WO2019102224A1

WO2019102224A1 - Cell penetrating polymers and uses thereof

Info

Publication number: WO2019102224A1
Application number: PCT/GB2018/053413
Authority: WO
Inventors: Sebastien Perrier
Original assignee: The University Of Warwick
Priority date: 2017-11-24
Filing date: 2018-11-26
Publication date: 2019-05-31
Also published as: GB201719590D0

Abstract

Advances in the development of small molecules as highly potent anticancer drugs often face limitations related to their poor solubility, rapid elimination and limited stability in the body. The use of drug carriers may address these challenges by providing a protective shell that enhances solubility and retards clearance from the blood stream. Moreover, incorporation of drug delivery vectors can of introduce functionality and increase the selective accumulation at the specific target site. So-far-neglected, but particularly interesting, are organic nanotubes (NTs) formed by cyclic peptide-polymer conjugates. We present a specifically designed system comprising a self-assembling cyclic peptide core, a functional polymer shell and a highly potent organoiridium candidate drug.

Description

CELL PENETRATING POLYMERS AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to methods for drug delivery. Specifically, the present invention relates to a self-assembling drug delivery system comprised of pharmacologic drugs complexed to a self-assembling cyclic peptide-polymer conjugate.

BACKGROUND TO THE INVENTION

Advances in the development of small molecules as highly potent anticancer drugs often face limitations related to their poor solubility, rapid elimination and limited stability in the body.^{1 2} The use of drug carriers may address these challenges by providing a protective shell that enhances solubility and retards clearance from the blood stream. Moreover, incorporation of drug delivery vectors can of introduce functionality and increase the selective accumulation at the specific target.³ Tuneable in size, the devices can be optimized for passive targeting to tumours via the enhanced permeability and retention (EPR) effect.⁴ In addition, most carriers can be functionalized by a variety of ligands (carbohydrates, peptides, proteins, antibodies, aptamers) to allow for active targeting of specific cells.⁵ Overall, drug carrier systems offer potential for improving the therapeutic efficiency of drugs and reducing their side effects.

It is therefore not surprising that a plethora of potential drug delivery vectors has been reported including, for example, spherical polymer micelles or vesicles.⁶ So-far-neglected, but particularly interesting, are organic nanotubes (NTs) formed by cyclic peptide-polymer conjugates. The nanotubes are built around a core made of b-sheet-forming cyclic peptides (CPs).⁷ CPs presenting an even number of alternating D- and L- amino acids adopt a flat conformation in which the alignment of amide functionalities allows the formation of hydrogen bonds between cyclic peptides, promoting their stacking into rod-like, cylindrical assemblies.⁸ Cylindrical objects exhibit a longer time of residence in the body than spheres of comparable size,⁹ and rod-like structures can show higher activity than spherical particles¹⁰ when loaded for example with active peptides,¹¹ antibodies¹² or proteins.¹³

It has been shown that because of their increased aspect ratio, elongated nanoparticles exhibit longer circulation times and can enhance cellular uptake and tumour accumulation in vivo.¹⁶ Filomicelles,¹⁷ polymer brushes¹⁸ and PEGylated tobacco mosaic viruses¹⁹'²⁰ are among organic tubular structures that have already been studied in vivo and shown promising results. Discher et al. have, for example, studied filomicelles and compared their behaviour to that of their spherical counterparts in vivo.²¹ They have shown that the cylindrical structures not only circulate in the blood for a considerable amount of time and manage to reach and enter tumour tissues, but also enable much higher loading of the anticancer agent Paclitaxel in comparison with the spherical ones while maintaining similar survival rates in mice, thereby leading to superior therapeutic efficiency. Milliner et al. have studied the pharmacokinetics of unimolecular cylindrical polymer brushes in rats, showing that they exhibit long term blood circulation, and that the aspect ratio of the brushes has a considerable impact on their pharmacokinetic parameters.²² They further studied this system in mouse xenografts, demonstrating that the brushes undergo EPR effect and tend to passively target tumour tissues.²³ The main limitation of such large stable objects is their relatively poor clearance from the system, usually imputable to recognition by the mononuclear phagocytic system (MPS) translating to high accumulation in organs such as the spleen and the liver.²⁴

One way to circumvent this issue is to explore the use of materials which undergo a supramolecular self-assembly, for example by directed hydrogen bonds.²⁵ Supramolecular polymers,²⁶ especially those who self-assemble in aqueous media,²⁷ have started to gain considerable attention in the field of nanomedicine. They allow for a bottom-up design strategy that enables extensive functionalisation of the building blocks, resulting in broad libraries of assembled materials. Examples of such systems include systems based on host- guest interactions,²⁸ or on the stacking of peptide amphiphiles into fibres.^29-31 One of their possible major advantages over other nanoparticles is their supramolecular nature which provides a considerable stability, but also allows them to eventually break up into unimeric entities small enough to be cleared out of the system, hence avoiding undesired organ accumulation.

An emerging class of elongated drug carriers which feature such a supramolecular assembly process are nanotubes formed of cyclic peptide-polymer conjugates.³² Cyclic peptides formed of an even number of alternating D- and L- amino acids have been shown to adopt a flat conformation leading to self-assembly into nanotubes through antiparallel b-sheet formation.³³ Conjugation of water-soluble polymers to these peptides enables control over the size and the functionality of the nanotubes. A few reports on such systems as drug carriers have shown promising properties in experiments on cell systems,³⁴'³⁵ but their in vivo behaviour has yet to be explored.

Despite all the above mentioned advantages of cyclic-peptide nanotubes (CPNTs) and the advances in the synthesis of functional materials, so far only a handful of examples of their use as drug delivery vectors has been reported.^{14 16} Blunden et al. have described the synthesis of such nanotubes bearing RAPTA-C, a moderately active ruthenium anticancer drug. They demonstrated that the attachment of the drug helped to increase its activity against cancer cells.¹⁴

Here, we synthesized a poly(hydroxypropyl methacrylamide) (pHPMA)-based cyclic-peptide polymer conjugate and demonstrated its ability to self-assemble into nanotubes. A non assembling polymer which does not contain the peptide core was also synthesised as a control. The in vitro behaviour of both conjugate and control polymer, as well as their pharmacokinetics and biodistribution in rats were studied and compared.

Furthermore, we present a specifically designed system comprising a self-assembling CP core, a functional polymer shell and a highly potent organoiridium candidate drug. Particular attention was paid to the use of biocompatible components in the synthesis and the effective attachment of the metallodrug through efficient drug conjugation suitable for cancer therapy. In this context, poly(2-hydroxypropyl methacrylamide) (pHPMA) was chosen, since it has attracted particular interest for drug delivery applications over the past few decades.^{17 18} Several systems derived from pHPMA have been studied in detail and are currently undergoing clinical trial.^{19 20}

In addition to comprehensive optimisation of the delivery vector, the choice of a compatible and potent drug is critical for designing effective therapeutics. Recently, organoiridium catalysts have been shown to exhibit high potency towards a wide range of cancer cells²¹ and through careful choice of ligands, the efficiency of these complexes can be improved by three orders of magnitude, reaching sub-micromolar values.²² Depending on the cell line and the complex, activity was shown to be ca. 5 to 10 times higher than that of the clinical drug cisplatin, and more than 200 times higher than RAPTA-C. The attachment of this type of complex to a polymeric carrier can be achieved through incorporation of a suitable metal binding ligand on the polymer chains.

Based on these considerations, we synthesized a cyclic peptide-pHPMA conjugate and loaded it with an organoiridium anticancer complex. The organoiridium fragment was attached through ligation to a pyridine-containing comonomer in the polymer shell. The ability of the drug-loaded conjugates to self-assemble into nanotubes in solution was thoroughly established by scattering techniques, and their cytotoxicity in vitro was assessed and compared to that of the free drug. For the first time, it was also tested alongside and a drug- loaded polymer control that does not contain the cyclic peptide core, in order to clearly assess the impact of the self-assembly on the cytotoxicity. Finally, cellular accumulation of the three compounds was studied both qualitatively and quantitatively and the mechanism of action was explored. SUMMARY OF INVENTION

The invention provides, a compound comprising a cyclic peptide and one or more polymer arms, wherein the polymer arms comprise a polymer obtaina ble by polymerisation of at least one monomer according to Formula I:

Formula I wherein R is a linker selected from : alkyl, aryl, amine, amide, carbonate, ester, ether, imide, sulfide, disulfide, sulfone, sulfoxide, ureide or a combination thereof; and Ar is an optionally substituted aryl or optionally substituted heteroaryl.

The inventors have found that cyclic peptides functionalised with polymers comprising at least one of the monomers according to Formula I can be employed as a particularly effective means of introducing drugs into cells, in particular cancer cells. Without being bound by theory, it is believed that the cyclic peptides with their polymeric arm(s) assemble into tube-like structures that have a particular affinity towards cells, especially cancers cells. The compound of the invention interacts with other such compounds in order to form such macromolecular nanotubes, wherein the polymer arm(s) extend radially from the cyclic peptide.

The term "alkyl" is intended to take its usual meaning and often will be selected from Ci to Cio alkyl. Moreover, one or more of the hydrogens may be replaced with a halogen such as chlorine or fluorine, usually fluorine. More often, the alkyl group is a Ci to C6 alkyl and most often the alkyl group is selected from methyl, ethyl, propyl or butyl.

The term "aryl" is intended to take its usual meaning, generally covering one or more aromatic or partially conjugated ring species. Typical examples include, but are not limited to : phenyl, cyclohexadienenyl, cyclopentadienyl, naphthyl, or combinations thereof. Most often, the aryl group will be phenyl. The term "heteroaryl" is intended to take its usually meaning, usually the heteroaryl species will be selected from the aryl species listed above wherein said species further comprise one or more heteroatoms; usually oxygen, nitrogen or sulfur but most often nitrogen. Typical examples include pyridinyl, furyl, or combinations thereof. Typical substituents that can be added to the aryl or heteroaryl groups include: hydroxy, halogens, alkyl and amino groups.

The skilled person will appreciate that the size of the linker "R" can be adapted in length by providing one or more of the claimed species. Often, this includes providing one or more of the moieties listed above together with one or more alkyl groups.

Typically, "Ar" is selected from those aryl and heteroaryl species described above. However, it is often the case that "Ar" is selected from pyridinyl, cyclopentadienyl or phenyl. Often, "Ar" is pyridinyl. It is believed that such groups are preferred because they are effective at binding organometallic complexes often used as drugs in therapy.

Further, it is often the case that R is a linker comprising ureide i.e. it comprises a group according to Formula II:

Formula II

Further, it is often the case that the at least one monomer is ((3-(pyridine-4- ylmethyl)ureido)ethyl) methacrylate (PUEMA) :

Whilst there is no particular restriction on the choice of polymerisation technique used to produce the polymer described in relation to the invention, it is often the case that the polymerisation is a free radical polymerisation, typically reversible addition-fragmentation chain-transfer polymerisation (RAFT).

Moreover, whilst it is not necessary to include a comonomer in the polymerisation process, it is often the case that the polymer is a copolymer further comprising comonomers. The copolymers may be block copolymers, random copolymers, alternating copolymers or a combination thereof. The choice of comonomer is not particularly limited provided that it does not adversely affect the binding properties of the compound of the invention with the drug for delivery. However, usually the one or more comonomers are hydrophilic in order to enhance the biocompatibility and water soluble properties. Typical examples of suitable comonomer include, but are not limited to: hydroxypropyl methacrylate, 2-hydroxyethyl methacrylate, 4-hydroxybutyl methacrylate, glycerol monomethacrylate or combinations thereof. Usually, the comonomer is hydroxypropyl methacrylate. Where a comonomer is employed and the polymer arm(s) are copolymers, it is usually the case that the monomer according to Formula I is positioned near a proximal end of the copolymer chain (near the cyclic peptide). Without being bound by theory, this is believed to be advantageous because, it allows adjacent polymer arms (such as those provided by neighbouring compounds in a nanotube structure) to effectively "shield" a drug bound to the monomer unit with the polymer, close to the cyclic peptide. However, the monomer according to Formula I may be incorporated towards the distal end of the polymer in some embodiments. Often, the copolymer comprises a polyether.

The polymer arm(s) typically have a molecular weight of less than 50,000 gmol ¹. However, more often the molecular weight in the range 1,000 to 30,000 gmol^-1, more typically 2,000 to 25,000 gmol^-1, even more typically 3,000 to 20,000 gmol^-1, more typically still 6,000 to 16,000 gmol^-1, and most typically in the range 8,000 to 12,000 gmol^-1. The polydispersity of the polymer is not particularly restricted but it will usually be in the range 1.0 - 2.0, more often 1.0 to 1.5.

The cyclic peptide need only comprise one polymer arm but it is often the case that two or more polymer arms are attached to the cyclic peptide. The number of arms attached to the cyclic peptide can be used to vary the concentration of drug associated with a given compound.

The cyclic peptide used in the invention is not particularly restricted provided that it is capable of being functionalised with the one or more polymer arms and can cooperate with other cyclic peptides so as to form a nanotube. Cyclic peptides can coordinate with other cyclic peptides to form nanotube structures in a number of ways and there is no particular restriction as to how this occurs. This may be achieved through equipping cyclic peptides with b-residues; equipping cyclic peptides with a-residues and g-residues; or incorporating e-amino acids into the cyclic peptide to name a few methods. However, often, the amino acids in the cyclic peptide sequence alternate between a D- and L- configuration. Often, the cyclic peptides are b-sheet forming cyclic peptides. A composition comprising compounds with a range of different cyclic peptides are envisaged. However, it is particularly preferred that the cyclic peptide comprises a mixture of leucine, lysine, terpines and asparate. Usually the cyclic peptide will be of less than 20 amino acids in length and more usually less than 16 amino acids in length. Usually, the cyclic peptide will have a length in the range of 6 to 12 amino acids, more often in the range of 7 to 9 amino acids and often, the cyclic peptide comprises 8 amino acids. Typically, the cyclic peptide is selected from: cyclo(L-Trp-D-Leu- L-Lys-D-Leu)2 or cyclo(L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu-L-Lys-D-Leu) wherein one or more of the "Trp" moieties can be replaced with an "Asp" moiety.

The nanotubes that compounds of the invention assembly into are not particularly restricted in length or width. However, the nanotubes are usually of a length of less than lOOnm, more often in the range lOnm to 50nm, more often in the range of 15nm to 30nm. Similarly, the nanotubes are usually of a width of less than lOOnm, more often in the range lOnm to 50nm, more often in the range of 15nm to 30nm.

There is provided in another embodiment of the invention, a composition comprising the compound according to the first aspect of the invention and one or more drugs. Typically, the one or more drugs are metallic or organometallic drugs. Without being bound by theory, it is believe that the "Ar" group is particularly effective at binding to metallic or organometallic drugs.

There is no particular restriction on the choice of metallic or organometallic drugs used in the invention but it is often the case that the metallic or organometallic drugs are those comprising platinum, iridium, rhodium, ruthenium, osmium or combinations thereof. More typically, the metallic or organometallic drugs is selected from organo-iridium compounds, organo-platinum compounds or combinations thereof.

There is also provided, in a second aspect of the invention, a method of preparing the compound according to the first aspect of the invention, the method comprising the steps of: i) polymerising at least one monomer according to Formula I in the presence of a chain transfer agent to form a polymer; ii) reacting said polymer with a cyclic peptide.

Whilst there is no particular restriction on the choice of chain transfer agent used in the process, the chain transfer agent typically comprises a reactive carboxylic acid moiety. Often, the chain transfer agent is (4-cyanopentanoic acid)yl ethyl trithiocarbonate (CPAETC).

There is provided in a third aspect of the invention, a compound according to the first aspect of the invention for use in therapy. Often, the compound according to the first aspect of the invention is used in drug delivery. Typically, the composition of the invention (comprising the compound according to the first aspect of the invention and one or more drugs) is for use in the treatment of cancer. Typical cancers include: ovarian cancer, lung cancer, testicular cancer, stomach cancer leukaemia, melanoma, prostate cancer, colon cancer, breast cancer, or combinations thereof.

There is provided in a fourth aspect of the invention, a method of treating cancer comprising the steps of administering the composition of the invention (comprising the compound according to the first aspect of the invention and one or more drugs) to a patient.

DESCRIPTION OF THE INVENTION

The present invention provides a novel nanoparticle drug delivery system that is able to improve the pharmacokinetic properties, the cellular selectivity and the cellular uptake of pharmacologic drugs. More specifically, the present invention provides a self-assembling drug delivery system comprising a metallic or organo-metallic drug complexed to a self-assembling cyclic peptide-polymer conjugate.

The invention provides, as a further aspect:

1. A cell-penetrating copolymer comprising the free radical polymerization product of the pyridine-functional monomer of formula (iii) wherein the linker R is selected from alykyl, aryl, amine, amide, carbonate, ester, ether, imide, sulfide, disulfide, sulfone, sulfoxide or ureide.

formula (iii) - pyridine functional monomer

2. A cell-penetrating self-assembling nanoparticle comprising :

a. a cyclic peptide core

b. at least 1 polymer arm comprising the polymerization product of the monomer of formula (iii)

3. A cell-penetrating self-assembling nanoparticle according to 2 where the pyridine functional monomer is (3-(pyridine-4-ylmethyl)ureido)ethyl)methacrylate (PUEMA)

Formula (iv) - (3-(pyridine-4-ylmethyl)ureido)ethyl)methacrylate (PUEMA)

The invention provides, as a another aspect:

4. A self-assembling nanoparticle drug delivery system comprising a copolymer produced as the result of polymerizing a pyridine-functional monomer of formula (iii), wherein the linker R is selected from alykyl, aryl, amine, amide, carbonate, ester, ether, imide, sulfide, disulfide, sulfone, sulfoxide or ureide.

5. A self-assembling nanoparticle drug delivery system according to 4 wherein the copolymer comprises a copolymer of (i) and a hydrophilic comonomer

6. A self-assembling nanoparticle drug delivery system according to 4 wherein the hydrophilic comonomer is a hydroxyl-functional monomer, selected from hydroxypropyl methacrylate, 2-hydroxyethyl methacrylate, 4-hydroxybutyl methacrylate or glycerol monomethacrylate

7. A self-assembling nanoparticle drug delivery system comprising :

a cyclic peptide core

b. at least one polymer arm or arms comprising copolymers according to 4 - 6

8. A self-assembling drug delivery system according to 4 and 7 which further comprises a complexed drug molecule

9. A self-assembling drug delivery system according to 8 wherein the conjugated drug is a metallic or organometallic drug

10. A self-assembling drug delivery system according to 8 and 9 wherein the metallic or organometallic drug is selected from organo-iridium compounds or organo-platinum compounds

11. A self-assembling drug delivery system according to 8 wherein the complexed drug is an anti- cancer drug 12. A self-assembling drug delivery according to any of 4-11 wherein the pyridine functional monomer is (3-(pyridine-4-ylmethyl)ureido)ethyl)methacrylate (PUEMA)

The invention provides, as a still further aspect:

13. A method of preparing a self-assembling nanoparticle drug delivery system, the method comprising the following steps:

c. Copolymerizing a pyridine-functional monomer of formula (iii) with a hydrophilic comonomer in the presence of a chain transfer agent, affording a biocompatible polymer with a reactive end group

d. Reacting the biocompatible polymer with a reactive end group with a cyclic peptide to form a conjugate with a cyclic peptide core and at least 1 polymer arms

14. A method according to 13 wherein the chain transfer agent is a RAFT agent

15. A method according to 13 where in the reactive end group of the chain transfer agent and therefore the resulting biocompatible polymer is a carboxylic acid

16. A method according to 13 and 14 wherein the chain transfer agent is (4-cyano pentanoic acid)yl ethyl trithiocarbonate (CPAETC)

17. A method according to 13 wherein the hydrophilic comonomer is selected from hydroxypropyl methacrylate, 2-hydroxyethyl methacrylate, 4-hydroxybutyl methacrylate or glycerol monomethacrylate

DESCRIPTION OF FIGURES

SCHEME 1 discloses synthesis of 2-(3-(Pyridin-4-ylmethyl)ureido)ethyl)methacrylate (PUEMA) monomer.

SCHEME 2 discloses synthesis of the HPMA-PUEMA copolymers and cyclopeptide-(HPMA- PUEMA)2 conjugates. Conjugate 2: CP-(pHPMA-co-PUEMA)2 and polymer 3: pHPMA-co- PUEMA. (i) HPMA, PUEMA, VA 044, DMSO/H20. (ii) cyclo(D-Leu-Lys-D-Leu-Trp)2, HTBU, NMM, DMSO.

SCHEME 3 discloses synthesis of drug-loaded compounds. Complexation of organoiridium complexes 4a and 4b to conjugate 2 and polymer 3. SCHEME 4 discloses the deprotection of the dye conjugated cyclic peptide.

SCHEME 5 discloses the conjugation of pDMA to the dye conjugated cyclic peptide.

SCHEME 6 discloses the synthesis of deprotected cyclic peptide H2N-CP-SH (3).

FIGURE 1 discloses characterising drug attachment to CP-polymer conjugates. 1H NMR characterization of the attachment of complex 4a onto conjugate 2, affording conjugate 2a.

FIGURE 2 discloses characterization of supramolecular structures. A) shows static light scattering profile of conjugate 2a in solution in PBS at different concentrations. B) shows small angle neutron scattering profile of conjugate 2a (orange dots) and its fit using a cylindrical micelle model (black line).

FIGURE 3 discloses viability of A2780 cells in presence of the drug-free compounds.

FIGURE 4 discloses antiproliferative activity of the compounds (continuous: conjugate, dashed : polymer) in A) A2780 B) PC3 and C) MDA cells, and cellular fluorescence intensity associated with rhodamine as determined by flow cytometry after incubation of the compounds for 3h at 4 °C, 3h at 37 °C and 24h at 37 °C in D) A2780 E) PC3 and F) MDA cells. Data represents geometric mean of fluorescence ± SD for two independent experiments done in triplicates: *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 5 discloses confocal images of PC3 human prostate carcinoma cells treated with rhodamine-labelled conjugates C5 for 24 h at 37 °C at a concentration of 20 pM. Lysosomes were stained using Lysotracker ® Green DND-26. Scale bar 20 pm.

FIGURE 6 discloses shows antiproliferative activity in A2780 cells. IC50 values for free organoiridium complexes, drug-bearing polymers, and drug-bearing conjugates using Ir-Cp* (dark) and Ir-Cpxph (light) as the drug.

FIGURE 7 discloses a comparison of antiproliferative activity between healthy and cancerous cells. A) shows antiproliferative activity of free drug Ir-Cpxph, drug-bearing polymer 3b and drug-bearing conjugate 2b in A2780 (cancer, dark) and HOF (healthy, light) ovarian cells. B) shows selectivity index of the Ir-Cpxph compounds, determined between A2780 and HOF.

FIGURE 8 discloses the cellular accumulation of Iridium. Iridium accumulated in A2780 cells after 24h of exposure to the free drug Ir-Cpxph (orange squares), the drug bearing polymer 3b (green diamonds) and the drug bearing conjugate 2b (purple circles) at equipotent IC50 conditions.

FIGURE 9 discloses an investigation of mechanism of cellular entry. A) shows iridium content of the membrane, cytosol, cytoskeleton and nucleus fractions of A2780 cells after 24 hours of exposure to the Ir-Cpxph compounds at equipotent IC50 concentrations. B) shows cellular accumulation of Ir after 4 h of exposure to the Ir-Cpxph compounds at equipotent IC50 concentrations at 4°C and 37°C.

FIGURE 10 discloses Plasma concentration versus time profiles of ¹⁴C- la belled polymer P4* (orange squares) and conjugate C3* (purple circles) following intravenous administration to rats at 12 mg/kg (mean ± SD, n = 4-5 rats).

FIGURE 11 discloses Distribution of ¹⁴C in organs, 24h after intravenous administration of conjugate (purple) and polymer (orange) at 12 mg/kg (mean ± SD, n = 4-5 rats).

FIGURE 12 discloses the ^XH NMR spectrum of linear peptide ( 1) (400 MHz, CDCI3).

FIGURE 13 discloses the ^XH NMR spectrum of protected cyclic peptide (2) (400 MHz, TFA-d).

FIGURE 14 discloses the ^XH NMR spectrum of the deprotected cyclic peptide (3) (400 MHz, TFA-d).

EXAMPLES

Example 1 : Synthesis and characterization of self-assembling cell-penetrating nanotubes and non-self assembling controls

The well-studied monomer building block A/-(2-hydroxypropyl)methacrylamide (HPMA) was chosen as the main monomer for designing the polymeric drug used in this work. In order to provide a binding site for the ligation of anticancer iridium complexes, pyridine was chosen as the binding ligand, since it can readily replace the chloride ligand present on the selected organoiridium precursor. Moreover, organoiridium pyridine complexes themselves exhibit good anticancer activity.²³ 2-(3-(Pyridin-4-ylmethyl)ureido)ethyl)methacrylate (PUEMA) was chosen as an exemplar monomer for the incorporation of pyridine functionality. Synthesis of 2-(3-(Pyridin-4-ylmethyllureidolethyllmethacrylate (PUEMA1 monomer (Scheme !)

2-Isocyanatoethyl methacrylate (2.2 g, 14.15 mmol) and 4-aminomethyl pyridine (1.53 g, 1 eq., 14.15 mmol) were mixed in dry DCM (10 mL) and left to stir at room temperature for 10 min. The solvent was evaporated under reduced pressure and PUEMA was collected as a white powder. Yield : 95% (3.53 g). ^-NMR (CDCI3, 300 MHz, ppm) : d = 8.52 (d, 2H, CH- N- CH pyridine), 7.19 (d, 2H, CH-C-CH pyridine), 6.09 (s, 1H, CH vinyl), 5.59 (s, 1H, CH vinyl), 5.13 (broad t, 1H, N H urea), 4.97 (broad t, 1H, N H urea), 4.38 (d, 2H, NH-C/-/2-pyridine), 4.25 (t, 2H, O -CH2), 3.53 (q, 2H, 0-CH2 -CH2), 1.93 (s, 3H, CH3). ¹³C-DEPT-NMR (CDCI3, 75 MHz, ppm) : 166.9, 157.5, 149.2, 148.3, 135.3, 125.5, 121.4, 63.4, 42.4, 39.0, 17.6. FTIR: (v, cm ¹): 3313 (N-H stretch, urea), 1720 (C=0 stretch, methacrylate), 1623 (C=C stretch, alkene), 1585 (C=0 stretch, urea). MS (ESI): [M+Na]⁺ calculated : 286.1, found : 285.9.

Synthesis of the HPMA-PUEMA copolymers and cvcloDeDtide-(HPMA-PUEMA12 conjugates

PUEMA-HPMA copolymers were synthesised using reversible addition-fragmentation chain transfer (RAFT) polymerization (Scheme 2).^24-25 Briefly, Chain transfer agent (CTA, here CPAETC or E(CPAETC)₂), monomers (HPMA, PUEMA, RhMA), initiator (VA 044) and solvent (70/30 DMSO/H20) were introduced into a flask equipped with a magnetic stirrer and sealed with a rubber septum (see Table SI for detailed conditions). The solution was degassed by bubbling nitrogen through it for 15 min, and then put in an oil bath at 44°C for the indicated time. Conversions were determined by 1H NMR.

Kinetic measurements of the copolymerization using CPAETC showed that PUEMA was consumed significantly faster than HPMA, indicating a higher reactivity. The direct consequence of this observation is that the functional monomer tends to be incorporated first, meaning that most of the pyridine ligands for metallodrug attachment will ultimately be found towards the s-chain end of the polymer.

The copolymer 1 was attached to the cyclic peptide by reacting the amine groups present on the cyclic peptide with the carboxylic acid end-group of 1, using O-(benzotriazol-l-yl)- /V^/V^/V'-tetramethyluronium hexafluorophosphate (HBTU) as a coupling reagent and the cyclic peptide-polymer conjugate 2 was purified by dialysis. Interestingly, since the attachment occurs at the s-chain end of the polymer, the pyridine units used for organoiridium attachment are located on average close to the cyclic peptide core, allowing the HPMA-richer shell to provide shielding of the drug from the environment. A non-self-assembling equivalent of the conjugate 2 was synthesized using a bifunctional CTA (E(CPAETC)2). The final compounds were well defined, with dispersities of 1.19 and 1.12 for the conjugate 2 and the polymer 3, respectively (Table 1).

Table 1: Summary of polymers used in this work.

Mn, th^a Mn, GPC^b _B[J

Entry Material

(g.mol ¹) (g.mol ¹)

Ϊ p( H PM A51 -co- PU EM A3.5) 8400 14 800 1.16

2 CP-(p(HPMA51-co-PUEMA3.5))2 17800 29200 1.19

3 pHPMA93-co-PUEMA7 15700 21400 1.12

³ Determined by ¹H NMR. ^b Determined by SEC using DMF (0.1 % LiBr) as eluent, calibrated with pMMA standards.

Complexation of oraanoiridium anticancer drugs

The attachment of the selected iridium complexes to the polymer 3 and cyclic peptide- polymer conjugate 2 was achieved following a ligand exchange procedure previously used to synthesize pyridine analogues of the chloride-containing drugs.²³ The chloride ligand of the iridium complexes 4a and 4b was first removed using silver nitrate, followed by complexation to the pyridine units of the polymer chains (Scheme 3). The complexes used in this work were the [(Cp*)Ir(phpy)CI] (a bbreviated as Ir-Cp*, 4a), which contains pentamethylcyclopentadienyl (Cp*) and C,N- chelated phenylpyridine (phpy) as ligands,²⁶ as well as the more hydrophobic [(Cp^xp^h)Ir(phpy)CI] (Ir-Cp^xp^h, 4b), in which the Cp* is replaced by an extended phenyltetramethylcyclopentadienyl (Cp^xp^h) ligand.²⁷

An excess of the iridium complex 4 (3 mol equiv per pyridine site) was used to maximise the drug loading onto the polymer and peptide-polymer conjugate. After purification by size exclusion chromatography, the drug-bearing compounds (conjugates 2a and 2b, and polymers 3a and 3b) were characterized by NMR spectroscopy (Figure 1). The shifts of characteristic peaks of the complex and of the pyridine units as well as the broadening of the signals associated with the complex clearly indicate full complexation of the drug to the polymer.

Characterization of supramolecular nanotubes In order to confirm the self-assembly of the conjugates into tubular structures, small angle neutron scattering (SANS) and static light scattering (SLS) measurements were performed on the drug-bearing conjugate 2a (Figure 2) in solution in deuterated PBS.

The SANS data were fitted with a cylindrical micelle model (Figure 2B), which accounts for both the overall elongated shape provided by the self-assembled cyclic peptide core (characteristic q ¹ dependency at low q values: cylinder form factor) and the polymer arms (Gaussian chain form factor at high q values).²⁸ By fitting geometrical parameters, reasonable values were obtained using a radius of 5 A for the peptide core, in accordance with previously reported results (see Supplementary Information).⁸'²⁹ However, the maximum length of these tubes cannot be fully determined by these SANS measurements, as the scattering intensity is still increasing at the lowest measured q values and does not show the formation of a plateau which is indicative of a limited length. Static light scattering (SLS) measurements were then carried out since this technique allows access to a larger window of observation (Figure 2A). SLS experiments showed that the molecular weight of the assemblies was not affected by the concentration of the solution (within the tested range) and this molecular weight was determined to be 9.74.10⁵ ± 0.37.10⁵ g.mol ¹ for the drug-bearing conjugate. Using the molecular weight of the unimer and the previously reported distance between adjacent peptides,⁷'²⁹'³³ the average length of the objects can be determined as 21.8 ± 0.9 nm, corresponding to 46 assembled conjugates (see Supplementary Information).

Example 2 : cellular compatibility of self-assembling cell-penetrating nanotubes and non-self assembling controls in the absence of oraanoiridium anticancer compounds

The self-assembling cell-penetrating nanotubes and non-self-assembling controls were tested for cellular compatibility using a growth inhibition assay with human A2780 ovarian carcinoma cells. Human A2780 ovarian carcinoma cells were obtained from the European Collection of Cell Cultures (ECACC) used between passages 5 and 18 and were grown in Roswell Park Memorial Institute medium (RPMI-1640) or Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% v/v of foetal calf serum, 1% v/v of 2 mM glutamine and 1% v/v penicillin/streptomycin. HOF human ovarian fibroblasts were obtained from ScienCell Research Laboratories, and maintained in fibroblast medium supplemented with 2% v/v of foetal calf serum, 1% v/v of penicillin/streptomycin and 1% v/v of growth factor serum. They were grown as adherent monolayers at 37°C in a 5% C02 humidified atmosphere and passaged at approximately 70-80% confluence.

Growth Inhibition Assay Briefly, 5000 cells were seeded per well in 96-well plates. The cells were pre-incubated in the corresponding drug-free media at 37°C for 48 h before adding different concentrations of the compounds to be tested. The polymer exposure period was 24 h. After this, supernatants were removed by suction and each well was washed with PBS. A further 72 h was allowed for the cells to recover in drug-free medium at 37°C. The SRB assay was used to determine cell viability. Absorbance measurements of the solubilised dye (on a BioRad iMark microplate reader using a 470 nm filter) allowed the determination of viable treated cells compared to untreated controls. IC50 values (concentrations which caused 50% of cell growth inhibition), were determined as duplicates of triplicates in two independent sets of experiments and their standard deviations were calculated. The drug-free control samples (polymer alone and cyclic peptide-polymer conjugates) were non-toxic in the tested range of concentrations (Figure 3).

Example 3 : Further cellular compatibility studies of self-assembling cell-penetrating nanotubes and non-self assembling

In view of the above data, a further self-assembling PUEMA-containing conjugate C3 using a cyclo(D-Leu-Lys-D-Leu-Trp)2 cyclopeptide core was prepared as a model for further in vitro, pharmacokinetic and biodistribution studies. A non-self-assembling polymeric equivalent P3 to the conjugate, described above, was used as a control.

Mn, th^a Mn, GPC^b

Entry Material eb

(g.mol ¹) (g.mol ^_1)

C3 CP-(p(HPMA₅₅-co-PUEMA₃.5))2 19200 24600 1.18

P3 pHPMAgs-co-PUEMAy 15700 21400 1.12

The biocompatibility of the compounds was tested in vitro on three cell lines (A2780 human ovarian carcinoma, PC3 human prostate carcinoma and MDA-MB-231 breast cancer) by performing cell growth inhibition assays for 72h. In all three cell lines, incubation with up to 500 pg/mL of the compounds did not result in any noticeable reduction of the cell viability (Figure 4, A-C).

The amount of compound associated with the cells was then quantified using flow cytometry ( Figure 4, D-F). A rhodamine monomer was copolymerised with HPMA and PUEMA following similar procedures as the materials described above (see Supplementary Information), to afford rhodamine-labelled conjugate C5 and polymer P6 (Figure S6). Corrections factors were used to enable comparison between the two compounds (see Supplementary Information, Figure S7 and Table S7). Cells were incubated in presence of the compounds for 3h and 24h at 37°C. In both cases and in all three cell lines, the polymer control associated significantly less than the conjugate (p < 0.0001). For example in A2780, the amount of conjugate C5 measured in the cells was nearly double that of polymer P6 after 3h; the discrepancy increased to 3.5 times more conjugate after 24h incubation. We attribute this result to the difference in size and aspect ratio between the two compounds, given the fact that the conjugates self-assemble in these conditions to form a cylindrical assembly with an average number of aggregation of 34, whereas the polymer P6 remains as a single unit. Particle shape³⁴ and size^{35 37} are thought to play a non-negligible role in cellular uptake, with larger particles exhibiting increased uptake up to a certain size, above which the uptake generally decreases. Depending on the study, percentage of uptake tends to peak between 20 and 100 nm, with particles with a diameter of either less than 10 nm or more than 100 nm entering the cells less than those of intermediate size. This effect is found across particles of very different nature, including coated iron oxide,³⁵ silica³⁶ and polymeric³⁷ nanoparticles. The present results are in line with these findings, with 16 nm-long nanotubes entering the cells to a higher extent than the single polymer chains.

It is also interesting to note that cellular association increases with time, indicating uptake occurs to a higher extent than excretion. In the case of the conjugates, a 3-fold increase in fluorescence in MDA cells was observed when varying the incubation time from 3h to 24h. Similar increases were found in other cell lines, with 3.8x in PC3 and 3.9x in A2780. This effect was also observed for the polymer, although to a lesser extent: increases in fluorescence between 3h and 24h incubation of 2.4x in A2780, 1.6x in PC3 and 2. Ox in MDA were recorded. In summary, these results indicate that the compounds accumulate in the cells over time (uptake > exocytose), which is commonly observed for nanosized objects (ref).^{38 39} In order to probe whether the mechanism of internalisation was energy-dependent, the experiment was also performed at 4°C, temperature at which these pathways are blocked. In all three cell lines, both compounds showed no accumulation after 3h in these conditions, indicating that the mechanism of cellular entry relies on endocytosis or other energy- dependent pathways.

Intracellular localisation of the conjugate was confirmed by confocal imaging, using the rhodamine-labelled compound C5 (Figure 5). Following PC3 cells incubation with the conjugate at 20 pM for 24h, rhodamine staining inside the cells confirmed that the compound was readily taken up by the cells and not simplyassociated with the membrane. Lysotracker ® green was added together with the conjugate to assess organelle localisation. The merged images of the red and green channels clearly demonstrate a noticeable amount of colour coincidence of the conjugates with the lysosomal compartments, which is in agreement with the flow cytometry data indicating energy-dependent entry pathways.

Example 4: Anticancer activity and cellular accumulation of self-assembling cell- penetratina nanotubes and non-self assembling controls after complexina of oraanoiridium anti-cancer compounds

The antiproliferative activity of the self-assembling cell-penetrating nanotubes and non-self assembling compounds controls with conjugated organoiridum compounds was determined for A2780 human ovarian cancer cells using the same assay as above. IC50 values (concentrations at which 50% of cell growth is inhibited) are given Figure 6.

Whilst the drug-free control samples (polymer alone and cyclic peptide-polymer conjugates) were nontoxic, the organoiridium-containing samples showed high activity. For both drugs, the IC50 of the loaded polymers was slightly higher (however still in the same order of magnitude) than that of the free drug : 1.90 ± 0.22 pM for the polymer 3a compared to 1.15 ± 0.04 pM for Ir-Cp*, and 1.80 ± 0.09 pM for the polymer 3b compared to 0.95 ± 0.03 pM for Ir-Cp^xp^h. For Ir-Cp*, no major difference was observed between the polymer and the conjugate (1.90 ± 0.22 pM for the polymer 3a compared to 1.70 ± 0.03 pM for the conjugate 2a). However the IC50 of the conjugate 2b (0.61 ± 0.02 pM) in A2780 was 3x lower than that of the drug-loaded polymer 3b (1.8 ± 0.09 pM). This substantial increase in activity between the polymer and the nanotubes suggests that the self-assembly has a noticeable impact on the behaviour of the carrier. Furthermore, the conjugate 2b was twice as potent as the free drug Ir-Cp^xp^h. For all the studied compounds the IC50 values were lower for Ir-Cp^xp^h, which is a more hydrophobic complex than for Ir-Cp*. This result shows the same trend as previously reported data for the complexes themselves^{26 27} (the present values are slightly different due to the updated protocol involving confirmation of Ir concentrations by ICP-OES measurements). These organoiridium complexes have not been previously conjugated to delivery vectors, but it is clear from previous studies that increasing the hydrophobicity of the cyclopentadienyl ligand enhances their antiproliferative activity.²²'²⁷'³⁰ The present report suggests that this is also the case when the complexes are conjugated to polymers and cyclic peptide-polymer conjugates. For this reason, further studies focussed on the more potent compounds bearing Ir-Cp^xp^h.

The antiproliferative activity of the three compounds (Ir-Cp^xp^h as free drug, loaded onto the polymer (3b), and the conjugate (2b)) was then determined against human ovarian fibroblasts (HOF), a model for healthy, non-cancerous cells (Figure 7), and compared to that against A2780 ovarian cancer cells.

All compounds are less toxic in normal HOF cells than in A2780 (Figure 4A). The selectivity index (SI, the ratio between the IC50 in HOF and the IC50 in A2780) is 4.7 and 3.3 for the free drug and the polymer, respectively (Figure 4B). Interestingly, it is significantly higher for the conjugate (10.7), suggesting a considerable degree of selectivity towards cancer cells, highlighting an advantage of using these new materials as delivery vectors.

The increased activity of the conjugate 2b compared to the polymer 3b and the free drug in A2780 cells may be related either to enhanced cellular accumulation or to a more efficient mode of action, for example through a different partitioning of the drug amongst the cell organelles. The possibility of enhanced accumulation (the balance of the uptake and efflux equilibrium), was investigated by exposing A2780 cells to the Ir-Cp^xp^h compounds (free drug, polymer and conjugate) at their respective IC50 values. At regular intervals over 24h, cells were collected and digested in nitric acid to determine the amount of iridium accumulated using inductively coupled plasma mass spectrometry (ICP-MS) (Figure 8).

Figure 5 shows that the kinetics of uptake are different for the three compounds: the maximum amount of iridium accumulated is reached after only 2 h in the case of the conjugate 2b, after which it remains the same, while for the polymer 3b, the amount is still increasing after 24 h. In the case of the free drug, the amount of iridium peaks at 4 h, before decreasing slightly. Such cellular efflux is common for organometallic complexes.^{31 32} These differences in the rate and profile of uptake suggest that the conjugate interacts differently with the cells. After 24 h of exposure to the free drug, the drug-bearing polymer and the drug-bearing conjugate under equipotent conditions, each at their IC50 concentrations (0.95 pM, 1.80 pM and 0.61 pM, respectively), 21.6 ± 0.7 ng, 28.7 ± 1.6 ng and 9.3 ± 0.2 ng of iridium per million cells were accumulated, respectively. Taking the differences of the IC50 values into account, similar percentages of the total amount of iridium administered are retained : 7.7 ± 0.2 % of the initial amount was accumulated for the drug, 6.5 ± 0.4 % for the polymer and 6.5 ± 0.1 % in the case of the conjugate. These values are similar to those observed previously for organometallic drugs.²³'³²

The results suggest that the increased cytotoxicity of the conjugate 2b compared to the other compounds is not due to enhanced uptake of iridium by the cells, and are further supported by the amount of iridium accumulated after exposure under equimolar conditions. The cells were incubated in the presence of the three compounds at the lowest IC50 (0.61 pM). Under these conditions, similar amounts of iridium were accumulated ( 10.1 ± 0.1 ng per million cells for the free drug, 10.5 ± 0.2 ng for the polymer and 9.3 ± 0.8 ng for the conjugate). We conclude that the attachment of the drug to the polymer or the conjugate does not affect the extent of the accumulation of iridium in the cells, which suggests that the nanotubes rather exhibit a more effective mode of action, for example through a different partitioning of the drug amongst the cell organelles.

In order to evaluate this partitioning, equipotent uptake experiments were repeated and the cell pellets collected after 24 h. The iridium content of the membrane, cytosol, cytoskeleton and nucleus fractions was determined by fractionation of the cell compartments, and the results are shown in Figure 9A. The total amount of iridium in each fraction follows the trend previously observed for whole cells: the amount of iridium increases in the order conjugate < drug < polymer. The percentage of iridium in the membrane fraction increases slightly in the order drug (56 %) < polymer (65 %) ~ conjugate (69 %), which may indicate that the polymer- conjugated drugs favour an endocytosis-mediated pathway, since endosomes and lysosomes are collected in the membrane fraction. To confirm that the polymer and conjugate follow an energy-dependent mechanism, accumulation experiments were undertaken at 4°C, conditions known to block endocytosis processes.³³ At 4°C the free drug accumulated to an extent of 3.8 ± 0.2 ng Ir per 10⁶ cells, which corresponds to about 15% of the amount accumulated at 37°C, suggesting that energy-independent pathways (such as passive diffusion) play at least a partia l role in the cellular accumulation of the free drug, in accordance with previous reports.³² The polymer 3b accumulated to a lesser extent, with 1.9 ± 0.3 ng Ir per 10⁶ cells. In contrast the conjugate 2b did not accumulate significantly in these conditions, supporting the hypothesis that cell entry involves energy-dependent mechanisms.

Example 5 : Plasma pharmacokinetics and

n biodistribution self-

cell- penetratinq nanotubes and non-self-asser

q controls

In order to characterise the in vivo behaviour of our compounds, both the cyclic peptide- polymer conjugates and control polymer were radiolabelled, taking advantage of the hydroxyl groups present on pHPMA to attach ¹⁴C-ethanolamine (see details in Supplementary Information). The obtained compounds C3* and P4* were purified by size exclusion chromatography (SEC) and extensively dialysed to remove any radiolabel excess. Effective labelling was confirmed by scintillation counting of SEC fractions and HPLC analysis (see Supplementary Information, Figures S9 and S10). The radiolabelled polymer P4* and conjugate C3* were injected intravenously to male Sprague Dawley rats at 12 mg/kg and blood samples were taken at regular intervals for 24h to determine the plasma concentration versus time profiles (Figure 10). Non-compartmental pharmacokinetic parameters are summarized in Table 1. The initial volume of the central compartment (Vc) was close to blood volume, which is typical of IV injections. The non-assembling polymer P4* showed rapid elimination from systemic circulation, in accordance with previously reported results on HPMA copolymers.^{40 42} The elimination half-life of the nanotubes was only slightly longer than that of the polymer control, indicating a similar rate of elimination from the system after the distribution phase. However the total exposure in the case of the nanotubes was significantly higher than for the polymer ( p < 0.0001), as shown by the difference in the AUC (area under the curve), which was found to be more than three times higher for the conjugates. We attribute this discrepancy to the larger size of the nanotubes, which allows them to partially avoid immediate renal clearance. Their elongated shape could also The increased exposure is in agreement with the reduced clearance (3 ± 0.2 mL/h for the nanotubes vs 12 ± 0.4 miyh for the polymer) and reduced terminal volume of distribution (70 ± 2 mL for the nanotubes vs 225 ± 35 mL for the polymer). The observed volume of distribution of the nanotubes is lower than for small molecular weight linear polymers, but higher than reported values for PEGylated dendrimers (as low as 25 mL after 30 h),⁴³ stars (approximately 60 mL after 7 days)⁴⁴ or small brushes (60 mL after 24 h) ⁴⁵

Table 1: Calculated pharmacokinetic parameters and urine recovery after intravenous administration of conjuragte C3* and polymer P4* to rats at 12 mg/kg (mean ± SD, n = 4-5 rats). **p < 0.01, ****p < 0.0001.

Conjugate (C3*) Polymer (P4*) tl/2 (h) 16.1 ± 1.3 13.4 ± 2.0

AUC (Mg/mL.h) 1120 ± 62 331 ± 10****

Vc (mL) 15.0 ± 1.0 16.6 ± 1.0

Vd, (mL) 70 ± 2 225 ± 35**

Cl (mL/h) 3 ± 0.2 12 ± 0 4****

Urine (% dose) 62 ± 7 72 ± 8

The percentage of dose recovery in urine was high for both the polymer (72 ± 8 %) and the conjugates (62 ± 7 %), indicating that the majority of both compounds is ultimately excreted from the body. The molecular weight cut-off for renal filtration is generally estimated to be around 50 kDa,⁴⁶ which is well below the molecular weight of the nanotubes (estimated to be 615 kDa by SLS) but above the mass of the polymer and the unimers. Hence, this result suggests that the labelled compounds found in urine are fragments of the initial nanotubes, either degraded chemically (free radiolabel), or physically (unimeric conjugates or very short tubes). To understand the fate of both compounds after administration, here specifically to verify that they are both largely excreted from the body within 24h, their accumulation in major organs (liver, spleen, pancreas, kidneys, heart, lungs, brain) was quantified by measuring the residual ¹⁴C present in the tissues harvested 24h after IV injection. Figure 11 shows the percentage of injected ¹⁴C recovered in each organ. Levels of accumulation were very low across all examined organs, with the highest amount found in the liver (3.1 ± 0.4 % for the conjugate, 1.3 ± 0.3 % for the polymer). Such low levels of organ accumulation are typical of small molecular weight HPMA copolymers.⁴²

The very low organ uptake, together with the high urine excretion, and the intermediate value of Vd (lower than for a small molecular weight polymer but higher than for dendrimers or stars or small brushes) may indicate that the nanotubes are in fact constituted of a mixture of slowly disassembling structures. A more advanced study is required to fully elucidate the mechanism of clearance, but one hypothesis is that the initially assembled structures exhibit prolonged circulation (as evidenced by the higher exposure of the nanotubes compared to the non-assembling polymer) and the resulting unimeric conjugates are ultimately cleared out of the body without organ accumulation.

Example 6 pDMA polymerisation

For the synthesis of the pDMA homopolymer, PABTC (50.06 mg, 0.210 mmol, 1 eq.), DMA (1.041 g, 10.50 mmol, 50 eq.), benzyl methacrylate (1.05 mmol, 5 eq.), VA-044 azo initiator (1.49 mg, 4.60 pmol, 0.0219 eq.) and a 1 :4 co-solvent of 1,4-dioxane (0.421 mL)and deionised water (1.264 mL) respectively were all weighed into a vial with a magnetic stirrer and sealed with a rubber septum. The solution was mixed thoroughly and deoxygenated by bubbling nitrogen for ca. 10 min. The vial was then placed in an oil bath set at 70°C for 20 hours. Samples conversion, calculated from ^XH NMR, during the polymerisation were taken using a degassed syringe. After the polymerisation, the mixture was cooled and opened to air. ^XH NMR a nd GPC of these polymers were taken to determined, conversion and molecular weight. The solvent was evaporated using the aid of nitrogen flow, then the polymer was resuspended in dioxane and precipitated in hexane (repeat 3 times) and dried in a vacuum oven. The product was a yellow solid.

Yield = 88% ( 1.0483 g); ^XH NMR (CDCb, 300 MHz, ppm) : 0.89 (3H, H3C-CH2-RAFT agent), 1.08 (3H, H₃C-(C(0)-OH)-RAFT agent), 1.85- 1.46 (4H, H₃C-CH₂-CH₂-RAFT agent + 2H, - CH-CH2- polymer backbone), 2.62 (-CH-CH2- polymer backbone), 2.80-3.25 (6H, -N- (CH₃)₂). Mn = 5,000 g mol ¹, D = 1.13 (THF SEC, Agilent EasyVial PMMA and PS calibration). CP-(pDMA)2 - Control conjugate

CP-(pDMA)2 (13) : The cyclic peptide cyclo(D-Leu-Lys-D-Leu-Trp)2, CP, was synthesised using literature protocol. CP ( 15 mg, 13.88 pmol, 2.2 eq.) was dissolved in DMF (0.5 mL) with the aid of sonication. In a separate vial, pDMA ( 11) (0.174 g, 30.54 pmol, 2.2 eq .), HATU (0.0116 g, 30.54 pmol, 2.2 eq.) and DIPEA (0.0108 g, 83.28 pmol, 6 eq.) were dissolved in 0.5mL DMF and shaken for 30 minutes then added to the CP solution. The combined solution was shaken for 2 days at room temperature. The solvent was evaporated with the aid of a nitrogen flow. The product then purified using centrifuge dialysis tubes with a molecular weight cut-off of 10 kDa (Merck Millipore). The purification of the excess polymer can be monitored via SEC traces (Yield = 58% ( 101 mg).

Cyclic peptide-dye conjugates

Dye conjugation to the cyclic peptide

Cy3-CP-protected : The partially deprotected cyclic peptide was synthesised according literature protocol. This cyclic peptide was dissolved in 0.5 mL of DMF with the aid of sonication. N,N-Diisopropylethylamine (DIPEA) (0.0117 g, 90.7 pmol, 6 eq.) was added to the CP solution and mixed. Cyanine3 NHS ester (purchased from Lumiprobe GmbH) (O.Ol lg, 17.4 pmol, 1.15 eq.) was added to the CP solution and stirred for 3 days. The reaction was followed via Liquid Chromatography Mass Spectrometry (LC-MS). The purified peptides were characterized by mass spectrometry. Yield : 78% (20.6 mg).

Cy3-CP-deprotected : Boc groups were removed in using a deprotection solution of TFA/TIPS/H20 (18 : 1 : 1 vol, 5 mL). The dye conjugated Boc protected CP (20.632 g) was agitated for 3 hours in the deprotection solution, then triturated using ice-cold diethyl ether and washed twice more with ice-cold diethyl ether. The pink precipitate was collected and dried under vacuum. Yield : 94% (25.7 mg).

Conjugation of pDMA to the dye conjugated cyclic peptide

Cyclic peptide-dye conjugate (Cy3-CP-dep, 6) (6.2 mg, 4.17 pmol, 1 eq.) was dissolved in DMF (0.5 mL) with the aid of sonication. In a separate vial, pDMA (11) (51.2 mg, 9.16 pmol, 2.2 eq.), HATU (3.48 mg, 9.16 pmol, 2.2 eq.) and DIPEA (3.23 mg, 25.0 pmol, 6 eq.) were dissolved in 0.5mL DMF and shaken for 30 minutes then added to the CP solution. The combined solution was shaken for 3 days at room temperature. The solvent was evaporated with the aid of a nitrogen flow. The product then purified using centrifuge dialysis tubes with a molecular weight cut-off of 10 kDa (Merck Millipore). The purification of the excess polymer can be monitored via SEC traces. Yield = 8% (4.390 mg). Linear peptide ( 1), protected cyclic peptide (2), and deprotected cyclic peptide CP- NH₂ (3).

Linear peptide ( 1)

H₂N-L-Lys(Boc)-D-Leu-L-Glu(OtBu)-D-Leu-L-Trp(Boc)-D-Leu-L-Glu(OtBu)-D-Leu-COOH.

Fully protected linear octapeptide was prepared via solid phase peptide synthesis (SPPS) on a Prelude Automated Peptide SynthesizerTM (Protein Technologies Inc.) using 2-ch lorotrityl chloride resin as the solid support. The first Fmoc protected amino acid was coupled to the resin using DIPEA (4 eq.) in DCM, followed by capping of unreacted resin sites using a solution of MeOH : DIPEA: DCM (7: 1 :2, v/v/v). Deprotection of the Fmoc group of the amino acids was done using 20% piperidine in DMF. Subsequent amino acids were coupled using Fmoc-amino acids (5 eq.), HCTU (5 eq.) and NMM (10 eq.) in DMF. In the last step, the linear octapeptide was cleaved from the resin (while keeping protecting groups on) by a solution of 20 vol % l, l,l,3,3,3-hexafluoro-2-propanol (HFIP) in DCM.

Ή NMR in CDC (400 MHz, ppm) : d = 8.00 (m, 1H), 7.52-7.26 (m, 4H), 4.95 (m, 1H), 4.68-4.37 (m, 6H), 4.17 (m, 1H), 3.14 (m, 2H), 2.38-1.23 (m, 64H), 0.95-0.68 (m, 24H).

MS (ESI-ToF) (m/z) : [M + H]⁺ 1355.8 (calculated : 1355.8), found 1498.9., [M + Na]⁺ 1377.8 (calculated : 1377.8).

Protected cyclic peptide (2)

Linear peptide (420 mg, 0.310 mmol) was cyclized by stirring at room temperature for 5 days in the presence of 1.2 equivalents of DMTMM-BF₄ (122 mg, 0.372 mmol) in 100 mL DMF. The solution was then concentrated to 10 mL under vacuum and then precipitated with cold methanol to obtain a white powder as protected cyclic peptide 2 (yield : 176 mg).

Ή NMR in TFA-d (400 MHz, ppm) : d = 8.14 (m, 1H), 7.69-7.32 (m, 4H), 5.25 (m, 1H), 4.92-4.65 (m, 7H), 3.16 (m, 2H), 2.62- 1.07 (m, 64H), 1.02- 0.72 (m, 24H).

MS (ESI-ToF) (m/z) : [M + Na]⁺ 1359.8 (calculated : 1359.8).

Deprotected cyclic peptide CP-Nhh (3)

Removal of the -Boc and -tBu protecting groups was achieved by adding a mixture of trifluoroacetic acid (TFA, 5 mL), triisopropylsilane (TIPS, 0.28 mL) and water (0.28 mL) to the protected cyclic peptide 2 (176 mg) and stirring for 3 hours. The resulting solution was then precipitated in ice cold diethyl ether and wash twice with ice cold diethyl ether to give an off-white powder as the deprotected cyclic peptide CP-NH2 3 (yield : 118 mg).

Ή NMR in TFA-d (400 MHz, ppm) d = 8.16 (m, 1H), 7.71-7.22 (m, 5H), 5.31-4.63 (m, 8H), 3.40-3.13 (m, 4H), 2.65-2.48 (m, 4H), 2.31-1.40 (m, 22H), 1.06-0.61 (m, 24H). MS (ESI-ToF) (m/z) : [M + H]+ 1025.6 (calculated : 1025.6), [M + Na]+ 1047.5 (calculated : 1047.5).

Linear peptide ( 1), protected cyclic peptide (2), and deprotected cyclic peptide HhN-CP-SH (3).

Linear peptide ( 1)

H2N-L-Cys(Trt)-D-Leu-L-Trp(Boc)-D-Leu-L-Lys(Boc)-D-Leu-L-Trp(Boc)-D-Leu-COOH

MS (ESI-ToF) (m/z) : [M + H]⁺ 1616.9 (calculated : 1616.9).

Protected cyclic peptide (2)

Linear peptide (827 mg, 0.511 mmol) was cyclized by stirring at room temperature for 5 days in the presence of 1.2 equivalents of DMTMM-BF₄ (201 mg, 0.614 mmol) in 100 mL DMF. The solution was then concentrated to 10 mL under vacuum and then precipitated with cold methanol to obtain a white powder as protected cyclic peptide 2 (yield : 380 mg).

MS (ESI-ToF) (m/z) : [M + H]⁺ 1598.8 (calculated : 1598.8).

Deprotected cyclic peptide H2N-CP-SH (3)

Removal of the -Boc and -Trt protecting groups was achieved by adding a mixture of trifluoroacetic acid (TFA, 2 mL), triisopropylsilane (TIPS, 0.11 mL) and water (0.11 mL) to the protected cyclic peptide 2 (200 mg) and stirring for 3 hours. The resulting solution was then precipitated in ice cold diethyl ether and wash twice with ice cold diethyl ether to give an off-white powder as the deprotected cyclic peptide H2IM-CP-SH 3 (yield : 168 mg).

MS (ESI-ToF) (m/z) : [M + H]+ 1056.6 (calculated : 1056.6). a. Synthesis of pPEGA-PDS

PABTC-PDS (CTA) (42.5 mg, 0.104 mmol, 1 eq.), poly(ethylene glycol) methyl ether acrylate ( 1.0 g, 2.083 mol, 20 eq.), ACVA (initiator) (1.46 mg, 0.0521 mmol, 0.05 eq .) and 1,4-Dioxane ( 1.0 ml) were introduced into a vial equipped with a magnetic stirrer and sealed with a rubber septum. The solution was degassed with constant stream of nitrogen for 15 min, the flask was then put in an oil bath set at 70 °C. The polymerizations were stopped after 3.5 h by cooling the flask and opening it to air. The polymer was purified by precipitation in cold diethyl ether and dried under vacuum. b. Synthesis of conjugate HhN-CP-pPEGA

H2N-CP-SH (5 mg, 4.73*10^-6 mol) and pPEGA-PDS (85 mg, 9.47*10 ⁶ mol) were dissolved in 1 mL DMF. The reaction was left for 2 days at room temperature. Then the DMF solution was added dropwise into 20 mL methyl tert-butyl ether. The conjugate FhN-CP-pPEGA was isolated by centrifugation and dried under vacuum. c. Synthesis of p(DMA-co-Boc-Phe-EMA)-PDS

PABTC-PDS (CTA) (23.5 mg, 1 eq.), DMA (200 mg, 35 eq.), Boc-Phe-EMA (104.5 mg, 5 eq), V601 (initiator) ( 1.33 mg, 0.1 eq.) and 1,4-Dioxane (0.6 ml) were introduced into a vial equipped with a magnetic stirrer and sealed with a rubber septum. The solution was degassed with constant stream of nitrogen for 15 min, the flask was then put in an oil bath set at 80 °C. The polymerizations were stopped after 3 h by cooling the flask and opening it to air. The polymer was purified by precipitation in cold diethyl ether and dried under vacuum. d. Synthesis of p(DMA-co-Phe-EMA)-PDS

p(DMA-co-Boc-Phe-EAm)-PDS (150 mg) was dissolved in 5 mL DCM and 1 mL TFA was added afterwards. The deprotection process was left for 2 h. And the solution was dispersed into 45 mL cold diethyl ether to obtain off-white precipitates. The product was isolated by centrifugation and dried under vacuum. e. Synthesis of conjugate H2N-CP-p(DMA-co-Phe-EMA)

H2N-CP-SH ( 10 mg, 1 eq) and p(DMA-co-Phe-EMA)-PDS (87 mg, 1.5 eq) were dissolved in 2 mL DMF. The reaction was left for 2 days at room temperature. Then the DMF solution was added dropwise into 10 mL water and purified using centrifuge dialysis tube with a molecular weight cut-off of 10 k 6 times to remove the unreacted polymer. Finally the aqueous solution was freeze-dried to obtain a white powder as conjugate H2N-CP-p(DMA-co-Phe- EMA).

Example 7

Synthesis of the cyclic peptide (N3-CP-NH2) Synthesis of linear peptide: Solid phase synthesis of linear peptide H2N-L-Lys(N3-pent)-D- Leu-L-Trp(Boc)-D-Leu-L-Lys(Boc)-D-Leu-L-Trp(Boc)-D-Leu-COOH was performed on a 2- chlorotrityl resin (0.50 g, resin loading 1.1 mmol-g-l) in a 10 mL sinter-fitted syringe. The resin was allowed to swell for 30 minutes using anhydrous dichloromethane (DCM, 4 mL). After draining the DCM, a solution containing Fmoc-D-Leu-OH (2 eq., 0.39 g, 1.1 mmol), DIPEA (4 eq./amino acid, 0.57 g, 4.4 mmol) in DCM (2 mL) was bubbled with N2 for 15 min then added to the resin and agitated for 2 h at room temperature. Following draining of the solution, the resin was washed with a mixture of DCM / DIPEA / methanol (17 : 1 : 2, 3 x 4 mL) to cap any unreacted sites on the resin, then washed with DCM (3 x 4 mL), DMF (3 x 4 mL) and DCM (3 x 4 mL) once more, after which the resin was dried under reduced pressure. Loading content was determined by deprotecting a sample of the dried resin (5 mg) by agitating in 20 % piperidine in DMF ( 1 mL, 25 min). The resulting solution was diluted by a factor 100 with DMF and UV-Vis was used to correlate molarity with the absorption of the Fmoc group at l = 301 nm with e = 7800 M-l -cm- 1. The loaded resin (0.40 g, 0.29 mmol) was transferred to a sintered syringe, and swollen in DCM for 30 min. Following the draining of the DCM, the resin was washed with DMF, and the Fmoc groups were removed by addition of 20% piperidine in DMF (2 x ( 10 mL, 5 min)). After removal of the deprotecting solution, the resin was washed with DMF (3 x 4 mL), DCM (3 x 4 mL) and DMF (3 x 4 mL). The second added amino acid was Fmoc-L-lys(N3-pent). Therefore, the amino acid (1.5 eq., 0.43 mmol), HATU (1.5 eq., 0.43 mmol) and DIPEA (3 eq., 0.87 mmol) were dissolved in DMF (2 mL) and degassed with N2 for 15 min before adding it to the resin. The coupling reaction was allowed to proceed at ambient temperature for 16 h. For subsequent coupling reactions, solutions containing the Fmoc-amino acid (3 eq., 0.87 mmol), HBTU (3.1 eq., 0.9 mmol) and DIPEA (6 eq., 1.74 mmol) in DMF (2 mL) were prepared the same way and the time for the coupling reaction was reduced to 3 h. Deprotection and addition steps were repeated in between each coupling step to obtain the desired octapeptide. After completion of the amino acid coupling reactions and the removal of the final Fmoc protecting group using 20 % piperidine in DMF, the peptide was cleaved from the resin using a solution of 20% HFIP in DCM (3 x (8 mL, 10 min)). The resin was washed with DCM (3 x 4 mL) and the filtrate was concentrated under reduced pressure to yield the linear peptide as an off-white solid.

Cyclization of linear peptide: Linear peptide (400 mg, 0.26 mmol) was dissolved in DMF (90 mL) and bubbled with N2 for 20 min. DMTMM-BF4 (1.2 eq., 103 mg, 0.31 mmol) was dissolved in DMF ( 10 mL), bubbled with-. N2 for 20 min and added dropwise to the linear peptide solution. The mixture was stirred under an atmosphere of N2 for 5 days. The DMF solution was reduced to a volume of ~ 5 mL under reduced pressure, and methanol (50 mL) was added resulting in the precipitation of the cyclic peptide. After centrifugation and discarding the supernatant, the pellets were resuspended in methanol and centrifuged, again. The final pellets were resuspended in methanol, combined and the solvent was evaporated under reduced pressure to yield the Boc-protected cyclic peptide in the form of a white powder.

Deprotection of cyclic peptide: Boc-protected cyclic peptide (200 mg, 0.133 mmol) was treated with a cleavage cocktail consisting of TFA : triisopropylsilane : water (18 : 1 : 1 vol. %, 2 mL) for 2 h. The Boc-deprotected cyclic peptide was isolated from the cleavage cocktail by precipitation in ice-cold diethyl ether. After centrifugation the supernatant was discarded and the pellet was washed with diethyl ether and centrifuged 2-fold after which the solvent was evaporated under reduced pressure to yield a white powder. Any residual remaining carbonate adducts on the tryptophan were removed by heating the product to 40°C under vacuum.

Synthesis of the polymer

Poly(o-nitrobenzyl methacrylate) (NHS-pNBMA25)

N-hydroxylsuccinimide-containing CTA (27.2 mg, 7.5 x 10-5 mol, 1 eq.), o-nitrobenzyl methacrylate (0.5 g, 2.3 x 10-3 mol, 30 eq.), AIBN (initiator) (1.2 mg, 7.2 x 10-6 mol, 0.1 eq.) and DMF (2.26 ml) were introduced into a vial equipped with a magnetic stirrer and sealed with a rubber septum. The solution was degassed with constant stream of nitrogen for 10 min, the flask was then put in a thermostated oil bath set at 65°C. The polymerization was stopped after 12 h by cooling the flask and opening it to air. The conversion (82%) was determined by 1H-NMR (in CDCI3). Residual monomer and initiator were removed by repeated precipitation into diethyl ether and centrifugation. The supernatants were discarded and the product was dried under vacuum.

Poly(poly(ethylene glycol) methyl ether acrylate) (NHS-pPEGA27)

N-hydroxylsuccinimide-containing CTA (15.0 mg, 4.2 x 10-5 mol, 1 eq.), poly(ethylene glycol) methyl ether acrylate (1.0 g, 2.1 x 10-3 mol, 50 eq.), V601 (initiator) ( 1.0 mg, 4.2 x 10-6 mol, 0.1 eq.) and 1,4-Dioxane (1.0 ml) were introduced into a vial equipped with a magnetic stirrer and sealed with a rubber septum. The solution was degassed with constant stream of nitrogen for 10 min, the flask was then put in a thermostated oil bath set at 65°C. The polymerizations were stopped after 4 h by cooling the flask and opening it to air. The conversion (54%) was determined by 1H NMR. Residual monomer and initiator was removed by repeated precipitation into cold diethyl ether and centrifugation. The supernatants were discarded and the product was dried under vacuum. Poly(poly(ethylene glycol) methyl ether acrylate) (BCN-pPEGA27)

NHS-pPEGA27 (500 mg, 3.7 x 10-5 mol, 1 eq.) was further reacted with extra strained alkyne N-[(lR,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-l,8-diamino-3,6- dioxaoctane (BCN-NH2) (0.024 mg, 7.4 x 10-5 mol, 2 eq.) in DMF with catalytic TEA overnight. The reaction solution was precipitated in cold diethyl ether. The supernatants were discarded and the product was dried under vacuum to afford BCN functionalized PPEGA27.

Synthesis of the Cyclic peptide polymer conjugates (pNBMA25-CP-pPEGA27)

Synthesis of pNBMA25-CP

Poly (o-nitrobenzyl methacrylate) (NHS-pNBMA25) (53.0 mg of solution, 9.0 x 10-6 mol, 2 eq.) was mixed with a stock solution (19.7 mg/ml in DMF) of the cyclic peptide (0.3 ml of solution, 6.0 mg, 4.5 x 10 6 mol, 1 eq.). To ensure the amino groups on the CP are deprotonated, 4-Methylmorpholine (40 mg/ml solution in DMF, 0.07 ml of solution, 2.8 mg, 2.8 x 10-5 mol, 6 eq.) was added and the reaction was stirred for 24 h at room temperature. The product was purified by precipitation and centrifugation in a mixture of Diethyl ether and Acetone (ratio : 2 : 5).

Synthesis of pNBMA25-CP-pPEGA27

pNBMA25-CP (60.0 mg of solution, 8.6 x 10-6 mol, 1 eq.) was mixed with BCN-pPEGA27 (171 mg, 1.3 x 10 5 mol, 1.5 eq .) and keep the reaction overnight in room temperature. The product was purified by precipitation and centrifugation in methyl tertiary-butyl ether, and dried in vacuum oven to afford the conjugates.

Preparation of DOX-Loading tubisomes

pNBMA25-CP-pPEGA27 (10.0 mg) was resolved in 1.0 mL DMF, and DOX-HCI (5.0 mg) was resolved in 1.0 mL water in the presence of TEA. Then, the water solution was dropwisely added into the DMF solution. Then 8 mL water was slowly added into the mixture solution, after stirring for 24 h in the dark environment, the resulting solution was dialyzed in the deionized water until the unencapsulated DOX was totally removed .

References

(1) Wang, A. Z. ; Langer, R. ; Farokhzad, 0. C., Nanoparticle Delivery of Cancer Drugs. Annu, Rev. Med. 2012, 63 (1), 185- 198.

(2) Petros, R. A. ; DeSimone, J. M., Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discovery 2010, 9 (8), 615-627. (3) Lammers, T. ; Kiessling, F. ; Hennink, W. E.; Storm, G., Drug targeting to tumors: Principles, pitfalls and (pre-) clinical progress. J. Controlled Release 2012, 161 (2), 175- 187.

(4) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K., Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J. Controlled Release 2000, 65 (1-2), 271-284.

(5) Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P., Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery. Chem. Soc.

Rev. 2013, 42 (3), 1147-1235.

(6) Chou, L. Y. T. ; Ming, K. ; Chan, W. C. W., Strategies for the intracellular delivery of nanoparticles. Chem. Soc. Rev. 2011, 40 (1), 233-245.

(7) Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.; Khazanovich, N., Self assembling organic nanotubes based on a cyclic peptide architecture. Nature 1993, 366 (6453), 324-327.

(8) Chapman, R.; Danial, M.; Koh, M. L.; Jolliffe, K. A.; Perrier, S., Design and properties of functional nanotubes from the self-assembly of cyclic peptide templates. Chem. Soc. Rev. 2012, 41 (18), 6023-6041.

(9) Geng, Y.; Dalhaimer, P.; Cai, S. S.; Tsai, R.; Tewari, M.; Minko, T.; Discher, D. E., Shape effects of filaments versus spherical particles in flow and drug delivery. Nat. Nanotechnol. 2007, 2 (4), 249-255.

(10) Truong, N. P.; Quinn, J. F. ; Whittaker, M. R. ; Davis, T. P., Polymeric filomicelles and nanoworms: two decades of synthesis and application. Polym. Chem. 2016, 7 (26), 4295- 4312.

(11) Muraoka, T.; Koh, C. Y. ; Cui, H. G. ; Stupp, S. L, Light-Triggered Bioactivity in Three Dimensions. Angew. Chem. Int. Ed. 2009, 48 (32), 5946-5949.

(12) Mandal, S.; Eksteen-Akeroyd, Z. H. ; Jacobs, M. J. ; Hammink, R. ; Koepf, M . ; Lambeck, A. J. A.; van Hest, J. C. M. ; Wilson, C. J.; Blank, K. ; Figdor, C. G. ; Rowan, A. E., Therapeutic nanoworms : towards novel synthetic dendritic cells for immunotherapy. Chem. Sci. 2013, 4 (11), 4168-4174.

(13) Kumar, S.; Anselmo, A. C.; Banerjee, A.; Zakrewsky, M.; Mitragotri, S., Shape and size- dependent immune response to antigen-carrying nanoparticles. J. Controlled Release 2015, 220, Part A, 141-148.

(14) Blunden, B. M. ; Chapman, R. ; Danial, M. ; Lu, H. X. ; Jolliffe, K. A.; Perrier, S. ; Stenzel, M . H., Drug Conjugation to Cyclic Peptide-Polymer Self-Assembling Nanotubes. Chem. Eur. J. 2014, 20 (40), 1274512749. (15) Wang, Y.; Yi, S.; Sun, L.; Huang, Y.; Lenaghan, S. C.; Zhang, M., Doxorubicin-Loaded Cyclic Peptide Nanotube Bundles Overcome Chemoresistance in Breast Cancer Cells. J. Biomed. Nanotechnol. 2014, 10 (3), 445-454.

(16) Chen, J.; Zhang, B.; Xia, F.; Xie, Y.; Jiang, S.; Su, R.; Lu, Y.; Wu, W., Transmembrane delivery of anticancer drugs through self-assembly of cyclic peptide nanotubes. Nanoscale 2016, 8 (13), 7127-7136.

(17) Strohalm, J.; Kopecek, J., Poly[N-(2-hydroxypropyl)methacrylamide]. IV.

Heterogeneous polymerization. Angew. Makromol. Chem. 1978, 70 (1), 109-118.

(18) Kopecek, J.; Kopeckova, P., HPMA copolymers: Origins, early developments, present, and future. Adv. Drug Delivery Rev. 2010, 62 (2), 122- 149.

(20) Sanchis, J. ; Canal, F.; Lucas, R. ; Vicent, M . J., Polymer-drug conjugates for novel molecular targets. Nanomedicine 2010, 5 (6), 915-935.

(21) Duncan, R., Polymer conjugates as anticancer nanomedicines. Nat. Rev. Cancer 2006, 6 (9), 688701.

(22) Liu, Z.; Romero-Canelon, I.; Qamar, B.; Hearn, J. M. ; Habtemariam, A. ; Barry, N. P. E. ; Pizarro, A. M. ; Clarkson, G. J. ; Sadler, P. J., The Potent Oxidant Anticancer Activity of Organoiridium Catalysts. Angew. Chem. Int. Ed. 2014, 53 (15), 3941-3946.

(23) Liu, Z. ; Sadler, P. J., Organoiridium Complexes: Anticancer Agents and Catalysts. Acc. Chem. Res. 2014, 47 (4), 1174-1185.

(24) Liu, Z.; Romero-Canelon, L; Habtemariam, A.; Clarkson, G. J.; Sadler, P. J., Potent Half-Sandwich Iridium(III) Anticancer Complexes Containing C^AN-Chelated and Pyridine Ligands. Organometallics 2014, 33 (19), 5324-5333.

(25) Scales, C. W.; Vasilieva, Y. A.; Convertine, A. J.; Lowe, A. B.; McCormick, C. L., Direct, Controlled Synthesis of the Nonimmunogenic, Hydrophilic Polymer, Poly(N-(2- hydroxypropyl)methacrylamide) via RAFT in Aqueous Media. Biomacromolecules 2005, 6 (4), 1846-1850.

(26) Hong, C.-Y.; Pan, C.-Y., Direct Synthesis of Biotinylated Stimuli-Responsive Polymer and Diblock Copolymer by RAFT Polymerization Using Biotinylated Trithiocarbonate as RAFT Agent. Macromolecules 2006, 39 ( 10), 3517-3524.

(27) Liu, Z.; Salassa, L.; Habtemariam, A.; Pizarro, A. M.; Clarkson, G. J.; Sadler, P. J., Contrasting Reactivity and Cancer Cell Cytotoxicity of Isoelectronic Organometallic Iridium(III) Complexes. Inorg. Chem. 2011, 50 (12), 5777-5783.

(28) Liu, Z.; Habtemariam, A.; Pizarro, A. M.; Clarkson, G. J.; Sadler, P. J., Organometallic Iridium(III) Cyclopentadienyl Anticancer Complexes Containing C,N-Chelating Ligands. Organometallics 2011, 30 (17), 4702-4710.

(29) Pedersen, J. S., Form factors of block copolymer micelles with spherical, ellipsoidal and cylindrical cores. J. Appl. Crystallogr. 2000, 33 (1), 637-640. (30) Chapman, R.; Koh, M. L; Warr, G. G.; Jolliffe, K. A.; Perrier, S., Structure elucidation and control of cyclic peptide-derived nanotube assemblies in solution. Chem. Sci. 2013, 4 (6), 2581-2589.

(31) Liu, Z.; Habtemariam, A.; Pizarro, A. M. ; Fletcher, S. A.; Kisova, A.; Vrana,

O. ; Salassa, L. ; Bruijnincx, P. C. A.; Clarkson, G. X ; Brabec, V.; Sadler, P. X, Organometallic Half-Sandwich Iridium Anticancer Complexes. J. Med. Chem. 2011,

54 (8), 3011-3026.

(32) van Rijt, S. H.; Romero-Canelon, L ; Fu, Y. ; Shnyder, S. D. ; Sadler, P. J., Potent organometallic osmium compounds induce mitochondria-mediated apoptosis and S-phase cell cycle arrest in A549 non-small cell lung cancer cells. Metallomics 2014, 6 (5), 1014- 1022.

(33) Novohradsky, V.; Liu, Z.; Vojtiskova, M.; Sadler, P. J.; Brabec, V.; Kasparkova, J., Mechanism of cellular accumulation of an iridium(III) pentamethylcyclopentadienyl anticancer complex containing a C,N-chelating ligand. Metallomics 2014, 6 (3), 682-690.

(34) Puckett, C. A.; Ernst, R. J. ; Barton, J. K., Exploring the cellular accumulation of metal complexes. Dalton Transactions 2010, 39 (5), 1159-1170.

(35) Huang, X. ; Teng, X.; Chen, D. ; Tang, F.; He, J., The effect of the shape of mesoporous silica nanoparticles on cellular uptake and cell function. Biomaterials

2010, 31 (3), 438-448.

(36) Huang, J.; Bu, L. ; Xie, J. ; Chen, K.; Cheng, Z.; Li, X.; Chen, X., Effects of Nanoparticle Size on Cellular Uptake and Liver MRI with Polyvinylpyrrolidone-Coated Iron Oxide Nanoparticles. ACS Nano 2010, 4 (12), 7151-7160.

(37) Lu, F.; Wu, S.-H.; Hung, Y.; Mou, C.-Y., Size Effect on Cell Uptake in Well- Suspended, Uniform Mesoporous Silica Nanoparticles. Small 2009, 5 (12), 1408- 1413.

(38) Yin Win, K.; Feng, S.-S., Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs. Biomaterials 2005, 26 (15), 2713-2722.

Kulkarni, S. A.; Feng, S.-S., Effects of Particle Size and Surface Modification on Cellular Uptake and Biodistribution of Polymeric Nanoparticles for Drug Delivery. Pharm. Res. 2013, 30 (10), 2512-2522.

(39) Hinde, E. ; Thammasiraphop, K. ; Duong, H. T. T. ; Yeow, J. ; Karagoz, B. ; Boyer, C. ; Gooding, J. J. ; Gaus, K., Pair correlation microscopy reveals the role of nanoparticle shape in intracellular transport and site of drug release. Nat Nano 2016, advance online publication.

(40) Etrych, 1. ; Kovaf, L. ; Strohalm, J.; Chytil, P. ; Rihova, B. ; Ulbrich, K., Biodegradable star HPMA polymer-drug conjugates: Biodegradability, distribution and anti-tumor efficacy. J. Controlled Release 2011, 154 (3), 241-248. (41) Etrych, 1.; Subr, V.; Strohalm, J. ; Sirova, M. ; Rihova, B.; Ulbrich, K., HPMA copolymer-doxorubicin conjugates: The effects of molecular weight and architecture on biodistribution and in vivo activity. J. Controlled Release 2012, 164 (3), 346-354.

(42) Sadekar, S.; Ray, A.; Janat-Amsbury, M.; Peterson, C. M.; Ghandehari, H., Comparative Biodistribution of PAMAM Dendrimers and HPMA Copolymers in Ovarian-

Tumor-Bearing Mice. Biomacromolecules 2011, 12 (1), 88-96.

(43) Kaminskas, L. M. ; Kelly, B. D. ; McLeod, V. M. ; Boyd, B. J. ; Krippner, G. Y. ; Williams, E. D.; Porter, C. J. H., Pharmacokinetics and Tumor Disposition of PEGylated, Methotrexate Conjugated Poly- 1- lysine Dendrimers. Mol. Pharmaceutics 2009, 6 (4), 1190- 1204.

(44) Khor, S. Y. ; Hu, J.; McLeod, V. M. ; Quinn, J. F. ; Williamson, M. ; Porter, C. J. H.; Whittaker, M. R.; Kaminskas, L. M .; Davis, T. P., Molecular weight (hydrodynamic volume) dictates the systemic pharmacokinetics and tumour disposition of PolyPEG star polymers. Nanomedicine: Nanotechnology, Biology and Medicine 2015, 11 (8), 2099-2108.

(45) Milliner, M.; Dodds, S. J. ; Nguyen, T.-H. ; Senyschyn, D.; Porter, C. J. H. ; Boyd,

B. J.; Caruso, F., Size and Rigidity of Cylindrical Polymer Brushes Dictate Long Circulating Properties In Vivo. ACS Nano 2015, 9 (2), 1294-1304.

(46) Elsabahy, M. ; Wooley, K. L., Design of polymeric nanoparticles for biomedica l delivery applications. Chem. Soc. Rev. 2012, 41 (7), 2545-2561.

The invention also provides:

formula iii - pyridine functional monomer

2. A cell-penetrating self-assembling nanoparticle comprising :

c. a cyclic peptide core

d. at least 1 polymer arm comprising the polymerization product of the monomer of formula

(iii)

3. A cell-penetrating self-assembling nanoparticle according to item 2 where the pyridine functional monomer is (3-(pyridine-4-ylmethyl)ureido)ethyl)methacrylate (PUEMA)

formula iv - (3-(pyridine-4-ylmethyl)ureido)ethyl)methacrylate (PUEMA)

5. A self-assembling nanoparticle drug delivery system according to item 4 wherein the copolymer comprises a copolymer of (i) and a hydrophilic comonomer.

6. A self-assembling nanoparticle drug delivery system according to item 4 wherein the hydrophilic comonomer is a hydroxyl-functional monomer, selected from hydroxypropyl methacrylate, 2-hydroxyethyl methacrylate, 4-hydroxybutyl methacrylate or glycerol monomethacrylate.

7. A self-assembling nanoparticle drug delivery system comprising : e. a cyclic peptide core

f. at least one polymer arm or arms comprising copolymers according to items 4 - 6 self-assembling drug delivery system according to items 4 - 7 which further comprises a complexed drug molecule. self-assembling drug delivery system according to item 8 wherein the conjugated drug is a metallic or organometallic drug. A self-assembling drug delivery system according to items 8 and 9 wherein the metallic or organometallic drug is selected from organo-iridium compounds or organo-platinum compounds. A self-assembling drug delivery system according to item 8 wherein the complexed drug is an anti-cancer drug. A self-assembling drug delivery according to any of item 4-11 wherein the pyridine functional monomer is (3-(pyridine-4-ylmethyl)ureido)ethyl)methacrylate (PUEMA) A method of preparing a self-assembling nanoparticle drug delivery system, the method comprising the following steps:

g. Copolymerizing a pyridine-functiona l monomer of formula (iii) with a hydrophilic comonomer in the presence of a chain transfer agent, affording a biocompatible polymer with a reactive end group

h. Reacting the biocompatible polymer with a reactive end group with a cyclic peptide to form a conjugate with a cyclic peptide core and at least 1 polymer arms A method according to item 13 wherein the chain transfer agent is a RAFT agent. A method according to item 13 where in the reactive end group of the chain transfer agent and therefore the resulting biocompatible polymer is a carboxylic acid. A method according to item 13 and item 14 wherein the chain transfer agent is (4-cyano pentanoic acidjyl ethyl trithiocarbonate (CPAETC). A method according to item 13 wherein the hydrophilic comonomer is selected from hydroxypropyl methacrylate, 2-hydroxyethyl methacrylate, 4-hydroxybutyl methacrylate or glycerol monomethacrylate.

Claims

1. A compound comprising a cyclic peptide and one or more polymer arms, wherein the polymer arms comprise a polymer obtainable by polymerisation of at least one monomer according to formula I:

Formula I wherein R is a linker selected from: alkyl, aryl, amine, amide, carbonate, ester, ether, imide, sulfide, disulfide, sulfone, sulfoxide, ureide or a combination thereof; and Ar is an optionally substituted aryl or optionally substituted heteroaryl.

2. A compound according to claim 1, wherein Ar is pyridine.

3. A compound according to claim 1 or 2, wherein R is a linker comprising urea .

4. A compound according to claim 1, wherein the at least one monomer is ((3-(pyridine- 4-ylmethyl)ureido)ethyl)methacrylate (PUEMA) :

5. A compound according to any preceding claim, wherein the polymerisation is free radical polymerisation, typically radical atom transfer polymerisation (RAFT).

6. A compound according to any preceding claim, wherein the polymer is a copolymer further comprising one or more hydrophilic comonomers.

7. A compound according to claim 6, wherein the one or more hydrophilic comonomers are selected from: hydroxypropyl methacrylate, 2-hydroxyethyl methacrylate, 4- hydroxybutyl methacrylate, glycerol monomethacrylate or combinations thereof.

8. A compound according any preceding claim comprising two or more polymer arms.

9. A compound according any preceding claim, wherein the cyclic peptide is selected from: cyclo(L-Trp D-Leu-L-Lys-D-Leu)2, cyclo(L-Trp-D-Leu-L-Trp-D-Leu-L-Trp-D-Leu- L-Lys-D-Leu) or combination thereof.

10. A composition comprising the compound according to any preceding claim and one or more drugs.

11. A composition according to claim 10, wherein the one or more drugs are metallic or organometallic drugs.

12. A composition according to claim 11, wherein the metallic or organometallic drugs are selected from organo-iridium compounds, organo-platinum compounds or combinations thereof.

13. A method of preparing the compound according to any of claims 1 to 9, comprising the steps of:

i) polymerising at least one monomer according to formula I in the presence of a chain transfer agent to form a polymer;

ii) reacting said polymer with a cyclic peptide.

14. A method according to claim 13, wherein the chain transfer agent comprising a reactive carboxylic acid moiety.

15. A method according to claim 13, wherein the chain transfer agent is (4- cyanopentanoic acid)yl ethyl trithiocarbonate (CPAETC).

16. A compound according to any of claims 1 to 9 for use in drug delivery.

17. A composition according to any of claims 10 to 12 for use in therapy.

18. A composition according any of claims 10 to 12 for use in the treatment of cancer.

19. A method of treating cancer comprising the steps of administering a composition according any of claims 10 to 12 to a patient.