inasmuch as it may be used by those skilled in the art to carry out the invention, is hereby specifically incorporated herein by cross-reference.

EXAMPLES

Example 1—Derivatisation with Lipid 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, for ICG-carrying Nanoparticles with Applications in Near-infrared Imaging

Method

(71) Blockage of amine terminal groups of PGA: 10 mg PGA salt (MW: 50-100 kDa) were dissolved in 2 mL of 0.1M sodium phosphate buffer (pH 7.3). 2.5 mg of Sulfo-NHS acetate were dissolved in 200 uL 0.1M sodium phosphate buffer and this was added to the PGA solution, under constant stirring. The reaction was allowed to run under stirring at room temperature for 1 hr. The solution was dialysed for 24 hrs against de-ionised water and then freeze-dried for 24 hrs.

(72) Carboxyl group substitution (1:50) with lipid molecule: 3 mg sulfo-NHS and 4 mg EDC were dissolved in 5 mL PGA 2 mg/mL solution in water. The resulting solution was heated at 80° C. in a sonicating bath and a 5 mL 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine 140 μg/mL solution in water (also at 80° C.) was added drop-wise. The reaction was allowed to take place at 70° C. for 3 hrs. The product was dialysed against water for 12 hrs and the derivative was freeze-dried for 24 hrs.

(73) Nanoparticle fabrication: 10 mg of the lipid-derivatised PGA sodium salt (MW: 50-100 kDa) and 5 mg of ICG were dissolved in 10 mL DMSO. This solution was then added drop-wise to a 5 mL poly-vinyl alcohol (PVA, MW: 85-124 kDa) solution under constant bath sonication. The resultant mixture was dialysed (MWCO: 8 kDa) against deionised water for 12 h (overnight). The mixture was then probe-sonicated for 3 mins and subjected to 4 rounds of ultrafiltration (MWCO: 100 kDa, 1 round=1000 RPM for 15 mins). The suspension retained by the filter was ultracentrifuged at 38,000 g for 90 min at 12° C. The precipitated pellet was suspended in 3 ml deionised water and probe-sonicated for 3 min to break down any aggregates. Second dialysis (MWCO: 8 kDa) was performed to remove any free ICG remaining, for 12 H. The solution was filter-sterilised using a micro-syringe filter and was snap-frozen followed by freeze-drying for 24 H. Lastly, the dry sample was suspended in 3 mL PBS and stored at 4° C., protected from light.

(74) Result on loading efficiency: The derivatised formulation resulted in 34% loading efficiency for ICG, 4.25-fold higher than that achieved with original PGA (8% loading efficiency).

Example 2—Performance of the ICG-Carrying Nanoparticles Formed Using the Lipid-Derivatised PGA

Method

(75) The ICG-carrying nanoparticles were prepared as described in example 1, using PGA that was derivatised with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, by 1:50 substitution of PGA side-chain carboxyl groups (as described in Example 1)

Performance of Nanoparticles

Optical Properties of the ICG-carrying Nanoparticles Formed with the Lipid-derivatised PGA

(76) For time-depended fluorescence emission characterization, phosphate-buffered saline (PBS) solutions/suspensions containing free ICG, or ICG nanoparticles in the absence (ICGNP) or the presence (d.ICGNP) of cathepsin B (0.6 units/mL) were stored in an orbital incubator at 37° C., protected from light, for 2 days. The equivalent ICG concentration in all systems was 6 μg/mL. Fluorescence emission spectra of these systems were acquired over the course of 48 h. The excitation wavelength of 740 nm was used to obtain the fluorescence spectra. For the afore-mentioned experiments, spectra and fluorescence intensities were obtained using a CLARIOstar® High Performance Monochromator Multimode microplate reader (BMG LABTECH, UK).

(77) The results shown in FIG. 4 demonstrate that incorporation of ICG in the nanoparticulate formulation results in significant suppression of fluorescence emission, which is recovered by 53% in 24 h and by 69% in 48 h, only in the presence of cathepsin B (FIG. 4). Fluorescence emission of free ICG was suppressed after 24 h.

(78) The cathepsin B-induced increase in fluorescence emission observed at 24 h post-incubation of the nanoparticles demonstrate the utility of this formulation from a diagnostic perspective for the selective detection and imaging/delineation of highly aggressive tumours with elevated proteolytic enzyme activity.

Imaging

(79) BxPC-3 (human pancreatic) cells were seeded with Corning Matrigel® Basement Membrane Matrix (VWR, UK) to a final concentration of 50×10.sup.3 cells/well. After 24 h, the serum-containing growth medium was removed replaced with fresh serum-containing four different ICG systems: free ICG, ICG-containing nanoparticles (ICGNP), ICGNP with cathepsin B inhibitor (ICGNP+E-64) and ICGNP with cathepsin B (ICGNP+CB). The final concentrations in all systems was 15 μM in ICG, 0.6 units/mL in cathepsin B and 50 μM in E-64. Cells were incubated with these systems, protected from light, at 37° C. in a 5% CO2 humidified incubator for 24 hrs. The serum containing growth medium was then removed, and the cell matrices were washed twice with PBS. The plate was then incubated with PBS in the individual 3D culture-containing wells for 2 hrs. Before obtaining images with an NIR camera and starting the NIR-laserimaging and treatment, fresh serum-containing growth medium was added to individual cells.

(80) Imaging was carried out under NIR laser excitation at 0.8 mW/cm.sup.2 using a laser diode LDC 240 C (ThorLabs, UK), the systems are imaged using a NIR CCD camera C10600 ORCA-R2 (Hamamatsu, Japan) with a combination of filters: a 700 nm longpass filter, a 825 nm longpass filter and a 835 nm bandpass filter (Thorlabs, UK).

(81) The results presented in FIG. 5 demonstrated the utility of the polyglutamate-based formulation in NIR imaging by verifying its efficacy to produce strong detectable signal after 24 h in cathepsin B-rich systems using pancreatic cancer cell-containing 3D matrices (FIG. 5).

(82) These aptitudes demonstrate the potential of the formulation in intraoperative NIR treatment of residual microscopic disease or unresectable tumour segments.

Example 3—Co-Polymer of Poly Glutamic Acid with Tyrosine, for Hematoporphyrin (HP)-Carrying Nanoparticles with Applications in Sonodynamic Therapy

Method

(83) 10 mg of either poly-I-glutamic acid sodium salt (PGA) (10 mg) (MW: 50-100 kDa) or poly-L-glutamic acid-tyrosine co-polymer 4:1 (PGATyr) (MW: 20-50 kDa) was mixed with 5 mg HP and dissolved in 10 mL DMSO. The resulting mixture was added drop-wise to a 5 mL polyvinyl alcohol solution (PVA, MW: 124 kDa) 0.5 mg/mL and the mixture was left under constant stirring for 1 hr. The mixture was then dialysed in membrane tubing (MWCO: 8 kDa) against water for 24 hr. Subsequently, the suspension was ultracentrifuged for 90 min at 38,000 g, at 12° C. The precipitated pellet was suspended in 3 mL deionised water and probe-sonicated for 3 min to break down any aggregates. Second dialysis was performed to remove any free HP remaining, for 12 hr. The solution was filter-sterilised using a 0.2 μm-pore filter and was snap-frozen followed by freeze-drying for 24 hrs. The dry sample was suspended in 3 mL PBS and stored at 4° C., protected from light.

(84) For determining loading efficiency of the nanoparticles with hematoporphyrin (HP), a standard curve of the absorbance against HP concentration was used.

(85) For cellular uptake studies, prostate cancer cells were seeded in wells of 96-well plates at a concentration of 2×10.sup.4 cells/well and incubated for 24 hours. Cells were then treated with the following systems: Nanoparticles at 10 μg/mL based on HP, nanoparticles (10 μg/mL) with cathepsin B (10 units/mL), nanoparticles with E64, a cathepsin B inhibitor (1 mg/mL) and Free HP (10 μg/mL) at pH 7.4 in a hypoxic chamber (37° C., 1% Oxygen) and incubated for 48 hours. The formulation-containing medium was then removed, cell monolayers were washed with saline and fluorescence (excitation wavelength: 534 nm, emission wavelength: 626 nm) emission of the wells was measured using a microplate reader.

(86) For the SDT efficacy studies, LNCaP cells were seeded in wells of 96-well plates (cell density: 10.sup.4 cells/well) for 24 H. The systems were then incubated at either pH 7.4 or pH 6.4, in the presence or the absence of nanoparticles at 10 μg/mL or 5 μg/mL final HP concentration, respectively, in hypoxic environment. Cells were exposed to varying ultrasound conditions, as indicated in FIG. 7.

(87) The in vivo studies were carried out in SCID mice bearing subcutaneous LNCaP tumours. The study was initiated when tumours reached 65-100 mm.sup.3 volume. Mice were be treated with the formulation by tail-vein injection at nanoparticle concentration 6 mg/Kg based on HP. Ultrasound irradiation of the tumours, using a Sonidel SP100 sonoporator, was performed after 24 h (time-point of accumulation and confinement within the tumour mass, based on previous studies) and using ultrasound conditions optimised for in vivo SDT (3.5 W/cm.sup.2, 30% Duty Cycle, for 3.5 min). Tumour volume was recorded using a Peira TM600 tumour-measuring device. The weight of mice was also recorded.

(88) Result on loading efficiency: The preparation formed with the co-polymer of PGA and tyrosine resulted in 18% loading efficiency for HP, 3-fold higher than that achieved with original PGA (<3% loading efficiency), using this particular protocol.

Performance of Nanoparticles

(89) Digestion of the formulation by cathepsin-B, at tumour-mimicking conditions (acidic pH), leads to decreased nanoparticle size and subsequent increased cellular uptake (FIGS. 6b and 6c, respectively).

(90) Sonodynamic treatment, at both normoxic and hypoxic conditions, demonstrated ultrasound-induced cytotoxic effects only for the nanoparticle-treated prostate cancer cells, while toxicity of the formulation in the absence of ultrasound was minimal. We have also demonstrated significant cytotoxic effects of SDT under hypoxic conditions (FIG. 7), in which ROS production is expected to be limited.

(91) Our in vivo studies in immunodeficient mice, using the hematoporphyrin-containing PGATyr nanoparticles for SDT, showed a 60% decrease in LnCAP tumour volumes within 24 h, following IV administration of a single dose (FIG. 8, left hand side). No adverse effects were recorded and body weight was stable (FIG. 8, right hand side).