Photostable far-red emitting pluronic silicate nanoparticles: perfect blood pool fluorophores for biphotonic in vivo imaging of the leaky tumour vasculature

Abstract

For correct analysis of anti-angiogenic therapies, we need new fluorescent probes that do not diffuse across the leaky tumour vascular endothelium before any treatment. The latter makes estimations of the functional blood volume and changes in vascular permeability uncertain after treatment. Therefore, we present here a new non diffusible fluorescent probe for two photon microscopy, which is composed of a hydrophobic push–pull dye (1) encapsulated in the apolar core of Pluronic F127–silica nanoparticles (1@F127–SiO₂, d = 22 nm). In apolar media, (1) has intense emission in the red (Φ_f = 39% at 650 nm) and two-photon absorption properties in the NIR. The NP probe was successfully tested in vivo on mice bearing tumours without diffusion across the tumour vascular endothelium. The two photon excitation wavelength at 1000 nm is optimum for deep tissue excitation.

---

Introduction

Various diseases¹ affect the blood vasculature, in particular cancer. Hypoxic cancer cells induce angiogenesis that plays a major role in invasive tumour growth and metastasis.² In vivo optical imaging of tumour angiogenesis before and after a therapy gives valuable information on changes in the vascular morphology, the blood flow, the vascular permeability and abnormality in blood vessel endothelial cells, and their interactions with cancer or cells of the immune system. For these applications, only a few non-diffusible fluorophores are available that stay in the leaky tumour vasculature, permitting discrimination in changes of functional tumour blood volume and vessel permeability before and after treatment.^3,4 Indeed, most fluorophores diffuse across the tumour vascular endothelium and can therefore not be used to correctly estimate these changes. In that prospect, organic fluorescent nanoparticles (NPs) represent an interesting alternative to soluble and diffusive unimolecular organic dyes.^5–8 They offer considerable advantages including: easy dispersion in water, enhanced brightness, accrued photostability and phagocytic uptake. Modification of their surface permits active targeting and multimode imaging.^9–11

Amphiphilic block copolymers like Pluronic® F127, a triblock EO₁₀₀–PO₆₅–EO₁₀₀ copolymer made of hydrophilic ethylene oxide (EO) and hydrophobic propylene oxide (PO) moieties, are an important class of non-ionic surfactants used to form and stabilize fluorescent organic nanoparticles.¹² In the context of tumour vascular imaging, Pluronic® F127 based fluorescent NPs show great potential as blood pool dye for intravital microscopy. They have a long circulation time, probably due to the protective hydrophilic shell formed by the EO segments that prevents the adsorption of proteins, adhesion to tissues and recognition by the reticuloendothelial system.^5,13 Pluronic® micelles are known to dissociate upon dilution.¹⁴ Shielding of polymeric micelle with a silica shell remarkably improves the resistance to dissociation in salted buffer.¹⁵ This original micelle/silica coprotection strategy was used to encapsulate various hydrophobic dyes, giving access to robust, ultrafine, very bright and stable core–shell polymer–silica colloidal particles. It was shown to be very efficient at reducing aggregation, preventing self-quenching, and restoring an intense fluorescence in aqueous media. Dyes were either previously functionalized with triethoxysilane groups to be part of the silica shell,^16–18 or non-functionalized and encapsulated without leakage.^19–21 Paradoxically, the same system was also used to favour the aggregation and encapsulate dye showing aggregation induced emission.²² Thus, it is a very promising approach for developing vascular tracers,^20,22 although some diffusion and accumulation around tumours were observed.^17,21

Deep in vivo two-photon vascular imaging needs long wavelength fluorophores with their absorption and emission in the far-red/near-infrared region: 650–1300 nm. In this region, that corresponds to the optical transparency window of tissues, light photon absorption by endogenous molecules (haemoglobin, H₂O…) is minimal for maximum penetration depth^4,23 and photon scattering is reduced. These phenomena are, however, still high in tumours due to their high cell and cell organelles densities.^8,24 Therefore, the development of fluorophores with efficient two-photon absorption and emission properties in the far red/NIR is important, especially for imaging tumour tissues.^11,25

Based on this literature, we employed the micelle/silica co-protection strategy to directly encapsulate a new push–pull dipolar dye 1 (Scheme 1). A push–pull dipolar structure was chosen to take advantage of the large Stoke shift usually associated with emission originating from charge transfer transition. This may help to shift the fluorescence in the far red. Additionally, such structures are known to exhibit environment sensitive fluorescence²⁶ along with two-photon absorption in the near infrared, and possibly solid state fluorescence.²⁷

Scheme 1 Structure and synthesis of dye 1.

Experimental section

¹H and ¹³C NMR spectra were recorded in CDCl₃ solution on Bruker Avance 500 MHz instrument. Chemical shifts were denoted in ppm (δ), and calibrated by using residual undeuterated solvent CHCl₃ (7.24 ppm) for ¹H NMR and the deuterated solvent CDCl₃ (77.00 ppm) as internal standard for ¹³C NMR. Coupling constants are given in hertz. Splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), or multiplet (m). Analytical thin-layer chromatography (TLC) was performed on precoated silica gel plates (F254, 0.2 mm thickness). HRMS measurements were performed by ESI-TOF (Bruker Daltonics® Micro TOF-Q II). Tetraethoxysilane (TEOS) and diethoxydimethylsilane (DEDMS) were purchased from Aldrich.

Synthesis of 2

7-Bromo-9,9-diethylfluorene-2-carbaldehyde (1.7 g, 5.14 mmol), diphenylamine (1.3 g, 7.7 mmol) and Cs₂CO₃ (2.51 g, 7.7 mmol) were mixed in toluene (24 mL) under argon. Then Pd(OAc)₂ (0.06 g, 0.27 mmol) and P(tBu)₃ (130 μL, 0.54 mmol) were added in the mixture. The final solution was heated at 120 °C for 24 h. After cooling back to room temperature, the reaction mixture was washed with water, 1 M HCl, and water again, dried over Na₂SO₄ and concentrated. The crude product was purified by chromatography on silica gel eluting with ethyl acetate/petroleum ether (1 : 20 by volume) giving a yellow solid (1.82 g, yield: 85%). ¹H NMR (CDCl₃, 500 MHz) δ 9.99 (s, 1H), 7.78 (m, 2H), 7.70 (d, J = 8.3, 1H), 7.60 (d, J = 8.2, 1H), 7.24 (m, 4H), 7.11 (m, 8H), 1.97 (m, 4H), 0.31 (t, J = 7.3, 6H); ¹³C(¹H) NMR (CDCl₃, 125 MHz) δ 192.2, 152.9, 150.5, 148.9, 147.9, 147.6, 134.5, 134.3, 130.8, 129.3, 124.4, 123.1, 122.93, 122.91, 121.6, 119.1, 118.2, 56.1, 32.4, 8.5.

Synthesis of 1

To a stirred solution of 7-(diphenylamino)-9,9-diethylfluorene-2-carbaldehyde (0.2 g, 0.48 mmol) and indane-1,3-dione (0.07 g, 0.48 mmol) in anhydrous ethanol (4 mL) was added 1 drop of piperidine. The solution was stirred at 80 °C for 3 h. After cooling to room temperature, the title compound 1 precipitated as a red crystalline solid. It was recovered by filtration, washed with acetonitrile and dried. Yield: 0.20 g (76%). ¹H NMR (CDCl₃, 500 MHz) δ 8.57 (s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.91–7.87 (m, 2H), 7.86 (s, 1H), 7.70–7.66 (s, 2H), 7.61 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.18–7.14 (m, 4H), 7.03 (m, 4H), 6.98 (s, 1H), 6.95–6.92 (m, 3H), 2.02–1.95 (m, 2H), 1.85–1.78 (m, 2H), 0.30 (t, J = 7.3 Hz, 6H); ¹³C(¹H) NMR (CDCl₃, 125 MHz) δ 190.8, 189.5, 153.4, 150.3, 149.1, 147.9, 147.6, 147.3, 142.6, 140.0, 135.2, 135.1, 134.9, 134.7, 131.4, 129.3, 128.6, 127.3, 124.6, 123.2, 123.2, 122.7, 121.7, 119.1, 118.0, 56.2, 32.5, 8.6; HRMS ESI calcd for C₃₉H₃₂NO₂ [M + H]⁺: 546.2430, found 546.2428.

Synthesis of 1@F127–SiO₂ NPs

In a 5 mL round-bottom flask, Pluronic® F127 (200 mg) was dissolved in 2–3 mL of dichloromethane. 0.3 mg of dye 1 was added giving a dye to pluronic weight fraction of 0.15 wt%. The solution was subsequently stirred at room temperature for 40 min to obtain a homogeneous red solution. Dichloromethane was then evaporated with a gentle flow of argon to give a film. 3.12 mL of 0.85 N hydrochloric acid were added and the mixture was stirred until a stable and optically transparent solution was obtained. TEOS (360 μL) was then added to the solution and the stirring continued for 105 min. DEDMS (30 μL) was finally added to terminate the particle growth. The final suspension was kept stirring at room temperature for 24 h. The solution was dialyzed for two days against distilled water to remove hydrochloride as well as unreacted low-molecular weight components, using a membrane bag with a 14 000 cut-off molecular weight. The suspension was then filtered through a 0.2 μm syringe filter to remove large aggregates. The final volume is 3.6 mL.

Animal model

Human glioma cells (U87MG GFP+, ATCC cell line, Teddington Middlesex, UK) were injected subcutaneously at a concentration of 10⁸ cells per mL in the left and right ear of nude mice (BALB/c, Charles River, Écully, France). The total injected volume was 20 μL: 10 μL cell suspension and 10 μL matrigel® (with growth factors, BD Bioscience, Europe, Erembodegem, Belgium). Mice were used for two-photon experiments at approximately 2–3 weeks after glioma cell injection. In accordance with the policy of Clinatec (permit number: B38-185 10 003) and the French legislation, experiments were done in compliance with the European Parliament and the Council Directive of September 22, 2010 (2010/63/EU) on the protection of animals used for scientific purposes. The research involving animals was authorized by the Direction Départementale des Services Vétérinaires de l'Isère – Ministère de l'Agriculture et de la Pêche, and the Ministère de l'Enseignement Supérieur et de la Recherche, France permit number: 2015051914157522_v1 (PI: Boudewijn van der Sanden, PhD, permit number 38 09 40 for animal experiences). All efforts were made to minimize the number of mice used and their suffering during the experimental procedure. Nude mice were housed in ventilated cages with food and water ad libitum in a 12 h light/dark cycle at 22 ± 1 °C.

Two-photon microscopy

Two-photon microscopy was performed using a Trimscope II (LaVision BioTec, Bielefeld, Germany) equipped with a 16× water-immersion objective (NA 0.8, Nikon, France) and Imspector 2015 software. The excitation wavelengths of the pulsed infrared laser (Insight DeepSea, Spectra-Physics, Evry, France) were 1000 nm for the red NPs and 750 nm for the blue NPs. The fluorescence emissions of GFP+ glioma cells and the NPs were epi-collected simultaneously by photomultiplier tubes using the following emission filters: 630/20 (Semrock, USA) for the red NPs, 492/SP (Semrock, USA) for the blue NPs and 542/50 for GFP+ cells.

Results and discussion

Scheme 1 shows the chemical structure and the synthetic scheme used to obtain dye 1. Lithiation of 2,7-dibromo-9,9-diethylfluorene with a stoichiometric amount of n-butyl lithium, followed by reaction with dimethylformamide and hydrolysis generates 7-bromo-9,9-diethylfluorene-2-carbaldehyde in 89% yield. A Buchwald–Hartwig coupling reaction with diphenylamine in the presence of palladium acetate, tert-butylphosphine and caesium carbonate affords aldehyde 7-(diphenylamino)-9,9-diethylfluorene-2-carbaldehyde 2 in 85% yield.^28,29 Finally, dye 1 is synthesized by a Knoevenagel condensation with indane-1,3-dione.

1@F127–SiO₂ NPs are prepared by optimizing the reported method.¹⁵ First, encapsulation efficiency is investigated by preparing NPs with different dye feeding ratios. The dye loading increases with the dye feeding ratio, until reaching a maximum of 92% encapsulation efficiency for an optimal feeding ratio of 0.15 wt%. Then, the encapsulation efficiency decreases when the dye feeding ratio is increased over that value (Table S1, Fig. S1, ESI†). TEM analysis and dynamic light scattering measurements reveal a uniform size of 22 nm with homogeneous distribution (Fig. S2, ESI†), in perfect agreement with what is reported for similar systems.¹⁵ NPs size remains constant upon incubation with Dulbecco's phosphate buffered saline, even after two weeks, demonstrating the excellent colloidal stability of 1@F127–SiO₂ (Fig. S4, ESI†).

Absorption (Fig. S11, ESI†) and fluorescence (Fig. 1-bottom) spectra of 1 are recorded in solvents of various polarities. Broad absorption bands centred around 500 nm are observed, showing a weak solvatochromism distinctive of internal charge transfer transitions in push–pull dipolar molecules. In contrast, the emission spectra are characterized by a large Stoke shift and a considerable positive solvatochromism, with maxima shifting from 537 nm in cyclohexane to 754 nm in DMSO (Fig. 1). This represents a 5360 cm⁻¹ bathochromic shift in emission, larger than for PRODAN, a well-known environment sensitive dye, for the same solvent systems.³⁰ Additionally, fluorescence quantum yields increase from 11% in apolar solvent (toluene) to 51% in chloroform, a solvent of medium polarity, to 1% in polar solvent (DMSO, Table 1). Interestingly, 1 also displays solid state fluorescence with intense emission at 656 nm in crystal (Φ_f = 20%), but much lower and red-shifted in nano-aggregates in water/acetone mixture (Φ_f = 5% at 669 nm). The two photon excitation spectrum, recorded in chloroform in the range 740–960 nm using a femtosecond Ti-sapphire laser, reveals a broad absorption band perfectly superposing twice the linear absorption as expected for non-centrosymmetric compounds (Fig. S12, ESI†). The two-photon cross sections σ_max in the range 740–960 nm are moderate of the order of 220 GM, with a maximum estimated over 300 GM at 1000 nm comparable to similar push–pull fluorophores based on a fluorene core.^26,29

Fig. 1 Top – Picture highlighting the solvatochromism in emission. Bottom – Absorption (in cyclohexane) and emission (λ_exc = 490 nm) spectra in organic solvents of various polarity.

Table 1 Spectroscopic properties of 1 in different solvents and in the solid state, and of 1@F127–SiO₂ NPs

Solvent	λmax/nm	λem/nm	Δν^a/cm^−1	Φf^b/%
a Stokes shift.b Quantum yields (in %) determined by using rubrene in methanol (Φf = 27%) as the standard.
Cyclohexane	504	537	1219	2
Toluene	495	584	3079	11
Toluene/CHCl3	503	646	4425	38
CHCl3	508	674	4848	51
CH2Cl2	501	707	5818	33
Acetone	486	725	6783	2
DMSO	496	754	6899	1
Solid (crystal)	—	656	—	20
Aggregate	493	669	5225	5
1@F127–SiO2 in water	498	650	4696	39

---

Absorption, emission and two-photon absorption spectra of 1@F127–SiO₂ NPs in PBS are shown in Fig. 2. The linear absorption presents a maximum at 498 nm, whereas the emission spectrum shows a maximum in the far-red at 650 nm, alike to what is observed in crystal, but with a two-fold increase in quantum yield (Φ_f = 39% against 20%). In comparison with nano-aggregates, this is an eight-fold enhancement in quantum yield along with a 25 nm hypsochromic shift of the maximum. Such Φ_f value of 39% is among the largest reported for red/NIR emissive nanoparticles.^7,10,11 The emission properties of 1@F127–SiO₂ NPs in PBS/water are actually comparable to what is obtained in toluene/CHCl₃ (1 : 1) mixture (Table 1). Two-photon absorption experiments are also consistent with what was observed for 1 in chloroform. This clearly indicates that, in nanoparticles, hydrophobic segments of F127 and the protective silica layer provide an apolar environment that isolates the chromophore molecules from contact with polar medium (water) and preserves their optical properties. Stability experiments, performed by monitoring the fluorescence intensity changes upon incubation with DPBS, do not show any decrease over a 14 days period, suggesting excellent physical stability of the prepared NPs. Besides, the photostability of 1@F127–SiO₂ NPs is also improved in comparison with the chromophore in chloroform without protection (Fig. S3, ESI†). It is worth noting that the F127–SiO₂ NPs core might contain other hydrophobic fluorophores emitting in different colours and even multiple fluorophores.¹⁸ This is interesting when multi-colour imaging is necessary, e.g. to compare the tumour vasculature before and after a therapy.

Fig. 2 Absorption and fluorescence emission (red) spectra (left and lower axis) and two-photon absorption cross-section (right and upper axis) of 1@F127–SiO₂ in water.

Preliminary cytotoxicity studies are performed by incubating HeGP2 cells with NPs solutions at different dilutions. No obvious cytotoxicity is observed: the cell viability remains over 80% after 24 h incubation (Fig. S6†). This corroborates previous observation of moderate cytotoxicity at low concentration on NIH/3T3,²⁰ HEK293 or A431 cell lines.²¹ The potential of F127–SiO₂ NPs for real-time two-photon blood vasculature imaging is next investigated on mice bearing tumours in the ear. Red emitting 1@F127–SiO₂ NPs are compared to commercial blood pool fluorophores such as Rhodamine B dextran (70 kDa) and to similar NPs bearing a blue emitting dye. Rhodamine B dextran has comparable molecular weight than the most abundant blood plasma protein, albumin (65 kDa). The leakage of this protein is often observed in tumours and is related to a higher interstitial fluid pressure.³¹ Immediately after intravenous injection of NPs, bright fluorescence from the whole blood vasculature network of mice ear is observed. Fig. 3 shows images of the blood vessels at 360 μm in depth, obtained with red emitting 1@F127–SiO₂ NPs 1 h after injection and using a 1000 nm excitation wavelength. The main blood vessels but also small capillaries deep below the tumour can clearly be observed, whereas the penetration depth with the blue emitting NPs is limited to the surface of the mouse ear (Fig. S7, ESI†). This highlights the benefits of combined NIR excitation/red emission for deep in vivo imaging. Fig. 3 and S9 (ESI†) also clearly show no leakage of 1@F127–SiO₂ NPs into the extra vascular tumour space during 1 h after intravenous injection. In comparison, Rhodamine B dextran 70 kDa is diffusing in the extra-vascular tumour space, which blurs the two-photon image of the tumour vasculature as shown Fig. 3D (see also Fig. S10, ESI and films in ESI†). The absence of 1@F127–SiO₂ NPs diffusion enables a time consuming 3D mosaic acquisition of the tumour vasculature in time (Fig. 4 and S8, ESI†). Diffusion of a fluorophore into the extra-vascular space during the acquisition would have over-estimated the blood volume as a function of acquisition time, making impossible comparisons of the blood volume in between tumour areas and normal tissue regions before and after an anti-angiogenic therapy.

Fig. 3 (A) 3D two-photon image (z-projection standard deviations of fluorescence intensities, z-stack 91 slices, step-size 2 μm, imaging depth 360 μm, excitation wavelength 1000 nm) at 1 h after injection of NPs showing the vasculature (red signals) and the tumour cells (glioma U87 green GFP signals). (B) The same as (A) without the cancer cells. (C) and (D) One slice of the z-stack: (C) is before i.v. injection of Rhodamine-B dextran 70 kDa and (D) is at 1 h after injection with a diffusion of Rh-B dextran in the extravascular space of the tumor. See ESI† for films of the fluorophore perfusion and diffusion. Scale bar = 100 μm.

Fig. 4 3D two-photon microscopy image of the functional vasculature (red signal of circulating 1@F127–SiO₂ NPs) at the surface of tumor (glioma U87 GFP cells in green) growing subcutaneously in a mouse ear. The image is a 3D reconstruction using free software (Blender) after a 3D mosaic acquisition (x–y plane: 6 × 6 images of 512² pixels with 91 slices in the z-direction, step-size 2 μm, imaging depth 360 μm, total acquisition time 5600 s).

Conclusions

In summary, a new push–pull dipolar dye 1 was synthesised presenting outstanding solvatochromic properties and interesting two-photon absorption properties. While 1 is barely emissive in high polarity solvents, an intense red emission (Φ_f = 39% at 650 nm) is restored in aqueous solution upon encapsulation, thanks to the non-polar environment provided by the micelle/silica nanoparticles inside. 1@F127–SiO₂ NPs are perfect blood pool fluorophores for deep NIR-NIR imaging of leaky tumour vasculature. No diffusion in the extra vascular space was observed for minimal 1 hour. This enables long 3D mosaic acquisitions for analyzing effects of anti-angiogenic therapies at a microscopic level for the whole tumour area.

Acknowledgements

Z. Zheng thanks the Chinese scholarship council and ENS de Lyon for a PhD grant. F. Caraguel acknowledges INSERM for the fellowship grant: PlanCancer 2009–2013 modeling complex biological processes. The intravital microscopy platform Grenoble was partly funded by the French program “Investissement d'Avenir” run by the “Agence Nationale pour la Recherche”; grant “Infrastructure d'avenir en Biologie Santé – ANR11-INBS-0006”. We thank F. Chaput (LC ENS, Lyon) for TEM imaging and W. Guan (IGFL, Lyon) for MTT cytotoxicity assays.

Notes and references

1. M. Rahman, D. Xie, H. I. Feldman, A. S. Go, J. He, J. W. Kusek, J. Lash, E. R. Miller III, A. Ojo, Q. Pan, S. L. Seliger, S. Steigerwalt and R. R. Townsend, Am. J. Nephrol., 2014, 40, 399–407; M. J. Ormseth, A. M. Oeser, A. Cunningham, A. Bian, A. Shintani, J. Solus, S. B. Tanner and C. M. Stein, Arthritis Rheumatol., 2014, 66, 2331–2338; J. Flammer, K. Konieczka and A. J. Flammer, EPMA Journal, 2013, 4, 14.
2. S. Goel, D. G. Duda, L. Xu, L. L. Munn, Y. Boucher, D. Fukumura and R. K. Jain, Physiol. Rev., 2011, 91, 1071–1121.
3. S. Ameer-Beg, P. R. Barber, R. J. Hodgkiss, R. J. Locke, R. G. Newman, G. M. Tozer, B. Vojnovic and J. Wilson, Proc. SPIE, 2002, 4620, 85–95; S. T. Proulx, P. Luciani, A. Alitalo, V. Mumprecht, A. J. Christiansen, R. Huggenberger, J.-C. Leroux and M. Detmar, Angiogenesis, 2013, 16, 525–540.
4. Z. Han, A. Fu, H. Wang, R. Diaz, L. Geng, H. Onishko and D. E. Hallahan, Nat. Med., 2008, 14, 343–349.
5. M. Maurin, L. Vurth, J.-C. Vial, P. L. Baldeck, S. R. Marder, B. van der Sanden and O. Stéphan, Nanotechnology, 2009, 20, 235102; M. Maurin, O. Stéphan, J.-C. Vial, S. R. Marder and B. van der Sanden, J. Biomed. Opt., 2011, 16, 036001.
6. L. Xiong, F. Cao, X. Cao, Y. Guo, Y. Zhang and X. Cai, Bioconjugate Chem., 2015, 26, 817–821; J. Xiang, X. Cai, X. Lou, G. Feng, X. Min, W. Luo, B. He, C. Goh, L. Ng, J. Zhou, Z. Zhao, B. Liu and B. Z. Tang, ACS Appl. Mater. Interfaces, 2015, 7, 14965–14974; B. Chen, G. Feng, B. He, C. Goh, S. Xu, G. Ramos-Ortíz, L. Aparicio-Ixta, J. Zhou, L. Ng, Z. Zhao, B. Liu and B. Z. Tang, Small, 2016, 12, 782–792.
7. Y. Gao, G. Feng, T. Jiang, C. Goh, L. Ng, B. Liu, B. Li, L. Yang, J. Hua and H. Tian, Adv. Funct. Mater., 2015, 25, 2857–2866.
8. Y. Wang, R. Hu, W. Xi, F. Cai, S. Wang, Z. Zhu, R. Bai and J. Qian, Biomed. Opt. Express, 2015, 6, 3783–3794.
9. C. Wu, Y. Jin, T. Schneider, D. R. Burnham, P. B. Smith and D. T. Chiu, Angew. Chem., Int. Ed., 2010, 49, 9436–9440; F. Ye, C. Wu, Y. Jin, M. Wang, Y.-H. Chan, J. Yu, W. Sun, S. Hayden and D. T. Chiu, Chem. Commun., 2012, 48, 1778–1780.
10. K. Li, Z. Zhu, P. Cai, R. Liu, N. Tomczak, D. Ding, J. Liu, W. Qin, Z. Zhao, Y. Hu, X. Chen, B. Z. Tang and B. Liu, Chem. Mater., 2013, 25, 4181–4187.
11. P. Liu, S. Li, Y. Jin, L. Qian, N. Gao, S. Q. Yao, F. Huang, Q.-H. Xu and Y. Cao, ACS Appl. Mater. Interfaces, 2015, 7, 6754–6763.
12. A. V. Kabanov, P. Lemieux, S. Vinogradov and V. Alakhov, Adv. Drug Delivery Rev., 2002, 54, 223–233.
13. A. V. Kabanov, E. V. Batrakova and V. Y. Alakhov, J. Controlled Release, 2002, 82, 189–212.
14. N. Rapoport, Colloids Surf., B, 1999, 16, 93–111.
15. Q. Huo, J. Liu, L.-Q. Wang, Y. Jiang, T. N. Lambert and E. Fang, J. Am. Chem. Soc., 2006, 128, 6447–6453; S. Zanarini, E. Rampazzo, S. Bonacchi, R. Juris, M. Marcaccio, M. Montalti, F. Paolucci and L. Prodi, J. Am. Chem. Soc., 2009, 131, 14208–14209.
16. E. Rampazzo, S. Bonacchi, R. Juris, M. Montalti, D. Genovese, N. Zaccheroni, L. Prodi, D. C. Rambaldi, A. Zattoni and P. Reschiglian, J. Phys. Chem. B, 2010, 114, 14605–14613; D. Genovese, S. Bonacchi, R. Juris, M. Montalti, L. Prodi, E. Rampazzo and N. Zaccheroni, Angew. Chem., Int. Ed., 2013, 52, 5965–5968.
17. E. Rampazzo, F. Boschi, S. Bonacchi, R. Juris, M. Montalti, N. Zaccheroni, L. Prodi, L. Calderan, B. Rossi, S. Becchi and A. Sbarbati, Nanoscale, 2012, 4, 824–830.
18. E. Rampazzo, S. Bonacchi, D. Genovese, R. Juris, M. Montalti, V. Paterlini, N. Zaccheroni, C. Dumas-Verdes, G. Clavier, R. Méallet-Renault and L. Prodi, J. Phys. Chem. C, 2014, 118, 9261–9267.
19. Y. Zhang, M. Wang, Y.-G. Zheng, H. Tan, B. Y.-W. Hsu, Z.-C. Yang, S. Y. Wong, A. Y.-C. Chang, M. Choolani, X. Li and J. Wang, Chem. Mater., 2013, 25, 2976–2985.
20. J. Geng, C. C. Goh, N. Tomczak, J. Liu, R. Liu, L. Ma, L. G. Ng, G. G. Gurzadyan and B. Liu, Chem. Mater., 2014, 26, 1874–1880.
21. L. Yan, H. Wang, A. Zhang, C. Zhao, Y. Chen and X. Li, J. Mater. Chem. B, 2016, 4, 5560–5566.
22. J. Geng, C. C. Goh, W. Qin, R. Liu, N. Tomczak, L. G. Ng, B. Z. Tang and B. Liu, Chem. Commun., 2015, 51, 13416–13419.
23. S. A. Hilderbrand and R. Weissleder, Curr. Opin. Chem. Biol., 2010, 14, 71–79; S. Luo, E. Zhang, Y. Su, T. Cheng and C. Shi, Biomaterials, 2011, 32, 7127–7138; C.-H. Quek and K. W. Leong, Nanomaterials, 2012, 2, 92–112; E. M. Sevick-Muraca, Annu. Rev. Med., 2012, 63, 217–231.
24. D. Kobat, N. G. Horton and C. Xu, J. Biomed. Opt., 2011, 16, 106014; P. Sarder, S. Yazdanfar, W. J. Akers, R. Tang, G. P. Sudlow, C. Egbulefu and S. Achilefu, J. Biomed. Opt., 2013, 18, 106012; S. Koga, Y. Oshima, N. Honkura, T. Iimura, K. Kameda, K. Sato, M. Yoshida, Y. Yamamoto, Y. Watanabe, A. Hikita and T. Imamura, Cancer Sci., 2014, 105, 1299–1306; S. Peron, T.-W. Chen and K. Svoboda, Curr. Opin. Neurobiol., 2015, 32, 115–123; P.-P. Yang, Y. Yang, Y.-J. Gao, Y. Wang, J.-C. Zhang, Y.-X. Lin, L. Dai, J. Li, L. Wang and H. Wan, Adv. Opt. Mater., 2015, 3, 646–651.
25. X. Wang, A. R. Morales, T. Urakami, L. Zhang, M. V. Bondar, M. Komatsu and K. D. Belfield, Bioconjugate Chem., 2011, 22, 1438–1450; M.-Q. Zhu, G.-F. Zhang, C. Li, M. P. Aldred, E. Chang, R. A. Drezek and A. D. Q. Li, J. Am. Chem. Soc., 2011, 133, 365–372; J. Qian, D. Wang, F. Cai, Q. Zhan, Y. Wang and S. He, Biomaterials, 2012, 33, 4851–4860; J. Massin, A. Charaf-Eddin, F. Appaix, Y. Bretonnière, D. Jacquemin, B. van der Sanden, C. Monnereau and C. Andraud, Chem. Sci., 2013, 4, 2833–2843; Z. Guo, S. Park, J. Yoon and I. Shin, Chem. Soc. Rev., 2014, 43, 16–29; Z. Zhao, B. Chen, J. Geng, Z. Chang, L. Aparicio-Ixta, H. Nie, C. C. Goh, L. G. Ng, A. Qin, G. Ramos-Ortiz, B. Liu and B. Z. Tang, Part. Part. Syst. Charact., 2014, 31, 481–491; Y. Lv, P. Liu, H. Ding, Y. Wu, Y. Yan, H. Liu, X. Wang, F. Huang, Y. Zhao and Z. Tian, ACS Appl. Mater. Interfaces, 2015, 7, 20640–20648.
26. O. A. Kucherak, P. Didier, Y. Mély and A. S. Klymchenko, J. Phys. Chem. Lett., 2010, 1, 616–620.
27. J. Massin, W. Dayoub, J.-C. Mulatier, C. Aronica, Y. Bretonnière and C. Andraud, Chem. Mater., 2011, 23, 862–873; M. Ipuy, Y.-Y. Liao, E. Jeanneau, P. L. Baldeck, Y. Bretonnière and C. Andraud, J. Mater. Chem. C, 2016, 766–779.
28. A. Baheti, P. Tyagi, K. R. J. Thomas, Y.-C. Hsu and J. T. Lin, J. Phys. Chem. C, 2009, 113, 8541–8547.
29. A. R. Morales, A. Frazer, A. W. Woodward, H.-Y. Ahn-White, A. Fonari, P. Tongwa, T. Timofeeva and K. D. Belfield, J. Org. Chem., 2013, 78, 1014–1025.
30. G. Weber and F. J. Farris, Biochemistry, 1978, 18, 3075–3078.
31. G. Baronzio, G. Parmar, M. Baronzio and M. Kiselevky, Cancer Genomics Proteomics, 2014, 11, 225–237.

---

Footnote

† Electronic supplementary information (ESI) available: Experimental details, additional figures, films and characterizations. See DOI: 10.1039/c6ra17438h

---