WO2018002170A1 - Polymeric micelle –phthalocyanine nano-systems for photodynamic therapy and/or fluorescence-based imaging - Google Patents

Abstract

Polymeric micelle - phthalocyanine nano-systems are useful as therapeutic, diagnostic, and theranostic (therapeutic and diagnostic) nano-systems for photodynamic therapy and/or fluorescence-based imaging. In particular, there is provided a composition comprising polymeric micelles as a nanocarrier and one or more molecules of a phthalocyanine as a payload, wherein the phthalocyanine is of the formula (I) in which R₁-R₈ and R_a and R_b are as defined. The invention also provides certain groups of phthalocyanines of general formula (I) that are also novel.

Description

POLYMERIC MICELLE -PHTHALOCYANINE NANO-SYSTEMS FOR PHOTODYNAMIC THERAPY AND/OR FLUORESCENCE-BASED IMAGING

FIELD OF THE INVENTION

The present invention relates to polymeric micelle - phthaiocyanine nano-systems and their use as therapeutic, diagnostic, and theranostic (therapeutic and diagnostic) nano- systems for photodynamic therapy and/or fluorescence-based imaging. BACKGROUND OF THE INVENTION

Photodynamic therapy (PDT) is an emerging combination therapy, which in principle requires three interacting elements: 1) a iight-activatable compound, the so-called photosensitizer; 2) light of appropriate wavelengths; and 3) tissue oxygen. Upon exposure of a photosensitizer to specific wavelengths of light, it becomes activated from a ground state to an excited state. As the photosensitizer returns to the ground state, it releases energy, which is transferred to the surrounding tissue oxygen to generate reactive oxygen species (ROS), such as singlet oxygen ( Ό2 ) and free radicals. These ROS mediate cellular toxicity of targeted cells, inducing apoptosis (i.e. non-inflammatory programmed cell death). PDT has been investigated extensively in the laboratory for decades, and for over 30 years in the clinical environment. The approach is non-invasive or minimally invasive, enables accurate targeting, and repeated administration without total-dose limitations. The current clinical applications of PDT include the treatment of acne, non-melanoma skin cancer, head and neck cancer, Barrett's esophagus, and wet macular degeneration. Recently, endovascuiar PDT has emerged as a very promising therapeutic modality for the therapy of atherosclerotic cardiovascular diseases and the restenosis after injury and in short-term studies has shown efficacy in limiting

atherosclerotic plaque inflammation in animal models. Additionally, endovascuiar PDT has proven safe and well tolerated in phase-I clinical trials for atherosclerotic heart disease patients and for patients with atherosclerotic peripheral artery disease.

Photodynamic therapy simultaneously reduces plaque inflammation and promotes repopulation of plaques with a SMC-rich (smooth muscle ceil - rich) stable plaque cell phenotype, while reducing disease progression. These early healing responses suggest that endovascular PDT is a very promising therapeutic modality for the therapy of atherosclerotic cardiovascular diseases, acute coronary syndromes, strokes, and peripheral artery disease. Unfortunately, clinical trials on endovascular PDT for the therapy of atherosclerotic cardiovascular diseases in patients are limited. One of the main problems in photodynamic therapy, limiting the use of many potent photosensitizers, is the difficulty in preparing pharmaceutical formulations that enable their parenteral (iv) administration. Due to their low water solubility, very potent hydrophobic

photosensitizers cannot be injected intravenously.

In vivo fluorescence imaging is an emerging combination diagnostic modality, which in principle requires two interacting elements: 1) a light-activatabie compound, the so-called fluorophore; and 2) light of appropriate wavelengths. Upon exposure of a fluorophore to specific wavelengths of light, it becomes activated from a ground state to an excited singlet state, and as it returns to the ground state it emits fluorescence light, usually at longer wavelengths. This emission can be visualized by appropriate sensors, enabling fluorescence imaging. In vivo fluorescence imaging has accelerated scientific discovery and development in the life sciences as it enables labeling of specific biochemical components and the visualization of biological processes. Endovascular fluorescence- based imaging has recently emerged as a very promising diagnostic modality of atherosclerotic cardiovascular diseases, and has focused on imaging components associated with atherosclerotic plaque inflammation, by using either endovascular fluorescence imaging alone or in combination with: (a) intravascular ultrasound imaging (IVUS); and/or (b) optical coherence tomography imaging (OCT); and/or (c)

photoacoustics (optoacoustics) imaging (PA or OA); and/or (d) near-infrared

spectroscopy imaging ( RS). During endovascular fluorescence-based imaging, the diagnostic endovascular catheter illuminates the blood vessel wal l and col lects the subsequent fluorescence. Early implementations were performed in ID scans of the blood vessels along their length. 2D endovascular fluorescence-based imaging was later achieved by performing 360-degrees rotation in addition to the endovascular catheter pullback, and was recently successfully demonstrated in vivo in atherosclerotic rabbit animal models. The use of light in the Near-infrared (NIR) spectrum enables in vivo endovascular IR fluorescence-based imaging (endovascular NIRF -based imaging) for highest l ight tissue penetration in the NIR optical window and deep tissue imaging through blood, without considerable reduction in sensitivity, by using endovascular IRF imaging either alone or in combination with IV US and/or OCT and/or PA (OA ) and/or MRS.

Due to their low water solubility, very efficient hydrophobic fluorophores cannot be injected intravenously. Additionally, the targeted delivery of efficient fluorophores is a very chal lenging problem.

The use of nanotechnoiogy for medical purposes (nanomedicine) has grown

exponentially over the past few decades. Although the domain originally focused on anticancer therapy, recent advances have pointed out the tremendous potential of nanomedicine in the therapy and diagnosis of atherosclerot ic cardiovascular diseases. The use of nano-systems in the therapy, diagnosis, and therapy monitoring of atherosclerotic cardiovascular diseases has emerged as a very promising strategy for efficient drug delivery, achiev ing several advantages which include: (i) the improved del ivery of poorly water-soluble drugs; (ii) the targeted delivery of drugs by avoiding the reticuloendothelial system, and utilizing the enhanced permeability and retention effect (EPR effect), and active tissue-specific targeting; (iii) the transcytosis of drugs across epithelial c n d o t h c 1 i a ! barriers; (iv) the delivery of macromoleciile drugs to intracel lular sites of action; (v) the co-del ivery of two or more drugs or therapeutic modal ities for combined therapy; (vi) the visualization of sites of drug del ivery by combining therapeutic agents with imaging modal ities, and (vii) the real-time and fol low-up read of the in vivo efficacy of a therapeutic agent. In this respect, various nanoparticle types have already been successfully utilized in medicine as nanocarriers, including polymeric micelles.

Polymeric micelles are nanostructures formed by the spontaneous self-assembly of block copolymers in solvents that are selective for one of the blocks within the polymer. The self-organization of amphiphilic copolymers is driven by hydrophobic interactions, whereby the selective solvation of the hydrophilic block in aqueous solutions arranges the polymer molecules in the most efficient form with the lowest free energy. These mostly spherical nanostructures are called micelles. In recent years much interest has been given to the self-assembly of block copolymers in aqueous systems to develop new applications in the field of controlled delivery of hydrophobic drugs. Currently several micel le formulations are being tested in cl inical trials. More importantly, several micelle formulations are already approved for use in the clinic. One such an example is

Genexol-PM, a m e t hox y- po I y ( eth y 1 e n c glycol )-block-poly( D,L-lactide) (mPEG-PDLLA) micelle loaded with pacl itaxel which is currently used to treat breast cancer in South Korea. Genexol-PM has shown lower toxicity than Taxol, with its max imum tolerated dose identified as 2 to 3 times that of Taxol . The lower toxicity and higher efficacy of paclitaxel in combination with polymeric micelles shows the great potential of these advanced drug carriers.

Photosensitizers are generally classified as either porphyrinoid or non-porphyrinoid derivatives. Among non-porphyrinoid photosensitizers, neutral ly charged hypocrel l in, squaraine and BODIPY derivatives, or cation ic compounds such as c h a I c o ge n o p yry I i u m , phenothiazinium. and ben zo [ a] phenot h iazini u m dyes (which include methylene blue and toluidine blue) have been the predominant focus. However, due to the limited potency and various side effects associated with many of them, the development of non- porpliyrinoid photosensitizers for appl ication in medicine has lagged considerably behind that of porphyrmoid photosensitizers. Indeed, none have yet been approved for cl inical use, and some have now been abandoned.

The porphyrinoid derivatives are further classified as first, second and thi d generation photosensitizers. First-generation photosensitizers include hematoporphyrin derivative

(HpD) and porphimer sodium (Photofrin). A number of second-generation

photosensitizers, like phthalocyanines, have been developed to allev iate certain problems associated with first-generation photosensitizers, such as prolonged skin

photosensitization and suboptimal tissue penetration. These second-generation

photosensitizers are chemically pure, absorb light at longer wavelengths, and cause significantly less skin photosensitization post-treatment. In addition, second-generation photosensitizers must be at least as efficient as Photofrin, the current gold standard for PDT. Second-generation photosensitizers bound to nanocarricrs in order to become water soluble for parenteral (IV) administration, and for targeted accumulation within selective tissues are referred to as third-generation photosensitizers, and currently represent an active research area in the field.

Among porphyrinoid photosensitizers, porphimer sodium (Photofrin), palladium- bacteriopherophorbide (Tookad ), NPe6, motexafin lutetium (Antrin, Lutrin or Lu-Tex) and phthalocyanines hav e been clinically approv ed or are currently under non-cl inical or cl inical inv estigation.

Phthalocyanines constitute one of the most promising families of the second -generation porphyrinoid photosensitizers with intrinsic fluorescence. Phthalocyanines are a group of photosensitizers / fluorophores structurally related to porphyrins. They present a number of properties that make them ideal PDT / fluorescence compounds. Phthalocyanines are robust and very versatile molecules with a strong absorption at 670 - 770 nm (ε ~ I 0⁵ M^"1 cm^"1). They yield high singlet oxygen production and long-lived fluorescence. Studies using the silicon phthaloeyanine Pc 4 (λ = 675/690nm):

in both in vitro and in vivo studies and also in a successfully completed Phase-I clinical trial for the treatment of cutaneous neoplasms, are so far the most promising (Baron, E.D. et ai, Laser. Surg. Med. 2010, 42, 728-735).

General aspects of photosensitizers/fluorophores, and of phthalocyanines in particular. are collected in many monographs and scientific articles, such as:

(a) McKeown, N.B., Phthaloeyanine Materials, Cambridge University Press, Cambridge,

1998;

(b) Dolmans, D.G.J. ; Fukumura, D.; Jain, R. .; Nat. Rev. Cancer 2003, 3, 380-387; (c) Ormond, A.B., Freeman, U.S.: Materials 2013, 6, 817-840;

(d) Master, A., Livingston, M.; Sen Gupta, A.; J. Control. Release 2013, 168, 88-102;

(e) Menon, J.U., Jadeja, P., Tambe, P., Vu, K., Yuan, B., Nguyen, K.T., Theranostics 2013. 3, 152- 166;

(f) Kadish, K.M., Smith K.M., Guilard, R., Handbook of Porphyrin Science, World Scientific, Singapore. 2013;

amongst others. However, third-generation photosensitizers with intrinsic fluorescence (i.e. those second- generation photosensitizers with intrinsic fluorescence bound to nanocarriers) have never been used for the therapy and/or diagnosis and/or therapy monitoring and/or theranostics of cancer or atherosclerotic cardiovascular diseases.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provided a composition comprising polymeric micelles as a nanocarrier and one or more molecules of a phthalocyanine as a payload,

wherein the phthalocyanine is of the formula I:

in which

(a) M represents a metal or metalloid atom,

(b) Ri, R₂, R3, R i, R% Ri., R? and R are independently selected from the group consisting of hydrogen, halogen, (Ί i ?alkyl, -R9, -OR9, -SR9, or -NR9R10,

in which R9 and Rio independently represent hydrogen, C_1-12alkyl, or a phenyl group optionally substituted by one or more Rn groups independently selected from the group consisting of G i ?alkyl, halogen, -OR₁₂, SR i?, and -NR12R0,

in which R12 and R13 each independently represent hydrogen or G i ?alkyl, or one or more pairs of Ri and R2, R3 and R t, R5 and Re, and R? and Rs are attached to adjacent carbon atoms and together form, together with the ring to which they are attached, an aromatic fused ring system, and (c) Ra represents hydrogen or a group

wherein

A and B independently represent a single bond. -CH?- or -(C=0)-,

and

R i a, R-2a, Rsa, Rib, R?h and R ¾ independently represent hydrogen, C i-icalkyl. -C≡CH, -COOI 1, -NH₂, -CH2NH2, -SC≡N, CH₂SC≡ or -| (X H2CH2 ]„-OR, in which R represents hydrogen or i f.alkyl and n represents an integer of from 1 to 10.

The compositions of the invention are referred to generally herein as "nano-systems^"". The invention also provides a method for therapy and/or diagnosis and/or therapy monitoring and/or theranostics of a lesion of a tissue in a subject,

which method comprises the steps of

(a) identi fying a subject having or suspected of having the lesion;

(b) administering to the subject a composition according to the invention;

(c) causing or allowing polymeric micelles present in said composition to accumulate upon and/or within the lesion, if present; (d) exposing the tissue to electromagnetic radiation so as to cause the

phthalocyaninc molecules to produce one or more of fluorescence, phosphorescence, reactive oxygen species, heat, an optical signal and an acoustic signal .

For use in diagnosis and/or therapy monitoring and/or theranostics, the method may further comprise the step of detect ing fluorescence, phosphorescence, an opt ical signal and/or an acoustic signal produced by the polymeric micel les, such fluorescence, phosphorescence, optical signal and/or acoustic signal being indicative of the presence, site and/or condition of the lesion.

When used therapeutically, the polymeric micel les may produce reactiv e oxygen species and/or heat, in such a manner as to bring about the death of cells within the lesion and/or the passivation of the lesion and/or the stabilization of the lesion and/or the regression of the lesion and/or the therapy of the lesion.

In a related aspect, the invention provides a composition according to the first aspect of the invention, for use in therapy and/or diagnosis and/or therapy monitoring and/or theranostics of a lesion of a tissue.

The polymeric micel les preferably comprise block copolymers of po I y ( c a p ro I ac to n e ) and poly( ethylene glycol ). The block copolymers may hav e the general formula p( I -poly(r.-caprolactone)p-L-poly( ethylene glycol)_q-RpEG wherein

RPCL is the terminal group of the po I y ( r.-c a p ro I a c to n e ) block;

p is an integer of from 4 to 1 00;

L is a l inker, for example selected from the group consisting of ester, amide, carbonate or carbamate; q is an integer of from 1 0 to 250; and

RIM el is the terminal group of the poly( ethylene glycol ) block.

The phthalocyanine is preferably a silicon phthalocyanine, i .e. in formula

preferably represents Si.

In preferred embodiments, Rt, represents a group

in which one, two or all of Rib, R?b and Rab independently represent

-[OCH₂CH₂]_N-OR, in which R represents hydrogen or Chalky!, preferably methyl, and n represents an integer of from 1 to 10, more preferably from 1 to 4, and in particular 3.

The nano-systems of the invention may accumulate in atherosclerotic plaques and/or other lesions, without the need for conjugation with other targeting moieties, and so may be effective delivery vehicles for PDT agents (ie the phthalocyanine component of the composition) to those atherosclerotic plaques and/or other lesions. Thus, whilst in some embodiments the polymeric micelles may be conjugated or otherwise associated with tissue/ceii-targeting moieties such as those discussed herein, in other embodiments such t i ss u e/c e 11 - 1 a rge t i n g moieties arc unnecessary and the compositions do not contain any such t i ss u e/ce I 1 - 1 a rget i n g moieties.

The compositions of the invention can be administered by a variety of routes, including without limitation parenteral I y (e.g. intravenously). The compositions therefore enable the targeted deliv ery of the phthalocyanines, which are potent photosensitizers and fluorophores, to atherosclerotic plaques and/or other lesions. Without wishing to be bound by theory, it is bel ieved that when the polymeric micel les of the fi st aspect of the invention are dispersed in an aqueous medium, the phthalocyanine moieties are solubi l ised within the hydrophobic interior of the polymeric micelles and/or, particularly where the phthalocyanine molecules are amphiphilic in nature, within the hydrophil ic shell. The polymeric micel le thus acts as a carrier for the phthalocyanine pay load.

Certain phthalocyanines of formula (I) are believed to be novel, and represent a further aspect of the invention, which thus provides a phthalocyanine of the formula la:

wherein

(a) M represents a metal or metal loid atom,

(b) R i , R₂, R,3, R t, R.% R.. R? and Rs are independently selected from the group consisting of hydrogen, halogen, -R.>, -OR9, -SR9, or - R.jR i u,

in which R9 and Rio independently represent hydrogen, G i?alkyl, or a phenyl group optional ly substituted by one or more Rn groups independently selected from the group consisting o G ^alky!, halogen, -OR 12, SR 12, and -NRi₂Ri3,

in which Ri₂ and R13 each independently represent hydrogen or C i - i ^alkyl, or one or more pairs of R i and R₂, R3 and R i, R5 and R<„ and R? and Rs are attached to adjacent carbon atoms and together form, together w ith the ring to which they are attached, an aromatic fused ring system, and

(c) Ra represents hydrogen or a group

wherein

A and B independently represent a single bond, -CH2- or -(C=0)-,

and

Ria, R-2a, R½, Rib, R-2b and R₃b independently represent hydrogen, Ci-icalkyl. -C≡CH, -COOH, -NH2, -CH2NH2, -SC≡N, CH2SC≡ or -[OCH₂CH2]_n-OR, in which R represents hydrogen or Ci /.alkyl and n represents an integer of from 1 to 10.

Another group of phthaiocyanines of formula (I) that are believed to be novel, and hence that represent a further aspect of the invention are those of formula (lb):

wherein

(a) M represents a metal or metalloid atom. (b) at least one of Ri, R?, R3, R t. R5. Rf.. R? and Rs is independently selected from the group consisting of -R9, -OR9, -SR9, or -NR9R10,

in which R9 and Rio independently represent a phenyl group optionally substituted by one or more Rn groups independently selected from the group consisting of

Ci- aikyl, halogen, OR12, SR12, and -NR12R0,

in which R12 and R13 each independently represent hydrogen or

and the others of Ri, R2, R3, R i, R5, R6, R7 and Rs represent hydrogen or one or more pairs of the others of R 1 and R2, R » and R t, R5 and Rc, and R? and R« are attached to adjacent carbon atoms and together form, together w ith the ring to which they are attached, an aromatic fused ring system, and

(c) R_a represents hydrogen or a group

wherein

A and B independently represent a single bond. -CH2- or (C=0)-,

and

Ria, R2a, R3a, Rib, R2b and R *b independently represent hydrogen. Ci-ieaikyi, -C≡CH, -COOH, -NH2, -CH2NH2, -SC≡N,^™CH₂SC≡N or -[OCH₂CH₂]n-OR, in which R represents hydrogen or G f.alkyl and n represents an integer of from 1 to 10. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows one example of a block copolymer suitable for use in the present invention, and a schematic representation of a polymeric micelle formed therefrom.

Figure 2 shows the phthaiocyanine loading capacity of a series of compositions according to the invention.

Figure 3 shows the phthaiocyanine loading efficiencies of a series of compositions according to the invention.

Figure 4 shows UV Vis spectra of a composition according to the invention and of a particular phthaiocyanine compound of the invention in DMF. Figure 5 shows UV Vis spectra of another composition according to the invention and of another phthaiocyanine compound of the invention in DMF.

Figure 6 shows the in vitro cel l-kil ling efficacy of a composition according to the invention.

Figure 7 shows the in vitro cell-killing efficacy of two other compositions according to the invention.

Figure 8 show fluorescent microscopy images from RAW cells after incubation with compositions according to the invention.

Figure 9 shows a fluorescent microscopy image from mouse intestine 1 hour after injection of a composition according to the inveniton. DETAILED DESCRIPTION OF THE INVENTION

Definitions As used herein, and unless indicated otherwise or the context requires otherwise:

"aikyl" as a group or part of a group means, unless otherwise specified, an aliphatic hydrocarbon group which may be straight or branched, and which, unless otherwise specified, may have from 1 to 20 carbon atoms.

"metalloid" means a chemical element that is a non-metal but which has properties that are in one or more relevant respects comparable to those of metals. The most preferred example of a of metalloid is silicon. An example of a suitable metal is ruthenium. "regioisomers" means position isomers having the same functional group or substituent in di fferent positions; regioisomers have the same molecular formula but often different chemical and physical properties.

Micelle-forming polymers

The polymeric micelles used in the present invention generally comprise block copolymers made up of a relatively hydrophobic block and a relatively hydrophilic block. When the polymers are dispersed in an aqueous medium, the polymers spontaneously self-assemble to form micelles, with the relatively hydrophobic parts of the polymer molecules forming a hydrophobic core region of the micelle and the relatively

hydrophilic parts forming a hydrophilic shell. The phthalocyanine molecules, being hydrophobic, preferentially occupy the hydrophobic core. Such a structure is shown schematically in Figure 1 , which shows a micelle constructed from block copolymers of po I y( r.-cap ro I acton e ) and poly( ethylene glycol ). The poly(r.- caprolactone) is hydrophobic and forms the core of the micelle, and the poiy(ethylene glycol ) is hydrophilic and forms the external shell of the micelle. The phthalocyanine molecules occupy the pol y( r.-caprol actone ) core. Figure 1 also shows a typical chemical structure of a suitable block copolymer, which conforms to the preferred general formula described above:

Ri'( I -poly(r.-caprolactone)p-L-poly( ethylene giycoi)_q-RpEG in the illustrated example, RPCL is a benzyl group derived from an initiator used in the synthesis of the pol y(r.-caprolactone ) part of the polymer, RPEG is simply a methyl group, and L is an amide moiety. In order to promote full biodegradabil ity of the polymer, L is preferably - though not necessarily - a biodegradable moiety.

The value of the p, i.e. the average number of r.-caprolactonc units present in the polymer molecule, is preferably from about 4 to about 20, or from about 4 to about 1 0.

The value of q, i.e. the average number of ethylene glycol units present in the polymer molecule, is preferably from about 10 to about 1 00, or from about 20 to about 60.

Suitable polymers for use in the invention may be prepared by methods that are known and will be famil iar to those skilled in the art. Preferably, the polymer is prepared by a convergent synthesis, in which the relatively hydrophobic and the relatively hydrophilic polymers are first synthesized and are subsequently coupled together by formation of the l inker group L. In the ca.se of a polymer hav ing the preferred general formula: Ri>( I -poly(r.-caprolactone)p-L-poly( ethylene glycol )_q-Rpi , the synthetic method may involve the separate formation of a poiy(r.-caprolactone) molecule hav ing the formula:

R i'( I -po 1 y( r.-cap ro 1 ac ton e )_P-( C=0 )-X where X is a leaving group, and a poly( ethylene glycol ) molecule of the formula:

H:N-poly( ethylene glycol )_irRpi (, followed by coupling of those two molecules together through formation of an amide bond. Phthalocyanines

The phthalocyaninc used in the invention is most preferably a sil icon phthalocyaninc, i.e. a phthalocyaninc of the general formula (I) or (la) in which M represents silicon. Preferably, in the phthalocyaninc of general formula (I) or ( la ) R_a represents

and Rb represents

A or B, or more preferably both A and B, may represent -(C=0)-. One, two or all of Rib, R2b and R₃b may independently represent

-[ OCH2Cl l?]i]-OR. R is most preferably methyl, and n most preferably represents an integer of from 1 to 4, and in part icular 3.

Similarly, one, two or al l of R_la, R2a and R_3a may i ndependentl y represent

-[OCH?CH2]n-OR . Again, R is most preferably methyl, and n most preferably represents and integer of from 1 to 4, and in particular 3/.

In other embodiments, one, two or all of R_la, R2a and R ¼ independently represent

In other embodiments, both R_a and Ri, both represent hydrogen, i .e. the phthalocyan ine is a di yd oxy phthalocyani ne.

The groups Ri, R2, R3, R4, R5, Re, R? and Rx present at the periphery of the

phthalocyanine molecule may represent hydrogen. In certain embodiments, al l of those groups represent hydrogen. In other embodiments, including embodiments of

phthalocyan ines of general formula ( lb ), certain of the groups Ri, R2, R₃, R t, Rs, Rf», R? and R , in particular one or more, e.g. al l four, of Ri, R₃, Rs and R7, represent

hydro yphenyl . Particular phthalocyanines that may be used in the present invention are those described in the Examples herein with the following designations:

CosmoPHOS-UAM 2

CosmoPHOS-UAM 3

CosmoPHOS-UA 5

CosmoPHOS-UAM 13 as well as mixtures and regio isomers thereof.

Phthalocyanines for use in the invention may be prepared using methods and starting materials that are known in the art and will be familiar to those skil led in the art. Such methods are illustrated in the Examples described herein. Preparation of nano-systems

Nano-systems according to the invention may be prepared by any suitable methods. A preferred method for the preparation of nano-systems according to the invention, comprising polymeric micelles loaded with phthalocyanine. is a film hydration method. In such a method, the polymer and phthalocyanine are dissolved in separate volumes of organic solvent, and the solutions mixed in proportions corresponding to the desired po I y m e r p h t h a I o c y a n i n e ratio. The organic solvent is then evaporated to form a thin solid film of poimer within which the phthalocyanine is homogeneously distributed.

Dissolution of the solid film in an aqueous medium then leads to the formation of the desired micelles. Heating, agitation and/or filtering of the aqueous medium may be used as appropriate.

The loading of the phthalocyanine in the polymeric micelles may vary widely. For instance, the phthalocyan ine may account for (on a dry basis) from about 0.01% w/w to about 50% of the composition, or from about 0.01 to about 25%, for example about 0. 1 to about 2% or from about 2 to about 10% or from about 10 to about 20%.

The average number of phthalocyanine molecules per polymeric micelle may thus also vary widely. For instance, there may be from 1 to about 10, or from 1 to about 50, or from 1 to about 100 phthalocyanine molecules per polymeric micelle, or more.

Formulations of the nano-systems of the invention The compositions according to the invention may be formulated in any suitable dosage form, for example as a solution or suspension. Preferably the compositions are in a form suitable for injection. Such forms are typically solutions or dispersions, usually in an aqueous medium. Preferably the composition is in the form of a solution or dispersion in an aqueous medium, or is a lyophilised material. Such a composition allows for simple

administration of the composition by injection, e.g. intravenous injection or intramuscular injection, or other suitable means. Lyophilised material may for example be reconstituted with water, saline solution or similar media prior to administration.

In other embodiments, the composition may be suitable for topical administration, e.g. being formulated as gels (water- or alcohol-based), creams or ointments containing nano- systems of the invention. In other embodiments, the composition may be suitable for oral administration (per os), e.g. being formulated as tablets or capsules containing nano-systems of the invention.

In other embodiments, the composition may be suitable for direct administration to a lesion of a tissue by any suitable means. Applications of the nano-systems

The compositions according to the invention are of use in a method for therapy and/or diagnosis and/or therapy monitoring and/or theranosties of a lesion of a tissue in a subject. Thus another embodiment of the invention relates to a method for therapy and/or diagnosis and/or therapy monitoring and/or theranosties of a lesion of a tissue in a subject,

which method comprises the steps of

(a) identifying a subject having or suspected of having the lesion;

(b) administering to the subject a composition according to the invention;

(c) causing or allowing polymeric micelles present in said composition to accumulate upon and 'or within the lesion, if present;

(d) exposing the tissue to electromagnetic radiation so as to cause the

phthalocyanine molecules to produce one or more of fluorescence, phosphorescence, reactive o ygen species, heat, an optical signal and an acoustic signal.

Without wishing to be bound by theory, it is believed that the polymeric micelles are a suitable size that they arc absorbed by, or accumulate upon, the lesions automatically without the requirement for any targeting moieties.

The method may further comprise the step of detecting fluorescence, phosphorescence, an optical signal and/or an acoustic signal produced by the polymeric micelles, such fluorescence, phosphorescence, optical signal and/or acoustic signal being indicative of the presence, site and/or condition of the lesion. This enables the signal emitted by the phthalocyanine molecules to be used to identify the presence of lesions and their location and thus can be used as a method of diagnosis and or therapy monitoring and/or theranosties. Phthaloeyanines are photoactive and. after irradiation by a light source, may produce fluorescence, phosphorescence, reactive oxygen species, heat, an optical signal or an acoustic signal. Preferably the phthaloeyanines are capable of producing fluorescence and/or reactive oxygen species after irradiation by a light source.

By producing fluorescence, once accumulated in the lesions, the phthaloeyanines can be used to locate the lesions within the subject thus enabling diagnosis and are also therefore of use in directing targeted therapies. In one embodiment, the phthaloeyanines produce reactive oxygen species and/or heat, in such a manner as to bring about the death of cells within the lesion and/or the passivation of the lesion and/or the stabilization of the lesion and/or the regression of the lesion and/or the therapy of the lesion by photodynamic therapy (PDT). Hence the

compositions of the invention may be of use in theranostics, ie combining diagnosis of a condition or conditions, usual ly through imaging, with therapy of the same condition and therapy monitoring. Reactive oxygen species act by damaging the targeted tissue and, by generating heat, localised hyperthermia may be induced.

The compositions of the present invention may also be suitable for monitoring the efficacy of a therapy. As the phthalocyanine molecules fluoresce, they can be used to monitor the presence, and hence the treatment, of lesions during and after the therapeutic procedure. This can in turn be used to monitor the efficacy of the therapy both during the therapeutic procedure (real-time therapy monitoring) and at any time point after the therapeutic procedure (follow -up therapy monitoring).

The compositions of the invention may be administered by any suitable route, for example by oral, parenteral, intranasal, sublingual, rectal, transdermal, inhalation or insufflation routes, and direct administration to a lesion of a tissue. Preferably, the compositions are administered parenteral ly, most preferably by intravenous injection. The polymeric micel les of the invention are caused or al lowed to accumulate upon and/or within the lesion. This is most commonly brought about by means of a delay between the administration of the composition and subsequent activation. This delay provides sufficient time for the polymeric micelles to circulate within the subject and to

accumulate upon and/or within the lesions. Typical ly, the delay may be from several minutes to several weeks or months, e.g. from 10 minutes to three months, or from 10 minutes to four weeks, or from 1 0 minutes to 2 weeks, 1 week, 48 hours, or 24 hours, or from 24 hours to three months, or from 24 hours to four weeks, or from 1 week to three months, or from 1 week to four weeks.

The compositions of the present invention are useful in treating a number of diseases w ithin a subject. Without limitation, the compositions may be used for the therapy and/or diagnosis and/or therapy monitoring and/or theranostics of atherosclerosis, cancer and other conditions including, without limitation, inflammatory diseases such as

inflammatory bow el disease, rheumatoid arthritis and autoimmune conditions, as wel l as infectious diseases and inflammation arising from infectious disease.

The methods and compositions of the invention are typically used for the therapy and/or diagnosis and/or therapy monitoring and/or theranostics of lesions of any tubular t issue, for example blood vessels, lymphatic v essels, respiratory tract, gastrointestinal tract, bile ducts, urinary tract or genital tract. Activation of the phthalocyanine photosensitizer may be brought about by means of a catheter, for example an optical fiber catheter or a side- firing and all-round emission optical fiber catheter, or the l ike introduced into the tubular tissue. The methods and compositions of the invention may, however, also be suitable for the therapy of solid tumours, for instance by topical application to skin tumours or by intraoperative direct administration to solid tumours of internal organs/tissues.

Embodiments of the invention will now be described in greater detail, by way of illustration only, w ith reference to the follow ing Examples. Abbreviations

M.p. Melting point

DCM Dichloromethane

DMF Dimethyl fo rm a m i d e

SiPc Silicon phthalocyanine

SiPcCb Dichloro sil icon phthalocyanine

PM Polymeric micelle

PM-(SiPc)_n Nano-system comprising polymeric micel les and silicon phthalocyanine

LED Light emitting diode

EXAMPLE 1

Synthesis of the silicon phthalocyanine designated "CosmoPHOS-UAM 3"

3,4, 5-tris(mcthoxy(triethylcnoxy) (benzoic acid was obtained following a synthetic procedure previously reported in the literature [Brunsveld, L; Zhang, I I.: Glasbeek, M.; Vekemans, J.A.J.M.; Meijer, E.W. J. Am. Chem. Soc.2000, 122, 6175-6182; Oar, M.A.; Serin, J.M.; Dichtel, W.R.; Freeh et. J.M.J. Chem. Mat.2005, 17, 2267-2275; PersecV. Science, 2001, 328, 1009-1014].

SiPcCb was obtained from, a commercial source. 3,4,5-tris(methoxy(triethylenoxy))benzoic acid (974mg, 1.6m mo!) and SiPcCb (85% dye content, lOOmg, 0.16mmol) were mixed in a p re-dried, flask with 1.5m L of 2- methoxyethyl ether under argon atmosphere, and reflux cd for 6 hours. A fter cooling down to room temperature, the solvent was removed under reduced pressure.

Purification of the slurry residue by column chromatography on silica gel (DCM/MeOH, 20: 1 ) yielded the pure compound as a blue gummy solid (122mg, 0.069mmol, 43%).

M.p.: 120 °C

H NMR (300 MHz, CDCb), δ (ppm): 9.71 (m, 8H, H_Pc), 8.41 (m, 811, H_Pc), 4.19 (s, 4H, HAI), 3.73 (m, 2H, CH₂), 3.68 (m, 4H, CH₂), 3.61 (m, 2H, CH₂), 3.56 (t. J = 4.8 Hz, 6H, CH₂), 3.50 (m, 8H, CH₂), 3.45 (m, 24 H, CH₂), 3.38 (m, 12H, CH₂), 3.31 (m, 1811, CH₃), 3.25 (m, 6H, CH₂), 3.0 (t, J= 4.5 Hz, 811, Ar-OCH₂)

¹³C NMR (300 MHz, CDC!s), δ (ppm): 158.73, 150.51, 150.16, 140.13, 135.45, 131.51, 125.24, 124.09, 105.67.71.83, 71.75, 71.69, 70.49, 70.48, 70.41, 70.28, 70.16, 70.05, 69.06, 67.42.65.81, 58.92, 58.84, 15.24

FT-IR (film), v (cm¹): 2920, 2851 , 1729, 1678, 1429, 1336, 1126, 1083, 737.

UV/Vis (CHCb), >.,„ax (nm) (log ε): 686 (4.99), 656 (4.08), 615 (4.12), 359 (4.44).

(MeOH), λ,„*ν (nm) (log ε): 683 (5.7), 653 (4.8), 615 (4.89), 357 (5.2)

HRMS (MALDI-TOF, PEGNa 1500 + PEGNa 2000 + Nal), m/zi Caic. For

C₈8HiioN8Nai0₂₈Si: 1778.7121, (bund 1778.7065 [M+Na]^' EXAMPLE 2

Synthesis of the silicon phthalocyanine designated "CosmoPHOS-UAM 2"

3,4,5-tris(dodecyioxy)benzoic acid was obtained following reported synthetic procedures [Percec,V.; Ahn,C.H.; Cho,W.D.; Jamieson, A.M.; Kirn,.!.: Leman,T.; Schmidt, M; Gerle,M.; Prokhorova,S.A.; Sheiko,S.S.; Cheng,S.Z.D.; Zhang, A.; Ungar,G.; Yeardiey,D.J.P. J. Am. Chem. Soc. 1998, 120, 8619-8631 ; Percec,V.; Ahn,C.H.; Bera,T. K.; Ungar,G.; Yeardiey,D. J. P. Chem. Eur. J. 1999, 5, 1070-1083].

SiPcCb (85% dye content, 1 50mg, 0.245 mmol ), 3,4,5-tris(dodecyloxy)benzoic acid (827mg, 1.23mmoi), and 3,4,5-tris(methoxy(triethylenoxy))benzoic acid (746 mg, 1 .23 mmol ) were dissolved in 4m L of 2-methoxyethyl ether in a pre-dried flask. The reaction was re fluxed for 6 hours under argon atmosphere. After cooling down to room temperature, the solvent was evaporated under vacuum. The remaining slurry solid was purified by column chromatography on silica gel, using DCM and then a gradient of DCM/MeOH, from 60: 1 to 30:1, as eluent. The central fraction, corresponding to the final product, was collected and the solvent evaporated to yield the pure compound as a gummy blue solid (120mg, 0.066mmoi, 27%).

M.p.: 115 °C

Transition temperature (to liquid crystal mesophase): 56.53 °C.

¹ H NMR (300 MHz, CDCb), δ (ppm): 9.72 (m, 8H, H_Pc), 8.40(m, 811, H_Pc), 4.21 (s, 211, HA_I), 4.19 (s, 211, H_AR), 3.74 (m, 211), 3.68 (m, 411), 3.61 (m, 211), 3.56 (m, 411), 3.50 (m, 411), 3.44 (m, 1011), 3.38 (411), 3.34 (m, 211), 3.31 (m, 911, CH₃), 3.28 (m, 411, OCH₂), 3.26 (s, 411), 3.0 (t, J= 4.5 Hz, 411, OCH₂), 2.84 (t, J =6.1 Hz, 411, OCH₂), 1,18 (m, 6611, CH₂), 0.87 (m, 911, CH₃)

¹³C NMR (300 MHz, CDCb), δ (ppm): 158.94, 158.75, 151.04, 150.54, 150.19, 140.15, 140.01, 135.53, 131.46, 125.30, 124.95, 124.07, 105.69, 105.04, 72.82.71.87, 71.79, 71.71, 70.52, 70.54, 70.32, 70.20, 70.09, 69.10, 67.69, 67.45.31.93.31.88, 29.87, 29.69, 29.66, 29.60, 29.58, 29.55, 29.52, 29.36, 29.30, 29.20, 28.80, 25.78, 25.70, 22.69, 22.65, 14.12, 14.07

FT-IR (film), v (cm¹): 2922, 2852, 1729.1635, 1430, 1384, 1336, 1123, 1083, 914, 762, 737 UV/Vis (CHCb), /.,„_* (nm) (log s): 686 (5.4), 655 (4.48), 617 (4.55), 360 (4.8)

HRMS (MALDI-TOF, DCTB + PPG a 2000), m/z: Calc. for CioaHi-wNsOwSi: 1822.0029, found 1822.0035 [M j

EXAMPLE 3

Synthesis of the silicon phthalocyaninc designated "CosmoPHOS-UAM 5"

The synthesis of 4-ethynyibenzoic acid was achieved through a wel l-establ ished protocol reported in the literature [Hung,M.C; LiaoJ. L; Chen. S. A.; Chen.S.H.; Su.A.C. J. Am. Chem. Soc, 2005, 727. 14576- 14577: Louzao, L; SecoJ.M.; Quinoa, E.; Riguera,R.

Angew. Chem. Int. Ed. 2010, 49, 1430-1433; Pauly.A.C; Theato.P. P. J. Polym. Sci, Part A: Polym.Chem. 2010, 49, 21 1-224].

SiPcCh (85% dye content, 100 mg, 0. 163 mmoi), 3,4,5- tris(methoxy(triethyienoxy))benzoic acid (0.298mg, 0.489mmoi) and 4-ethynyibenzoic acid (72mg, 0.489 mmol ) were suspended in 1 .6m L of 2-methoxyethyl ether in a p re- dried flask. The mi ture was re fluxed under argon atmosphere for 5 hours. The solvent was evaporated and the slurry residue purified by column chromatography using as eluent first DC VI and then a gradient of DCM/MeOH from 60: 1 to 30: 1. The blue-greenish fraction was collected, yielding the pure compound as a glassy solid (70mg, ().054mmol. 33%). ¹ H NMR (300 MHz, CDCb), δ (ppm): 9.73 (m, 8H, H_Pc), 8.40 (m, 811, Hiv ), 6.36 (d, J = 8.4 Hz, 2H, HSAT), 5.08 (d, J= 8.4 Hz, 211, H₂AI), 4.17 (s, 211, ΜΛ,), 3.53 (m, 611), 3.52 (m, 611), 3.44 (m, 1211).3.38 (m, 811), 3.31 (m, 911, CH₃), 3.28 (m, 211), 3.25 (s, 411), 2.99 (t, J= 4.5 Hz, 411, OCH₂), 2.74 (s, 111, CM)

¹³C NMR (300 MHz, CDCls), δ (ppm): 158.68, 158.65, 150.48, 150.20, 140.07, 135.55. 131.48, 130.41, 127.24, 124.24, 105.62, 72.47, 71.87, 71.82, 71.73, 71.67, 70.56.70.45, 70.39, 70.29, 70.26, 70.15, 70.04, 69.05, 67.38, 61.71, 58.98, 58.90, 58.82, 15.24

I T-IR (film), v (cm¹): 2957, 2922.2852, 2340, 1959, 1728, 1676, 1463, 1289, 1122. 1082, 914, 863, 773, 762, 734, 686

UV/Vis (CHCb), λ,,,,ν (iim) (log s): 686 (5.5).655 (4.56), 617 (4.63), 359 (4.91), 271 (5.34)

HRMS (MALDI-TOF, DCTB-PMMANa 1000 + Nal), m/zi Calc. for

C69H₆8N₈NaiOi6Sii: 1315.4415. found 1315.4385 [M+Na]⁺; Calc. for CeHegNgOieSii: m/z: 1292.4517, found 1292.4513 [M ]

EXAMPLE 4

Synthesis of Dih ydroxy-Sil icon phthalocyanine

CosmoPHOS-UAM 3 (the product of Example 1) (lOmg, 0.0057mmol) was subjected to hydrolysis by treatment with concentrated H2SO4 (1.5mL) for 4 hours, followed by addition of a crushed ice/H₂0 mixture (15mL) and treatment with a concentrated ammonium hydroxide : pyridine solution (1 : 1 v/v) (5m L). The solution was stirred for another 20 hours and filtered. The solid obtained was washed ( 1 1?0, acetone, toluene and heptane) and vacuum-dried to yield the corresponding SiPc of general formula I in which both R_a and Ri, represent hydrogen (2.68mg, 0.0048mmoi, 82%), ie.e the phthalocyanine is a dihydroxy phthalocyanine. The characterization was in accordance with that p rev io u s I y m e n t i o n ed in literature [ Davidson..!. B.; Wynne,K.L; Macromolecules 1978, 11, 186.] Amongst other materials, the fol lowing can be prepared by the same procedure:

2, 9(10), 16(17), 23(24)-Tetra-feri-butyi dihydroxy-silicon phthalocyanine (as a mixture of regioisomers)

2,9(10),16(17),23(24)-Tetra-n-butoxy dihydroxy-silicon phthalocyanine (as a mixture of regioisomers)

2,9,10,16,17,23 ,24-Octa-n-butoxy dihydroxy silicon-phthalocyanine

EXAMPLE 5

Synthesis of the silicon phthalocyaninc designated "CosmoPHOS-UAM 13"

CoemoPHOS-UAM 13

Step 1 : Pd( PPh » ) i (45mg, 0.039mmol) was added to a solution of 4-iodo-phthaionitriie (lg, 3.937mmoi) in dry THF (35m L ) and the reaction mixture was stirred for 20 minutes. K2CO3 (544 mg, 3.937 mmol), 3 -fi yd rox y pli en y I boron i c acid (543 mg, 3.937 mmol ) and H2O ( 10 ml.) were added and the reaction mixture refluxed for 24 hours, after which it was cooled to room temperature. The THF was evaporated and the resulting solid suspended in H2O was filtered, washed with H2O, redissoived in THF and dried over Na2SO-i. After evaporation of the solvent the solid was purified by column

chromatography on silica gel, using heptane/EtOAc 2: 1 as eluent, yielding 1 (0.867g, 1.338mmol, 34%) as a white solid.

Ή NMR (300 MHz, CDC b), δ (ppm): 7.98 (p, J = 9.3 Hz, 1 11, H.\, ), 7.93 - 7.83 (m, 211, HAT), 7.38 (d, J = 7.5 Hz, 1 1, H.\, ), 7. 14 (d, J = 8.5 Hz, 1H,

6.95 (d, J = 9.5 Hz, 1 11, HAI), 4.93 (s, 1 11, OH ). ¹³C NMR (300 MHz, MeOD), δ (ppm): 141.48, 136.46.134.14.131.77.123.63,98.96. HRMS (MALDI-TOF, IX I B), m/z: Calc. for Ci₄H₈N₂ONa: 243.0528, found

243.0524 [M+Naj Step 2: Ammonia gas was bubbled through a solution of 1 (307mg, 13.9mmol) and

NaOMe (14mg, 0.48 m mo I) in d y MeOH (lOmL) for 1 hour. The reaction mixture was stirred for another 14 hours at reflux temperature, under ammonia atmosphere, after which MeOH was removed under reduced pressure. The resulting solid was washed with saturated HiC! solution and the product was filtered to yield 2 (322mg, 13.6mmol, 98%) as a pure white yellowis solid.

ΊΙ NMR (300 MHz, MeOD), δ (ppm): 8.25 (s, 111, H_Ar), 8.06 - 7.88 (m, 2H,

7.37 - 7.24 (m, 111, H_Ar), 7.17 (d, J = 7.7 Hz, 111,

7.12 (s, 111, H_Ar), 6.92 6.83 (m.111, ¹³C NMR (300 MHz, MeOD), δ (ppm): 159.02, 155.23, 148.95, 142.53, 129.88, 121.59, 117.97, 115.54.109.95.

HRMS (MALDI-TOF, DC I B), m/z: Calc. for C14H12N3O: 238.0980, found 238.0976 [M + Hj- Step 3: 2 ( 1 OOmg, 422mmol) was dissolved in recently distilled quinolonc ( 1.5ml.) and stirred under argon atmosphere for 10 minutes. S1CI4 (72μ1., 633mmol) was added dropwise to the reaction and the temperature was raised to 220° C for 1 hour. The solution was cooled to room temperature and the solid was filtered and washed with H₂0, MeOH and acetone to yield CosmoPHOS-UAM 13 (76.4mg, 321mmoi, 76%) as a dark green solid.

UV/Vis (DMSO), λ (nm) (log s): 691 (4.81), 621 (4.11), 460 (4.02), 363 (4.42)

MS (MALDI-TOF, DCTB), m/z Calc. for C56H34 8O6S1: 942.2371, found

942.2385[M]\ Calc. for CseftsNgOsSi: 925.2343, found 925.2351 [M-OH] EXAMPLE 6

Synthesis of benzyl -poly(8-caprolactone)-methoxypoly(ethyleneglycol)

( Ben-PCL-mPEG ) A library of benzyl -poly(8-caproiactone)-methoxypoiy(ethyieneglycoi) ( Ben-PCL- mPEG ) block copolymers were prepared by a convergent synthetic approach. This Example describes the preparation of a block copolymer formed from a

poly(i.-caprolactone) 8-mer and a methoxy poly( ethylene glycol ) with a molecular weight of ca. 2000. a) Preparation of benzyl-PCLs-OH olymer

A typical procedure for the synthesis of a benzyl -PCLs-OH polymer with a degree of polymerization of the PCL of 8.0 was as follows. A mixture of ε-caprolactone (25.00g, 0.2 mol), benzyl alcohol (2.96g, O.()27mol ) and stannous octoate (1 drop) was heated and the ring opening polymerization in the melt was allowed to proceed overnight at 130°C in a nitrogen atmosphere. The product was purified by dissolution in dichloromethane followed by precipitation in a 20-fold excess of cold (-20°C) diethyl ether. The Benzyl - PCL8-OH was filtered and dried overnight in vacuo at room temperature to give a white powder (yield: 96%).

¹ H NMR (300 MHz, CDCb), δ (ppm): 7.3 (s, aromatic protons benzyl alcohol), 5. 1 (s, CC¾0), 4.05 (m, CH2Ci¾0), 3.6 (t, CH₂C¾OH), 2.3 (m, 0C(0)C7/?), 1 .6 (m,

CH2CH2CH2CH2CH2), 1.3 (m, CH2CH2CH2CH2CH2). b) Preparation of benzoyl-PCLs-PNC

Benzyl -PCL8-OH (4.00g. 3.6mmol ) (product of step a) was dissolved in 20m L dry toluene in a nitrogen atmosphere. The solution was cooled to 0°C and triethy aminc

(0.70g, 7.2mmol ) and subsequently para-nitrophenyicarbonylchioride (PNC-Ci; 1.45g, 7.2mmol ) were added to the solution while stirring. After 1 hour, the mixture was filtered to remove the triethyleneaminc HC1 salt and precipitated into cold (-20°C) diethyl ether. The product was filtered and carefully washed with a small amount of cold (- 20°C) diethyl ether. The resulting product, benzyl -PC Ls-PNC, was dried in a vacuum oven and was obtained as a white powder (yield: 95%).

¹ H NMR (300 MHz, CDCls), δ (ppm): 8.3 (d, aromatic protons of PNC), 7.3 (m, aromatic protons of benzyl alcohol and PNC), 5. 1 (s, CC¾0), 4.2 (m, (T C77 OC(0)0), 4.05 (m, CH2C7/. ), 2.3 (m, OC(0)Ci¾), 1 .6 (m, CH2CH2CH2CH2CH2), 1 .3 (m,

CH2CH2CH2CH2CH2).

c) Preparation of mPEG2000-NH?.

mPEG-NH2 was synthesized according the procedure outlined by Elbert and Hubbell [ Biomacromoleeules (2001 ) 2(2), 430-44 1 ]. In a typical procedure mPEG2000-OH (50. Og, 25mmol ) was dissolved in 700m L of dry toluene and dried by the removal of 350m L of solvent by azeotropic distillation. After the solution was cooled in an ice-bath, 25m L of DCM and triethylamine (14.5mL, 1 00m mol ) were added. Subsequently, mesyl chloride (7.73m l., 1 00m mol ) was added drop-wise under stirring and al lowed to react overnight at room temperature. The solution was filtered and the product was

precipitated in a large excess of cold diethyl ether. After drying, the formed mPEG2000- mesylate was reacted with 1 00m L of an aqueous ammonia solution (25%) for 4 days at room temperature. Subsequently the ammonia was al lowed to ev aporate and the pH of the solution was raised to 1 3, using 1 M NaOH. The solution was extracted three times with 200m L DCM. The organic phases were combined and the solution was

concentrated. The mPEG2000-NH2 was isolated by precipitation in cold diethyl ether, and drying in vacuo (yield: 76%).

¹ H NMR (300 MHz, CDCb), δ (ppm): 3.65 (m, PEG protons), 3.37 (s, CH2OCH3), 2.94

(t, CH2CH2NH2). d ) Synthesis of Ben-PCL-mPEG

Benzyl -PC LN-P C (product of step b); l .OOg, 0.707mmol ) was dissolved in 20 m L of dry toluene. To the resulting solution, mPEG2()00-N l l2 (product of step c); 1.41g,

0.707mmol ) was added and the reaction mixture was stirred for I hour at room temperature under a nitrogen atmosphere. The mixture was poured into diethyl ether, filtered and careful ly washed at least six times with diethyl ether to remove p- nitrophenol . The product was dried in a vacuum oven and obtained as a white powder (yield: 97%).

¹ H NMR (300 MHz, CDCb), δ (ppm): 7.3 (m, aromatic protons of benzyl alcohol ), 5. 1 (s, CC /.0), 4.05 (m, CH2C7/ ) ), 3.64 (m, PEG protons), 3.38 (s, OCf¾), 2.3 (m, OC(0)C/¾), 1.6 (m, CH2CH2CH2CH2CH2), 1.3 (m, C H 2 C H C //. ' H 2 C H ) .

EXAMPLE 7

Preparation of nano-systems [P -(SiPc)_n1

(Polymeric icelle compositions of Silicon Phthalocyanines)

M icelles loaded with photosensitizer were formed by a film hydration method. In short, lOmg of block copolymers were dissolved in ImL of dichloromethane. 5mg of the corresponding photosensitizer (SIPc) was dissolved in ImL THF and different amounts corresponding to the desired polymer/photosensitizer ratios were mixed with the DCM block copolymer solution. By evaporation of the dichloromethane and THF a thin solid film is formed. The formed film contained both block copolymers and homogenously distributed photosensitizer. The block copolymer and photosensitizer film was subsequently dissolved in ImL PBS solution and filtered through a 0.2 urn syringe filter to remov e uneneapsulated photosensitizer. This procedure results in a photosensitizer loaded micelle solution of approx. 10 mg'm L.

Nano-systems [PM-(SiPc)_n] prepared by the method abov e, using the SiPc and PM systems described in Examples 1 , 2 and 7. respectiv ely are set out in Table 1. These nano-systems, identified by the Composition umbers 1 to 32, differ from each other in one or more of the following respects:

a) the number of caprolactone repeat units present in the polymer,

b) the nature of the initiator used to form the po 1 y ( c a ro lactone) (i.e. the group RPCL),

c) the nature of the phthalocyanine, and

d ) the loading of phthalocyanine in the polymeric micel les. Table 1

EXAMPLE 8

Loading efficiency and capacity of prepared compositions

Ail prepared composit ions were dissolved in DMF and the amounts of encapsulated photosensitizer were measured by UV/Vis spect ro photo m etry to determine loading capacity (wt% of Si Pc in micel les) and loading efficiency (% encapsulated with respect to feed %). The results are shown in Figure 2 and Figure 3.

EXAMPLE 9

Aggregation state of SiPcs in PM

The aggregation behaviours of the SiPcs in the prepared compositions was investigated. It was found by UV/Vis spectroscopy that Compositions 1 - 1 0, 16-20, 26-28 have the same spectrum as SiPcs dissolved in DMF, indicating that these Compositions did not show aggregation of the SiPcs in the micelles. Figure 4 shows the UV/Vis spectrum obtained for Composition 1 (solid line), compared to CosmoPHOS-UAM 3 in DMF (broken line ). Compositions 1 1 - 1 5. 2 1 - 1 5 and 29-3 1 did show aggregation with a clear red shift in the UV/Vis spectra. Figure 5 shows the UV/Vis spectrum of Composition 1 1 (sol id line), compared with CosmoPHOS-UA 2 in DMF (broken line).

IN VITRO DATA

Photo-c vtotox i c i t v of prepared compositions in RAW 264.7 cells The abil ity of the nano-systems to induce cel l kil ling was studied in mouse macrophage cell line RAW 264.7. Macrophages are the most relevant target cell type for studying the therapeutic goal - treatment of atherosclerosis - with these systems. For the experiment, a custom-made LED dev ice

for il luminating cel ls on 96-wel l plate was used. The illumination time was 10 minutes with LED l ight intensity 3.04 mW/cm². Cel ls without nano-systems and/or without light activation are always used as a control, as wel l as the control solutions in which nano-systems were made. For each sample, a wide range dilution series was tested. Cells were incubated 24 hours after LED-illumination before measuring the photo-cy totox icity. The IC50-value in RAW cells for

Composition 2 is 70 ng/μΐ (Figure 6). For both Composition 26 and Composition 27, the IC50-values is 35 ng/μΐ (Figure 7).

Uptake studies in RAW264.7 cells The abil ity of polymeric micelles to enter a macrophage cel l line was analyzed by fluorescence microscopy. Figure 8 shows fluorescent microscopy images from RAW cells after incubation with Compositions 26 and 27. Cells without nano-systems serve as controls (CTRL). Although not evident in the monochrome representation of Figure 8, the red fluorescence indicated that by a concentration of 1 0 ng/ iil al l the cells contain the fluorescent nano- systems.

IN VIVO DATA

Figure 9 shows a fluorescent microscopy image from a mouse intestine 1 hour after injection of Composition 2. Again, although not ev ident in the monochrome

representation of Figure 9, the localization of Composition 2 in the small intestine of the mouse is demonstrated by red fluorescence.

Claims

Claims

1. A composition comprising polymeric micelles as a nanocarrier and one or more molecules of a phthalocyanine as a payload,

wherein the phthalocyanine is of the formula I :

in which

(a) M represents a metal or metalloid atom,

(b) Ri , R₂, R₃, R t. R5, R-6, R-7 and Rs are independently selected from the group consisting of hydrogen, halogen, -R9, -OR9, -SR9, or -NR9R10,

in which R9 and Rio independently represent hydrogen, C i i ^alky!, or a phenyl group optionally substituted by one or more Rn groups independently selected from the group consisting of C_1-12alkyl, halogen, OR 12. -SR12, and -NR 12R ! ·,

in which R12 and R13 each independently represent hydrogen or G ^a!ky , or one or more pairs of Ri and R2, R3 and R t, R5 and Rc, and R? and Rx are attached to adjacent carbon atoms and together form, together with the ring to which they are attached, an aromatic fused ring system, and

(c) Ra represents hydrogen or a group

and

Rb represents hydrogen or a group

wherein

A and B independently represent a single bond, -CH₂- or -(C=0)-,

and

Ria, R2a, R-3a, R ib, R211 and Rsb independently represent hydrogen, G-iealkyl,

-C≡CH, -COOH, -NH2, -CH2NH2, -SC≡N. -CH₂SC≡N or -[OCH₂CH₂]n-OR, in which R represents hydrogen or Chalky! and n represents an integer of from 1 to 10.

2. A composition as claimed in Claim 1 , wherein the polymeric micelles comprise block copolymers of poly(caprolactone ) and poly(ethyiene glycol ).

3. A composition as claimed in Claim 2, wherein the block copolymers have the general formula

Rp( I -poly(i-.-caprolactone)p-L-poly( ethylene glycol Χ,-RPK,

wherein

RPCL is the terminal group of the pol y( r.-caprol actone ) block;

p is an integer of from 4 to 1 00;

L is a linker, for example selected from the group consisting of ester, amide, carbonate or carbamate;

q is an integer of from 10 to 250; and

RPEG is the terminal group of the poly(ethylene glycol ) block.

4. A phthalocyanine of the formula la:

wherein

(a) M represents a metal or metal loid atom,

(b) Ri, R₂, R3, R i, R5, R., R? and Rs are independently selected from the group consisting of hydrogen, halogen, -R9, -OR9, -SR9, or -NRgRio,

in which R> and Rio independently represent hydrogen, C u .alkyl, or a phenyl group optionally substituted by one or more Rn groups independently selected from the group consisting of Ci palkyl, halogen, OR 12, -SR12, and -NR12R13,

in which R12 and R13 each independently represent hydrogen or Ci ^alkyl, or one or more pai s of R i and R2, R3 and R i, R5 and Re, and R? and Rx are attached to adjacent carbon atoms and together form, together with the ring to which they are attached, an aromatic fused ring system, and

(c) Ra represents hydrogen or a group

wherein

A and B independently represent a single bond, -CH2- or (C=0)-,

and Ria, R?a, R3a, Rib, R?b and R₃b independently represent hydrogen, C i-u-alkyl. -C≡CH, -COOH, -NH₂, -CH2NH2, -SC≡N, -CH₂SC≡N or -[OCH₂CH₂]n-OR, in which R represents hydrogen or G ealkyl and n represents an integer of from 1 to 1 0.

5. A phthaiocyanine of the formula lb:

wherein

(a) M represents a metal or metalloid atom,

(b) at least one of Ri, R₂, R3, R4, R5, Re, R? and Rx is independently selected from the group consisting of -R9, -OR9, -SR9, or -NR9R10,

in which R9 and Rio independently represent a phenyl group optionally substituted by one or more Rn groups independently selected from the group consisting of C_1-12alkyl, halogen, OR i .\ -SR₁₂, and -NRi₂Ri₃,

in which Ri₂ and R13 each independently represent hydrogen or G ^alkyl, and the others of Ri, R₂, R₃, R t, R5, Rf>, R7 and Rx represent hydrogen or one or more pairs of the others of R i and R₂, R₃ and R t, R5 and Re, and R? and Rx arc attached to adjacent carbon atoms and together form, together with the ring to which they are attached, an aromatic fused ring system, and

(c) Ra represents hydrogen or a group

and

Rb represents hydrogen or a group

wherein

A and B independently represent a single bond, -CH₂- or -(C=0)-,

and

Ria, R2a, R½, Rib, R211 and Rsb independently represent hydrogen, G-iealkyl, -C≡CH, -COOH, -NH2, -CH2NH2, -SC≡N, -CH₂SC≡N or -[OCH₂CH₂]n-OR, in which R represents hydrogen or Chalky! and n represents an integer of from 1 to 10.

6. A composition or a phthalocyamne as claimed in any preceding claim, wherein M represents silicon.

7. A composition or a phthalocyanine as claimed in any preceding claim, wherein R_a represents

and Rb represents

8. A composition or a phthalocyanine as claimed in Claim 7, w herein A and B both represent -(C=0)-.

9. A composition or a phthalocyanine as claimed in Claim 7 or Claim 8,

wherein one, two or all of Rib, R2b and R¾ independently represent -[OCH₂CH₂]_n-OR, in which R represents hydrogen or Ci_6alkyl, preferabl y methyl, and n represents an integer of from 1 to 1 0, more preferably from 1 to 4, and in particular 3, and wherein one, two or al l of R_la, R-2a and R_3a independently represent

-[OCH₂CH₂]_n-OR, in which R represents hydrogen or C halky I, preferabl y methyl, and n represents an integer of from 1 to 1 0, more preferably from 1 to 4, and in particular 3, and/or one, two or al l of R_la, R2a and R_3a independently represent C i leaikyl .

1 0. A composition or a phthalocyanine as cl aimed in any preceding claim, wherein Ri, R2, R3, R i, R5, Re, R? and R« each represent hydrogen .

1 1 . A composit ion or a phthalocyanine as claimed in any one of Claims 1 to 9, wherein one or more of Ri, R₃, R5 and R? represent hydroxyphenyi.

12. A composition as claimed in any one of Claims 1 to 3, wherein R_a and Ri, both represent hydrogen.

1 3. A composit ion as claimed in any one of Claims 1 to 3 wherein the phthalocyanine molecules following exposure to electromagnetic radiation, produce one or more of fluorescence, phosphorescence, reactive oxygen species, heat, an optical signal and an acoustic signal.

1 4. A composition as claimed in any one of Claims 1 to 3 or Claim 1 3, in a form suitable for injection, such as a solution or dispersion in an aqueous medium, or a

iyoph i ! ised material , or a form suitable for topical adm in istration, such as a gel , cream or ointment, or a form suitable for oral administration, such as a tablet or capsule, or a form suitable for direct administration to a lesion of a tissue.

1 5. A composit ion as claimed in any one of Claims 1 to 3 or Claim 1 3 or Claim 14, for use in therapy and/or diagnosis and/or therapy mon itoring and/or theranostics of a lesion of a tissue.