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A novel POSS-PCU nanocomposite polymer as a biocompatible coating for quantum dots Sarwat Butool Rizvi, Shi Yu Yang, Mark A Green, Mo Keshtgar, and Alexander M. Seifalian Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00462 • Publication Date (Web): 06 Oct 2015 Downloaded from http://pubs.acs.org on October 14, 2015

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Bioconjugate Chemistry

Purification causes loss of MUA

QDs fall out of solution- hydrophobic

Hydrophobic QDs stabilise within the core of the polymer micelle MUA coated QD with a hydrohillic surface

POSS

emulsification forms polymer micelles with a hydrophobic core and hydrophillic surface

POSS terminated prepolymer chain

Polymer encapsulated QDs are photostable and biocompatible

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A novel POSS-PCU nanocomposite material as a biocompatible coating for quantum dots Sarwat B Rizvi1, Shi Yu Yang1, Mark Green2, Mo Keshtgar1,3 and Alexander M Seifalian1,3* 1

Centre for Nanotechnology & Regenerative Medicine, UCL Division of Surgery &

Interventional Science, University College London, London, United Kingdom, 2Department of Physics, King’s College London, London, United Kingdom. 3Royal Free Hampstead NHS Trust Hospital, London, United Kingdom.

*Correspondence: Professor Alexander M. Seifalian, Professor of Nanotechnology & Regenerative Medicine University College London Email: [email protected] Tel: +44 7985380797

Abbreviations MUA: Mercaptoundecanoic acid, POSS-PCU: Poly (carbonate-urea) Urethane Polyhedral Oligomeric Silsesquioxane, uQDs: MUA coated QDs (referred to as uncoated QDs), cQDs: POSS-PCU polymer coated QDs (referred to as coated QDs)


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Abstract Quantum dots (QDs) are fluorescent nanoparticles with unique photophysical properties that enable them to potentially replace traditional organic dyes and fluorescent proteins in various bio-imaging applications. However, the inherent toxicity of their cores based on cadmium salts, limits their widespread biomedical use. We have developed a novel nanocomposite polymer emulsion based on Polyhedral Oligomeric Silsesquioxane Poly (carbonate-urea) Urethane (POSS-PCU) that can be used to coat quantum dots to nullify their toxicity and enhance photostability. Here we report the synthesis and characterization of a novel POSSPCU nanocomposite polymer emulsion and describe its application for coating QDs for biological application. The polymer was synthesized by a process of emulsion polymerization and formed stable micelles of ~ 33nm in diameter. CdTe/CdS/ZnS QDs were efficiently stabilised by the polymer emulsion through encapsulation within the polymer micelles. Characterisation studies showed no significant change in the unique photophysical properties of QDs after coating. The polymer was biocompatible to HepG2, HUVECs and Mouse skeletal muscle cells at 2.5% after a 24 hours exposure on in vitro testing. Polymer encapsulated QDs showed enhanced photostability on exposure to high degrees of UV irradiation and air as well as significantly reduced cytotoxicity on exposure to HepG2 cells at 30µg/mL for 24 hours. We have therefore concluded that the POSS-PCU polymer emulsion has a potential to make a biocompatible and photostable coating for QDs enabling a host of biomedical applications to take this technology to the next level.

Keywords: Quantum Dots; POSS; Biocompatible coating; Amphiphilic polymer


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Introduction

Quantum dots (QDs) or semiconductor nanocrystals have been the focus of intense research over the past decade.

Their advantages over the traditional fluorophores in biological

sciences are well established1 and they promise to be the next generation fluorophores. QDs are composed of materials from the elements in the periodic groups II-VI, III-V or VI with a core/shell structure and a size ranging from 2-10 nm (200-10000 atoms)2. Their inorganic core is mostly based on salts of cadmium, tellurium and selenium surrounded by a wider band gap shell of semiconductor material (e.g. Zinc sulphide or Zinc selenide). The surrounding shell enhances the QDs’ quantum yield and photostability through protection of their surface against oxidation and removal of surface defects by entrapment of excitons to the core. The effects of quantum confinement give QDs unique photophysical properties such as size tunability, photostability, broad absorption with narrow emission spectra, lower excited state decay rates, and higher quantum yields (QY). Multiple coloured QDs emitting from the UV to the Near Infra-Red wavelengths can all be excited with a single wavelength of light as long as this wavelength is less than their absorption onset. This unique property of multiplexed imaging has important bearing in their application for biological imaging whereby multiple targets can be localised at one point in time with a single wavelength of light. Moreover, near infrared emitting QDs can revolutionise biomedical imaging as biological tissue is fairly transparent to near infra red wavelengths allowing relentless possibilities of deep tissue imaging. The potential biomedical applications of QDs include cancer localisation3-5, sentinel lymph node biopsy6, detection of micrometastasis7,8, image guided targeted drug delivery for chemotherapeutic agents9-12 and photodynamic therapy (PDT)13. QD technology forms one of the most promising frontiers in personalised medicine that would allow the diagnosis and treatment of disease at a truly molecular and cellular level. The greatest hindrance to the realisation of this application is their toxicity which stems from various physical properties including their nano-size, concentration, surface charge14-18, inorganic core components, surface coating, photochemical oxidation and mechanical stress. The bare nanocrystal is extremely toxic and while the ZnS shell offers some protection it is not sufficient to shield the core from oxidation due to the presence of oxidizing species in the biological environment as well the effects of UV radiation. Surface coating forms the most significant determinant of overall QD toxicity apart from providing three unique properties for their biomedical


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application. Firstly it makes the QDs more biocompatible through making it soluble in aqueous solutions. Secondly it shields the toxic core from the surrounding media therefore prevent their toxicity from damaging tissues and cells. Finally, it provides free surface reactive groups for biofunctionalization which are required for their eventual targeted application.

The use of amphiphilic polymers as a surface coating for QDs has generated considerable interest among researchers. Various polymers including poly(maleic anhydride-aly-1octadene)19,20, and block copolymers (e.g. polysterene-b-poly(acrylic acid))21, poly(methyl methacrylate)-poly(ethyleneoxide) and amphiphillic hyperbranched polyethylenimine2,22 have been used to coat QDs. Other amphiphylic polymers include octylamine-modified polyacrylic acid and polyethylene glycol (PEG)-modified lipids that prevent non-specific interaction of QDs with cells and RES uptake23,24. However, the prolonged stabililty of these polymers in the oxidative biological environment is uncertain. Mancini et al25 reported the quenching and chemical degradation of polymer coated QDs by ROS as they diffuse across the polymer coating, leading to chemical oxidation of the sulphur or selenium atoms on the QD surface. This ‘etching’ process leads to lattice defects causing fluorescence quenching followed by the release of soluble toxic chalcogenide species.

We have synthesized and characterized a novel nanocomposite polymer emulsion based on Polyhedral Oligomeric Silsesquioxane (POSS) chemically integrated into Poly (CarbonateUrea) urethane (PCU) and used it as a coating for semiconductor quantum dots (figure 1). Polyhedral oligomeric silsesquioxane (POSS) is a cage-like silsesquioxane molecule of approximately 1.5 nm in diameter, containing an inner inorganic framework composed of silicone and oxygen (SiO1.5)n , that is externally covered by organic substituents and could be regarded as the smallest possible silica particle26. Unlike silica and silicones, the organic constituents on the outer surface of POSS make it compatible with polymers, biological systems or surfaces26. As POSS is extremely hydrophobic it imparts amphiphilic properties to the polyurethane emulsion. The POSS integrated PCU emulsion is therefore an amphiphilic block copolymer that self-organises into micelles in an aqueous solvent to form a hydrophobic core and hydrophilic shell (figure 2). The advantages of using a POSS integrated copolymer for QD coating are based on the inherent properties of the POSS as it enhances the mechanical properties of PCU and imparts properties of greater biodegradative resistance to


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oxidation and hydrolysis27 as well as properties like antithrombogenicity28 lower immunogenicity and biocompatibility.


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Figure 1. Reaction scheme for the synthesis of the POSS-PCU emulsion


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A

B

C

Figure 2. POSS-PCU polymer micelle structure and QD encapsulation. A) Chemical Structure of a TransCyclohexane Chlorohydrin Isobutyl POSS -terminated polymer chain. B) Schematic diagram of the types of chains forming the pre-polymer and structure of a polymer micelle. C) Encapsulation of QDs by the polymer micelle. Several QDs stabilise in the core of the polymer micelle through hydrophobic interactions without causing significant change in the overall hydrodynamic diameter of the polymer micelle.


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The solid form of the nanocomposite (POSS-PCU) polymer has been awarded an international patent with a trade name of UCLNano™. It has found application in the development of biomedical devices like nose grafts, vascular grafts, heart valves and cardiac stents29-31 as well as nasolacrimal ducts and a tissue engineered trachea that has successfully been implanted in man.

In this study we used the emulsion form of the POSS-PCU

nanocomposite polymer to coat quantum dots for biomedical application. This paper reports the synthesis and characterization of this novel nanocomposite polymer emulsion and its application for coating QDs. For the purpose of this study mercaptoundecanoic acid (MUA) coated QDs are referred to as uncoated (uQDs) and POSS-PCU polymer emulsion coated QDs are referred to as coated (cQDs). The biocompatibility and cytotoxicity of the polymer alone and both polymer coated and uncoated QDs has also been analysed.

Results Synthesis of the POSS-PCU polymer emulsion As the TMXDI reacts with the single -OH group on the trans-Cyclohexane Chlorohydrin Isobutyl POSS, the latter forms a terminal group on the polymer chain (figure 2A) rather than pendant groups on a polymer backbone as in some other formulations of previously reported

POSS-PCU

27,28,32,33

. The polymer in our emulsion contains 2% POSS and therefore,

statistically, all polymer chains should be terminated by a POSS moiety. This is based on the fact that the molecular weight of the polymer is around 50 000, measured by Gel Permeation Chromatography (GPC), which at 2% POSS would equate to one mole of POSS per mole of polymer. However, it is possible that there are three types of polymer chains in the prepolymer (figure 2B), either bearing no POSS or a single POSS unit as a terminal moiety or rarely 2 POSS units at each end of the chain. As POSS is extremely hydrophobic in an aqueous emulsion, the polymer nanoparticles self-organise into micelles bearing a hydrophobic POSS core surrounded by poly (carbonate-urea) urethane chains, with the COOH groups on the DMPA as the hydrophilic interface (figure 2A). The free -COOH groups not only serve to ensure solubility in aqueous media but also provide reactive groups for liganding to biomolecules for a specific targeted action of polymer coated QDs. Figure 2C demonstrates the encapsulation of QDs by the POSS-PCU polymer micelle. After purification the MUA coated QDs (uQDs) fall out of solution and are easily stabilised within the hydrophobic core of the polymer micelle. POSS-PCU polymer emulsion is therefore an amphiphilic block copolymer capable of self-assembly into polymer micelles. This is similar


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to a core/shell structure previously reported for POSS grafted PVA nanoparticles34 except that POSS-PCU is non-biodegradable and resistant to oxidation and hydrolysis in the solid form27.

Characterization of the POSS-PCU polymer emulsion Dynamic Light Scattering is an excellent technique to assess the hydrodynamic diameter of nanoparticles. Most polymer micelles were found to be ~ 32.9 nm (+/- SD 4.3) in diameter with a smaller peak evident at 79nm (figure 3A). This result was correlated with TEM studies by counter staining the background with 2% uranyl acetate (figure 3B). TEM showed majority of the polymer micelles of approximately 33-34nm with a few larger micelles of 60120nm likely to be formed as a fusion of smaller ones.

A

B

Figure 3. Size distribution of QDs and POSS-PCU polymer micelles. A) DLS showed a peak hydrodynamic diameter of 32.9nm with polymer micelles ranging in size from 30-120nm. There is no significant change in the size of polymer micelles after coating (cQDs). Also all the uQDs are coated by the polymer as evidenced by the disappearance of the uQD peak after coating. B) TEM image of polymer micelles counterstained with 2% uranyl acetate shows sizes similar to the size range of the polymer micelles (30-105nm).

UV-Vis absorption and fluorescence emission studies (figure 4) revealed that the polymer absorbs in the UV range and emits at around 460nm and this explains its bluish white appearance after initial synthesis. Various dilutions of the polymer emulsion were prepared in PBS and incubated at 37°C for 7 days to mimic a physiological environment. UV-vis absorption and fluorescence studies showed no change in comparison to those placed at room temperature for the same period of time, indicating that the polymer remained stable in a physiological environment. The concentration of the polymer after synthesis was 34.4% and this was confirmed by determining the dry weight. The pH of the raw polymer emulsion was ACS Paragon Plus Environment

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9.3 but this dropped to physiological pH of 7.5 after dilution with PBS with no effect on the colloidal stability. Centrifugation of the diluted polymer (1.25%) caused large aggregates to settle down and the supernatant appeared to be a homogenous milky colloidally stable dispersion. The dry weight of the centrifuged polymer was 0.56%. Filtration of the diluted polymer (1.25%) yielded a dispersion that appeared less milky with a dry weight of 0.28%

Fluorescence emission (a.u)

and like the centrifuged polymer, remained colloidally stable on standing.

A

400 350 300 250 200 150 100 50 0

uQDs cQDs (POSS-PCU 2.5%) POSS-PCU 2.5%

500

600 700 Wavelength (nm)

800

B uQDs

4 3.5

Absorbance (a.u)

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cQDs (POSS-PCU 2.5%) POSS-PCU 2.5%

3 2.5 2 1.5 1 0.5 0 300

400

500 600 700 Wavelength (nm)

800

Figure 4. Photoluminescence and UV-vis absorption spectroscopy A) MUA coated CdTe/CdS/ZnS QDs (uQDs) emit at 680nm. A blue-shift of ~ 10nm is evident in the peak emission wavelength after POSS-PCU coating (cQDs). However, the POSS-PCU emulsion does not interfere with QD fluorescence. B) The absorption onset of QDs remains unaltered at 460nm after POSS-PCU coating.

Characterization of POSS-PCU coated QDs


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UV-vis absorption spectra of the uncoated QDs (uQDs) showed an absorption onset at 460nm and this remained unchanged after coating (figure 4B). Emission spectra of the polymer coated QDs (cQDs) showed that they emit at 680nm and this undergoes a slight blue shift of ~ 10nm after coating (figure.4A). TEM studies demonstrated that uQDs ranged in size from 36nm showing considerable clustering and aggregation (figure 5A). This effect was largely removed in the cQDs that maintained their size distribution but appeared spaced out. It was evident that several QDs were encapsulated in each polymer micelle. While TEM confirmed the non-aggregated nature of the cQDs, it could not be used to show their overall radius as polymer micelles are not visible on TEM without a uranyl acetate contrast that darkens the background and masks the QDs. We therefore used Dynamic Light Scattering (DLS) to compare the hydrodynamic diameter of the naked and coated QDs (figure 3A).

The

hydrodynamic diameter of the uQDS was ~ 4nm (+/_ 0.8) and this correlated with the core diameter on TEM. The hydrodynamic diameter of the cQDs closely matched that of the polymer micelles showing a peak at 32.9nm (+/_ 4.3nm) with no evidence of particles at 4nm indicative of free QDs. This indicated that the polymer micelles had encapsulated all the QDs in solution without undergoing a significant change in size. A smaller peak at 79nm (+/_ 19.8nm) was evident in the cQD sample and this was similar to that of polymer micelles. This may represent larger micelles though trapped QDs in these aggregates could not be excluded.


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A

B

Figure 5. TEM A) MUA coated QDs (uQDs) appear aggregated and clustered. B) POSS-PCU coated QDs (cQDs) appear well spaced out as several QDs are encapsulated by each polymer micelle. POSS-PCU coating is not electron dense and therefore not visible on TEM without staining.

Photostability of the coated QDs The fluorescence emission intensity of uQDs on sustained UV excitation in the presence of air showed a significant drop compared to the cQDs at the end of a 2 hour period (p-value

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