Orthogonally Bifunctionalised Polyacrylamide Nanoparticles: a Support for the Assembly of Multifuctional Nanodevices

Polyacrylamide nanoparticles bearing two orthogonal reactive functionalities were prepared by reverse microemulsion polymerisation. Water-soluble photosensitisers and peptide or carbohydrate moieties were sequentially attached to the new nanospecies by orthogonal conjugations based on copper-catalysed azide-alkyne cycloaddition and isothiocyanate chemistry.


Introduction
Growing interest in the application of nanotechnology in biomedical research has encouraged great efforts in devising improved materials for the generation of multifunctional nanodevices designed for targeted drug delivery, diagnostic imaging and biosensing.2][3][4][5][6][7][8][9] Polyacrylamide nanoparticles (PANPs) are hydrophilic and give stable colloidal dispersions in water, hence they are suitable for biologic applications.An appealing feature of polyacrylamide nanoparticles is their versatility, resulting from their chemical flexibility.3][14] Cargoes can be loaded on polyacrylamide nanoparticles either by encapsulation within the polymer network or by conjugation to reactive functionalities, which can be engineered on the surface of the particle by including suitably functionalised acrylamides in the polymerisation step. 15Thus, polyacrylamide nanoparticles have been decorated with a wide variety of molecules, including targeting agents (e.g.: peptides, carbohydrates, aptamers, oligonucleotides, lipids, etc.), 15,16 fluorescent dyes, reporter molecules, 17 drugs, or contrast agents. 18,191][22][23] The association of photosensitisers with polyacrylamide nanoparticles opened the way to the generation of light responsive nanodevices, which have applications as therapeutic, diagnostic and investigative tools.The incorporation of a photosensitiser into a nanoparticle is an effective way to overcome the poor water solubility displayed by many photosensitisers, to achieve higher loading of photosensitiser in the target cells, and in many instances to prolong the half-life of the photosensitiser in plasma, in particular when the surface of the nanoconjugate is coated with polyethylene glycol (PEG) to delay opsonisation. 24,257][28][29][30] Due to their size, nanosensors allow measurements to be performed within a single cell, with minimal perturbation of the system.In nanosensors, the polymer constituting the core of the particle entraps a fluorescent dye that responds to the variations of the concentration of a given analyte in the intracellular environment by modifying its fluorescence emission profile.Thus, changes in the fluorescence signal following the application of an external stimulus provide real time information on the fluctuations of the level of the analyte, and on the biological processes associated with such fluctuations.The use of nanosensors presents several advantages over the use of stand-alone fluorescent dyes: the choice of the fluorescent probe can be solely based on its sensitivity to the analyte of interest and on its emission profile, because the hydrophilic matrix circumvents all issues related to low water solubility, instability and toxicity of the dye.Once incorporated in the polyacrylamide matrix, even hydrophobic dyes can be used in aqueous solutions, and because interactions of the encapsulated probe with macromolecules are prevented, its degradation by enzymes and its association with proteins or membranes are reduced.For the same reason, the cell is protected from toxic effect exerted by the dye.
][34][35][36][37][38] In the course of our studies concerning the role of reactive oxygen species in cell signalling, we became interested in studying the mobilization of intracellular calcium following singlet oxygen production.We previously reported the synthesis of a nanosensor comprising of a calcium-sensitive fluorophore (calcium green 1, CG-1) embedded in a porphyrin-conjugated polyacrylamide nanoparticle (Fig. 1, A), as a self-standing nanodevice able to elicit a controlled perturbation of the cellular system (production of singlet oxygen) and to register the downstream events of the cellular response to such variation (calcium ion mobilization). 31ith the aim of improving cellular delivery of the nanosensors, we reasoned that a cell penetration enhancing moiety or a targeting agent could be conjugated to the surface of the nanodevice alongside the porphyrin, provided a second reactive functionality could be introduced on the polymer.(Fig. 1, B).In order to maintain control over the selectivity of the conjugations, the two reactive functionalities displayed on the new nanoparticles would need to display no overlapping reactivity, so that either one of them could be selectively conjugated to a given cargo.
The functionalisation of polyacrylamide nanoparticles with amino groups is widely documented. 8,17,18,31,40,41To generate these species, a suitable acrylamide containing an amino-group is copolymerised with acrylamide and N,N-methylenebisacrylamide as the cross-linker, under reverse-phase microemulsion polymerisation conditions.][42] Numerous examples of efficient decoration of amino-functionalised polyacrylamide nanoparticles with cargoes of diverse nature are reported in the literature. 8,17,18,31,43,44e have previously shown that porphyrins bearing an isothiocyanate group or a hydroxysuccinimide-activated carboxylic function can be attached to the surface of amino-functionalised polyacrylamide nanoparticles. 4,31The synthesis of nanoparticles bearing alkyne or azide groups, reactive under CuAAC conditions, was also reported: [45][46][47] in particular, we have shown that alkyne-functionalised polyacrylamide nanoparticles successfully undergo conjugation with TAMRA derivative and peptidocoumarins in the presence of tetrakis(acetonitrile) copper(I) hexafluorophosphate and TBTA. 48In these reports, the introduction of the functional groups on the particle surface was achieved via a common strategy, i.e. by adding the functionalised acrylamide to the polymerisation mixture.On the basis of these studies, we envisaged that the combined use of amino-and azido-(or alkyne)-bearing acrylamides during the polymerisation step would afford nanospecies displaying the two required orthogonal functionalities which, under the appropriate conditions, could selectively take part in conjugation with different cargoes.
Several examples of polymers prepared under controlled polymerisation conditions (living free-radical polymerisation) and displaying orthogonal functionalities have been reported in the literature, [49][50][51][52][53][54][55] but to the best of our knowledge it has never been exploited for the assembly of multifunctional polyacrylamide nanospecies under reverse phase microemulsion polymerisation.

Results and discussion
While N-(3-aminopropyl)methacrylamide hydrochloride is a commercially available compound, no acrylamide functionalised with an azide or a triple bond can be purchased.Therefore we first focused on the synthesis of two acrylamide monomers with comparable chain lengths, bearing the desired groups.Amino-azide 1 in its Boc-protected form was obtained starting from 2-(2-aminoethoxy)ethanol in two steps on a multi-gram scale. 56Boc-protected amino-alkyne 2 was also obtained in two steps from ethanolamine.The desired acrylamides 3 and 4 were prepared in moderate yields by reaction of 1 and 2 with acryloyl chloride following HCl-mediated Boc-deprotection.(Scheme 1).
Acrylamides 3 and 4 were efficiently incorporated into polyacrylamide nanoparticles under reverse-phase microemulsion polymerisation conditions. 27,48(Scheme 2, Box 1) The desired nanospecies 5 and 6 were obtained in 81 and 88% yield, respectively.IR spectra of the particles obtained showed a band at 2111 Fig. 1 Scheme 1 cm À1 for 5, which is diagnostic for the stretching of N-N bond in the azido groups, and a very weak band at 2090 cm À1 for 6, generated by the stretching of the triple bonds.
A further confirmation of the presence of the desired reactive functionalities on the nanoparticles was provided by the successful conjugation of 5 and 6 with ''clickable'' porphyrins 7 and 8, respectively, under Cu(I)-catalysed triazole formation conditions. 57Alkyne-bearing porphyrin 7 and azide-bearing porphyrin 8 were obtained by coupling hydroxysuccinimidoester 9 with amines 10 and 11 in NaHCO 3 buffer.(Scheme 3) The choice of using 7 and 8 as zinc complexes was made in order to prevent the sequestration of copper ions by the macrocycle during CuAAC.The conjugation reaction occurred smoothly in water at room temperature, in the presence of CuSO 4 and sodium ascorbate.(Scheme 2, Box 2) Gel filtration on Sephadex G25 provided an expeditious mean to separate the nanoconjugates from the catalysts and non-reacted porphyrin. 58Nanoconjugates 12 and 13 were recovered in 95 and 83% yields, respectively, following precipitation with ethanol and filtration.
Having verified the usefulness of acrylamides 3 and 4 for the introduction of azides and terminal alkynes on polyacrylamide nanoparticles, we turned our attention to the preparation of doubly-functionalised species.To this end, we modified the protocol by replacing the 1% of functionalised acrylamide in the mixture of monomers with 0.5% of N-(3-aminopropyl)methacrylamide hydrochloride (APMA) and 0.5% of either 3 or 4. We were pleased to observe that in both cases the polymerisation afforded bifunctionalised nanospecies 14 and 15 (Scheme 2), whose average hydrodynamic diameter did not differ significantly from the diameter exhibited by the monofunctionalised particles.(Table 1).The presence of the alkyne and azide functionalities was again verified by IR, while the presence of the amino group was revealed by means of fluorescamine staining. 59ith the aim of assessing the versatility of the new bifunctionalised polyacrylamide nanospecies, and to verify the feasibility of orthogonal sequential double-conjugations, we selected a group of conjugatable molecules to decorate the particles.(Scheme 4) Bearing in mind that this work originated from the need of associating nanoparticles with both photosensitisers and cell-penetration or cell-targeting entities, we selected a small group of molecules representative of both groups.Porphyrins 7, 8, and 16, 60 and phthalocyanine 17 were chosen as examples of photosensitisers, whereas RGD-containing cyclo-pentapeptides 18 and 19 and 1-propargyloxymannose 20 as models for targeting moieties.Although RGD-containing peptides 8,46,[61][62][63][64][65] and mannose [66][67][68][69] have already been used in association with a variety of nanoparticles to improve their receptor-mediated targeting and cellular uptake, in this work they were selected as models to study the feasibility of the conjugation of peptides and carbohydrates to the new doubly-functionalised nanospecies.As shown in Scheme 3, all the molecules selected were engineered to display groups which would provide them with reactivity towards amines, azides, or triple bonds.With ''clickable'' porphyrins 7 and 8 already in our hands, we chose isothiocyanato porphyrin 16 as an amino-reactive photosensitiser.Porphyrin 16 was prepared in quantitative yield by metallation of the corresponding free base in 10 mM NaHCO 3 buffer.Although the conjugation of 16 to amino-functionalised nanoparticles does not require the presence of metal ions in the reaction mixture, the use of a zinc-porphyrin was necessary because the resulting nanoconjugate would be exposed to copper ions under the CuAAC conditions of the second ligation step.RGD-containing cyclopentapeptides were synthesised by Fmoc-SPPS, according to literature procedures. 70The azide group was incorporated in peptide 18 by coupling Fmoc-3-azidolysine 71 during chain elongation, whereas the isothiocyanate function of 19 was installed on the side chain of Lys by treatment of the corresponding amino-derivative with 1,1 0 -thiocarbonyldi-2(1H)pyridone. 72ecause amino-functionalised polyacrylamide nanoparticles have been reported to be prone to irreversible aggregation, 48 a behaviour that has been ascribed to the Michael addition of the amino-groups onto residual acrylamide moieties on the surface of the particle, we envisaged that the attachment of the aminoreactive cargo should be carried out before the CuAAC conjugation.(Scheme 2, Box 3).
Thus, nanoparticles 15 were first treated with isothiocyanate porphyrin 16 in the presence of 30 mM triethylamine.The resulting porphyrin-conjugated particles 21 were then exposed to azide-functionalised cyclopentapeptide 18 under the conditions described above.The doubly-decorated nanospecies 22 were obtained in 75% yield over two steps.Exchanging the reactive functionalities on the cargo molecules did not hamper the double conjugation, as we confirmed by exposing particles 15 first to cyclopentapeptide isothiocyanate 19 to afford conjugate 23, and then to porphyrin 8 to give nanospecies 24.The final nanoconjugate was obtained in 63% overall yield.(Scheme 4) A similar conjugate was obtained starting from particles 14, which reacted with cyclopentapeptide 19 in the presence of triethylamine, yielding 25, and subsequently with porphyrin 7 under CuAAC conditions to give conjugate 26, confirming that the reactivity of the azide group is maintained on the bifunctionalised nanoparticles.(Fig. 2) Treatment of nanospecies 27, obtained by reaction of porphyrin 16 on particles 14, with 1propargyloxymannose 20 12 under CuAAC conditions yielded conjugate 28 in 90% yield.Treatment of nanoconjugate 23 with azido-phthalocyanine 17 73 in 10% aqueous DMSO in CuAAC conditions, gave nanoparticles 29 in 91% yield.Fig. 2 summarises the nanoconjugates obtained by applying the protocols devised with bifunctionalised nanoparticles and different permutations of the conjugatable molecules we selected.

Characterisation of the nanoconjugates
The hydrodynamic size of the nanoconjugates was measured by photon correlation spectroscopy.(Table 1 and Table 2).The conjugated nanospecies showed a marked increase in hydrodynamic radius compared to the native particles.These observations are in agreement with our previous findings, 4 and can be explained on the basis of the increased thickness of the counterions corona tightly associated with the nanoparticle, caused by the covalent ligation of tri-cationic molecules on the surface of the gel. 74,75The presence of positive charges could also cause the swelling of particles because of the increased osmotic pressure in the proximity of the nanogel. 76The efficiency of the attachment of the photosensitisers to the particles was evaluated by UVvisible spectroscopy.The UV-visible spectra of particles 22 and particles 29 show the typical profiles of metal complexes of porphyrins and phthalocyanines.(Fig. 3) To reveal the presence of mannose on particles 28 we relied on a colorimetric test based on the reaction of carbohydrates with phenol in the presence of concentrated sulfuric acid, described by Masuko et al. 77 The presence of mannose is evidenced by the development of a broad band centred at 470-480 nm in the absorption spectrum.(Fig. 4) A similar assay has been previously used to estimate the loading of mannose on mesoporous silica nanoparticles. 68The presence of peptides 18 and 19 on nanoparticles was detected with the Sakaguchi test, 78 which reveals the presence of guanidine on the side-chain of Arg. (Fig. 5) An application of this assay to detect the presence of RGD-containing peptides anchored on a solid support has been reported previously. 79The tests described above were intended to be qualitative assays, aimed at verifying the effectiveness of the conjugation reactions.The results obtained confirmed that the presence of the first cargo on the particle does not prevent the attachment of the second one, indicating that the reactivity of the two groups is maintained throughout the whole conjugation process.
Following our original concept of obtaining doubly-conjugated Ca-sensing nanoparticles, we prepared nanoparticles 30, displaying alkyne and amine groups and entrapping To this end, particles 30 were first treated with porphyrin 16 in the presence of triethylamine, and then peptide 18, CuSO 4 and sodium ascorbate were added to the aqueous solution of the conjugated nanospecies, after gel filtration.The presence of both cargoes was verified as described above.The sensors show a linear response to the increasing calcium concentration over the range of 0-12 nM (y ¼ 0.942x + 134.85,R 2 ¼ 0.9799).These results are in agreement with data previously generated, confirming that embedding CG-1 within porphyrin-coated polyacrylamide nanoparticles does not impair its sensitivity to calcium ions in the surrounding environment. 31

Conclusions
This work shows that the insertion of two orthogonal functionalities on polyacrylamide nanoparticles can be achieved, and that the reactivity of such groups can be successfully exploited to sequentially load the particles with two different cargoes.To the best of our knowledge this represents the first example of the application of orthogonal conjugation on polyacrylamide nanoparticles.The insertion of the two reactive groups on the particle is achieved via standard reverse phase microemulsion polymerisation techniques, yet it gives access to nanodevices with a high degree of functionalisation and complexity.The mild reaction protocols and expeditious purification make this novel approach especially suitable for the conjugation of biomolecules  to nanoparticles; however we anticipate that this method will have a broad relevance, because it will be applicable to conjugate cargoes of different nature to a wide variety of polymeric nanoparticles.

Experimental
General remarks 1 H and 13 C NMR spectra were recorded on JEOL Eclipse 400 and JEOL Lambda 400 spectrometers (operating at 400 MHz porphyrin trichloride were prepared according to literature procedures. 60hemical reagents were purchased from Sigma-Aldrich, Fluka, Acros, Lancaster, Alfa Aesar, TCI Europe, and Novabiochem at the highest grade of purity available, and were used as received, unless otherwise stated.Dichloromethane and THF were dried by filtration through alumina and storage over   View Online activated molecular sieves. 80All other solvents were purchased from Fisher Scientific and used as received.Deionised water was obtained from a Millipore Milli-Q system.Gel filtrations were performed on Sephadex Ò G-25 medium, using pre-packed PD-10 columns (GE Healthcare, UK), using deionised water as the eluent.Filtrations through membranes were performed on Sartolon Ò polyamide filters, 25 mm, 0.20 mm pores.Solid phase extractions were performed on Supelco Discovery DSC-18 cartridges (1 g).RP-HPLC analyses were performed on a system consisting of a Perkin Elmer series 200 LC pump, and a Perkin Elmer 785A UV/vis detector.The separations were performed on a Gemini C18, 5 m, 150 Â 4.6 mm, 110 A (Phenomenex, UK), equipped with a SecurityGuard C18 (ODS) 4 Â 3.0 mm ID guard column (Phenomenex, UK) at a flow rate of 1 mL min À1 .The mobile phase consisted of 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B).Gradient: 0.0-10.0min 0-95% solvent B, 10.0-15.0min 95% solvent B, 15.0-15.1 min at 95-5% solvent B, 15.1-18.0min 5% solvent B.

General procedure for solid-phase peptide synthesis. H-D(tBu)fK (3-N 3 )R(Pmc)G-OH
The synthesis of the protected cyclopentapeptides was accomplished according to the Fmoc/t-Bu strategy manually in a 6 mL fritted SPE tube (Supelco, UK), equipped with a cap and plastic valve (Chromabond, UK).Loading of the resin was achieved by treating 2-chlorotrityl chloride polystyrene resin (1 g, 1.4 mmol) with a solution of N-a-Fmoc-glycine (832 mg, 2.8 mmol) and DIPEA (1950 mL, 11.2 mmol) in dry DCM (10 mL) for 60 min.Unreacted sites on the resin were capped by treatment with a solution of DCM/MeOH/DIPEA (3 : 1 : 0.5 v/v/v, 2 Â 15 min).The first residue loading was measured spectrophotometrically, and was estimated to be 1.37 mmol g À1 .Coupling reactions were performed by treating the resin (400 mg) with a solution of Fmoc-amino acid (2 equiv relative to the resin loading), PyBOP (1.9 equiv) and diisopropylethylamine (4 equiv) in DMF (10 mL g À1 resin) for 60 min.The efficiency of the coupling reactions was assessed by Kaiser test. 81N-Fmoc deprotection was performed by treatment piperidine/DMF (1 : 4 v/v, 10 mL g À1 resin, 4 Â 3 min).The cleavage was performed by treating the resin with TFA/MeOH (1 : 99 v/v, 10 Â 2 min).The cleavage solutions were neutralised by addition to pyridine/MeOH (1 : 9 v/v, 20 mL), concentrated to small volume and precipitated from ice-cold water.The precipitate was washed again with ice cold water (20 mL) and dried in vacuo.The residue was dissolved in the minimum of methylene chloride and precipitated from diethyl ether.The white solid was triturated diethyl ether to obtain the crude peptide that was used in the next step without further purification.The cleavage performed onto 500 mg of peptidyl resin afforded 150 mg of crude peptide (40% based on loading of first residue).RP-HPLC (detector:

General procedure for cyclization reactions. Peptide cyclo[D (tBu)fK(3-N 3 )R(Pmc)G]
A 0.5 mM solution of linear protected peptide 18, whose pH value was adjusted to 8-9 by addition of DIPEA, was treated with PyBOP (1 equiv).The solution was stirred at room temperature for 1 h.Solvent was removed under reduced pressure and the residue dissolved in the minimum of methylene chloride.Diethyl ether was added to precipitate crude peptide, which was triturated and washed with diethyl ether (3 Â 10 mL), and in the next step without further purification.RP-HPLC (l ¼ 220 nm): t R ¼ 11.Protected peptide 18 (72 mg) was treated with TFA/TIS/water (95/2.5/2.5, 1 mL) at room temperature for 2 h.The solvent were evaporated in reduced pressure and the residue was taken in a TFA (0.2 mL) and precipitated with diethyl ether (10 mL).Washing with diethyl ether was repeated 3 times, and the crude peptide was purified by Supelco Discovery DSC-18 cartridges (1 g), eluting with 0.1% TFA in acetonitrile/water (5/95).The solvent was evaporated and the residue was taken in MeOH.The solid residue was filtered off, and the TFA salt of the peptide was precipitated from the solution by addition of diethyl ether, yielding a white solid (58  General procedure for the synthesis of functionalised polyacrylamide nanoparticles: alkyne-functionalised nanoparticles 6 Degassed hexane (42 mL) was added to a stirred solution of Brij Ò 30 (3.08 g, 8.5 mmol) and dioctyl sulfosuccinate sodium salt (1.59 g, 3.6 mmol).The mixture was allowed to stir under argon until complete dissolution of the surfactants.A solution of acrylamide (527 mg, 7.6 mmol), 4 (13 mg, 0.08 mmol), and N,N 0methylenebisacrylamide (160 mg, 1.0 mmol) in water (1.8 mL) was added to the hexane mixture.10% aqueous ice-cold ammonium persulfate (30 mL) was added to the reaction vessel, followed by N,N,N 0 ,N 0 -tetramethylethylenediamine (15 mL), and the reaction mixture allowed to stir at room temperature under argon for two hours.Excess hexane was evaporated in vacuo, and the resulting white viscous liquid was washed with ethanol and centrifuged (7 Â 50 ml, 10 min, 4000 rpm).The solid was taken in ethanol and isolated by filtration through membrane, and dried in vacuo to yield the desired alkyne functionalized nanospecies as a white solid (621 mg, 88%).PCS z ave (nm): nm 40.0 (deionised water).

Alkyne-amine bifunctionalized nanoparticles embedding aminodextran-conjugated CG-1 30
The synthesis of PANPs displaying two reactive functionalities was performed according to the general procedure reported above, replacing 40 mL of water in the aqueous solution with 40 mL of a 5 mg mL À1 aqueous solution of aminodextranconjugated calcium green 1.The desired particles were obtained as a pale pink solid.(647 mg, 92%).PCS z ave (nm) 37.7.

Nanoconjugates 27
The conjugated nanospecies were obtained following the general procedure, using nanoparticles

Sakaguchi test
The assay is based on the method described by Michael et al., 79 with minor modifications.Solutions: Reagent A: 5% urea and 0.05% 1-naphthol in 95% ethanol.Reagent B: 0.7% bromine in 1.25 M NaOH. 5 mg mL À1 solutions of RGD-coated nanospecies in deionised water were used as the samples.Positive control samples were prepared using 1 mM Arg in deionised water.Deionised water and 5 mg mL À1 suspension of nanospecies 15 in deionised water were used as negative controls.Method: 250 mL of sample solution were added to 250 mL of reagent A, followed by 500 mL of reagent B in an eppendorf tube.The mixture was mixed on a vortex shaker and then it was allowed to stand at room temperature for 5 min.The solution was then transferred in a quartz cuvette (1 mL) and the absorption spectrum was recorded between 250 and 750 nm.The test was considered positive when I sample /I QÀ1 > 2 at 480 nm.

Phenol-sulfuric acid test
The assay was based on the micro-method described by Masuko et al. 77 Solutions: Reagent A: 5% phenol in deionised water.Reagent B: concentrated H 2 SO 4 (1.14 g mL À1 ). 5 mg mL À1 solutions of nanospecies 27 in deionised water was used as analyte solution.Positive control samples were prepared using 10 mM mannose in deionised water.Deionised water (control C) and 5 mg mL À1 suspension of nanospecies 15 in deionised water (control D) were used as negative controls.Method: 450 mL of reagent B were added to 150 mL of the analyte solution, followed by 90 mL of reagent A in a test tube equipped with a stopper.The mixture was mixed on a vortex shaker and then it was transferred into a graphite bath and allowed to stand at 90 C for 10 min.
The solution was then transferred in a quartz cuvette (1 mL) and the absorbance of the sample was recorded between 400 and 550.The test was considered positive when I sample /I QÀ1 > 2 at 450 nm.

for 1 H
and 100 MHz for13 C).CDCl 3 , DMSO-d 6 , and MeOH-d 3 were used as solvents.Chemical shifts (d) are reported in parts per million (ppm), referenced to either CHCl 3 ( 1 H, 7.26 ppm; 13 C, 77.16 ppm) or DMSO ( 1 H, 2.50 ppm; 13 C, 39.52 ppm) or MeOH ( 1 H, 3.31 ppm; 13 C, 49.00 ppm).Coupling constants (J) are recorded in Hz and significant multiplicities described by singlet (s), doublet (d), triplet (t), quadruplet (q), broad (br), multiplet (m), or doublet of doublets (dd).A Malvern Photon Correlation Spectrometer equipped with a 70 mW blue laser operating at 470 nm at an angle of 90 was used for the PCS measurements.The measurements were performed at room temperature, at a concentration of 5 mg mL À1 of nanospecies in 7 mM aqueous NaCl, unless otherwise stated, and the solutions were filtered through a 0.22 mm filter (Millex GP) previous to the measurements.CI, EI, and pAPCI mass spectra were performed at the EPSRC Mass Spectrometry Service (Swansea).ESI mass spectra were performed on a Varian 500-MS ion trap spectrometer, equipped with a Varian Prostar 212LC binary gradient pumping system and a Varian ProStar 410 autosampler.A standard Varian ESI source was used operating in +ve ion mode.Data was acquired and processed using Varian Workstation software.MALDI mass spectra were performed on a Bruker Reflex IV MALDI-TOF, operated in reflectron mode and monitoring positive ions.Data is acquired and processed using Bruker Compass software.1,8-Dihydroxy-9,10dihydroanthracen-9-one (dithranol) was used as the matrix.UV-visible spectra were recorded on a Varian Cary spectrophotometer.Fluorescence spectra were recorded on a Cary Eclipse Fluorimeter.Flash chromatography was carried out on silica gel 60 (MP Biomedicals, 32 À63 mm).Analytical TLC was carried out on aluminium sheets pre-coated with silica gel 60 (Fluka, 0.2 mm thick).Commercial solvents and reagents were used without further purification unless stated otherwise.Porphyrins 5-(4-isothiocyanatophenyl)-10,15,20-tris-[(4-methylpyridynium)yl]porphyrin trichloride and 5-[4-(succinimide-N-oxycarbonyl)phenyl]-10,15,20-tris-[(4-methylpyridynium)yl]

Table 1
NanoscaleThis journal is ª The Royal Society of Chemistry 2012 Downloaded by University of Hull on 21 February 2012 Published on 26 January 2012 on http://pubs.rsc.org| doi:10.1039/C2NR11947AView Online dextran-supported CG-1. 31(Scheme 5) The two conjugation steps required for the preparation of nanoconjugates 31 were performed sequentially, without isolating the intermediate conjugate.(Scheme 5)
Nanoparticles 5 (60 mg) and were dispersed in deionised water (9 mL).To the solution, porphyrin 7 (2 mg, 2.1 Â 10 À3 mmol) was added, followed by 10 mM aqueous CuSO 4 (250 mL, 2.5 mmol) and 100 mM aqueous sodium ascorbate (250 mL, 25 mmol).The resulting mixture was shielded from light and shaken overnight on a rotating shaker.The desired conjugated species were This journal is ª The Royal Society of Chemistry 2012 Nanoscale