Priming the pores of mesoporous silica nanoparticles with an in-built RAFT agent for anchoring a thermally responsive polymer

Accepted Manuscript Priming the pores of mesoporous silica nanoparticles with an in-built RAFT agent for anchoring a thermally responsive polymer Smrutirekha Mishra, James M. Hook, Leena Nebhani PII:

S1387-1811(18)30543-2

DOI:

10.1016/j.micromeso.2018.10.012

Reference:

MICMAT 9150

To appear in:

Microporous and Mesoporous Materials

Received Date: 11 August 2018 Revised Date:

28 September 2018

Accepted Date: 15 October 2018

Please cite this article as: S. Mishra, J.M. Hook, L. Nebhani, Priming the pores of mesoporous silica nanoparticles with an in-built RAFT agent for anchoring a thermally responsive polymer, Microporous and Mesoporous Materials (2018), doi: https://doi.org/10.1016/j.micromeso.2018.10.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Priming the pores of mesoporous silica nanoparticles with an in-built RAFT agent for anchoring a thermally responsive polymer Smrutirekha Mishra,a James M. Hookb and Leena Nebhani*a

Organically

modified

silica,

co-condensation,

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polymerization, organic-inorganic hybrid

Abstract

surface-initiated

RAFT

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Keywords:

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Smrutirekha Mishra,a Prof. Leena Nebhania* a Department of Materials Science and Engineering, Indian Institute of Technology Delhi, New Delhi-110016, India E-mail: [email protected] Dr. James M. Hookb b Mark Wainwright Analytical Centre and School of Chemistry, University of New South Wales, Sydney, NSW, 2052, Australia

RAFT mesoporous silica nanoparticles (MSNs) have been synthesized for the first time via co-condensation

of

an

organoalkoxysilane

RAFT

agent,

1-phenylethyl(3-

triethoxysilyl)propyl)carbonotrithioate, with tetraethoxysilane (TEOS) in aqueous basic

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cetyltrimethylammonium bromide. The stability of the organoalkoxysilane RAFT agent during the synthesis of MSNs was established using solution NMR and UV-visible

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spectroscopies. The RAFT-MSNs were then used for elaboration with N-isopropylacrylamide (NIPAM) via controlled surface-initiated RAFT polymerization. The success of the

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incorporation of the organoalkoxysilane RAFT agent into the silica network, and polymerization of PNIPAM was confirmed with TGA, FT-IR, UV-visible spectroscopy, and

29

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Si solid state NMR spectroscopy. The effect of organoalkoxysilane based RAFT agent

and PNIPAM grafting on the morphology, size and surface area of the resulting MSNs was investigated using SEM, TEM, XRD, DLS and BET analysis. The appearance of carbon signals in 13C solid state NMR, T signals in 29Si solid state NMR and C-H stretching signals in FT-IR establishes that we have successfully obtained organic-inorganic hybrid materials. The pore area calculated using Barrett-Joyner-Halenda (BJH) analysis was 87.8 m2.g-1 for 1

ACCEPTED MANUSCRIPT control-MSNs, 91.1 m2.g-1 for RAFT-MSNs and 132.9 m2.g-1 for PNIPAM-MSNs. The thermoresponsive behaviour of PNIPAM grafted inside the pores of MSNs was studied by dye as well as drug loading experiments at variable temperatures, followed by characterization using confocal laser scanning microscopy. The green and red fluorescence

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corresponding to fluorescein and doxorubicin respectively was strongly retained in the PNIPAM-MSNs after 24 h of incubation at 25°C while lack of fluorescence after 24 h incubation at 35°C, demonstrates the thermoresponsive behaviour of the polymer being

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grafted inside the pores of mesoporous silica nanoparticles.

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1. Introduction

Cutting-edge achievements have been made with the controlled synthesis of different types of surfactant templated mesoporous silica nanoparticles (MSNs) in recent years. The possibility of organically functionalizing[1] interior[2] and exterior[3] surfaces of

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MSNs utilizing various organoalkoxysilanes is one of the most interesting and challenging areas in the field of surface engineering today. MSN’s superlative properties such as high surface area, ordered pore structure and uniform pore size

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distribution has been the motivation for further modification, functionalization and application. Thus, depending on the functional organic groups present in or on MSNs,

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they have been widely used in catalysts,[4] sensors,[5] absorbents[6] and drug delivery.[7] Due to amorphous walls[8] and large inner surface area features, MSNs provide an effective platform for covalent inclusion of a variety functional groups. The main pathway for these processes includes post-functionalization[9] in which an organic group is covalently attached to the inorganic surface after the synthesis of the MSN, or by co-condensation[10] which involves incorporation of the organic group into the synthesis of the condensed porous inorganic structure. Babonneau et al.,[1] Fowler et al.,[11] and Simon et al.[12] highlight the elegance of co-condensation as a highly 2

ACCEPTED MANUSCRIPT convergent approach, when conditions are favourable for inclusion of organic groups, such as alkylamino, alkylthiol, phenyl, which eventually populate the interior and exterior surfaces of the MSN. Similarly, Lim et al.[13] functionalized MCM-41 mesoporous silica with vinyl groups by post-functionalization, as well as by co-

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condensation. The chemical modification/functionalization of the surface can be utilized as a prop for promoting the growth of polymeric chains.[14] Depending on the functional group present, a diverse range of monomers can be successfully

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polymerized on the MSNs, using several approaches for covalent bonding of polymeric chains. Broadly, they can be classified as "grafting-to"[15,16],"grafting-from"[14,17,18]. In

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the "grafting-to" approach, the intact polymer is grafted onto the silica surface, while in "grafting-from" approach, the polymer chain is grown from a suitably modified surface. In general “grafting to” is complicated by steric hindrance, while "graftingfrom" technique allows the growth of polymeric chains directly from the surface

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without any steric hindrance and thus leads to a higher grafting density. Ma et. al.[17] reported use of RAFT and “grafting-from” technique for polymerization of charged quaternary amines and poly(ethylene glycol) methyl ether methacrylate on the surface

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of MSNs via a two-step procedure. The polymer modified MSNs were utilized for in vivo drug release studies. Huang and co-workers[15] used “grafting-to” technique for

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modification of flash silica using variety of polymers and copolymers synthesized using RAFT polymerization. All the synthesized polymers contained trimethoxy functionality which was used for modification of silica. The grafting density of polymeric chains on silica particles was reported around 0.018-0.076 chains/nm2. MSNs have been consistently utilized for drug delivery applications because of the uniform pore size and pore volume.[19] One of the major challenges for this purpose, however, is to find appropriate gatekeepers to keep the loaded guest molecules intact, and to this end, thermoresponsive polymers[20] such as poly(N-isopropyl acrylamide) 3

ACCEPTED MANUSCRIPT (PNIPAM)[21] have been in high demand for such a role. Numerous reports [22-24] have been described for grafting PNIPAM onto silica surfaces. Brunella et al.[25] reported grafting onto the modified surface of MCM-41 mesoporous silica, in two stages: first by modifying the MSN with 3-methacryloxypropyltrimethoxysilane in which the

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trimethoxysilane group binds to the MSN surface, leaving the methacrylate group available for free radical polymerization with NIPAM. The polymer-coated pores were then loaded with the anti-inflammatory, ibuprofen, and the copolymer graft acted as

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the gatekeeper to hold the drug load intact. It was concluded that significant release of ibuprofen was observed at 40°C which is above LCST of PNIPAM while at 25°C the

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extent of release of drug is lower as polymer chains are in extended form. Another example by Hua et al.[26] in which thiol functionalized silica particles were polymerized with PNIPAM chains onto the surface by photopolymerization. Leaching of the silica from the solid enabled the preparation of hollow particles for applications

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in drug and gene delivery.

Addition of RAFT agents to the surface of MSNs via post-functionalization,[27-29] however, is limited by its lack of convergence and is therefore multistep in nature, involving first

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synthesizing MSNs followed by their functionalization with an organoalkoxysilane. In addition, not all silanols groups are available for further functionalization. A diverse range of

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morphologies can be obtained via co-condensation of TEOS with hydrophobic/hydrophilic organoalkoxysilanes in the presence of a template directing agent. For example, Zhang et al.[30] have reported the formation of different morphologies such as rods and spheres by using different alkyl substituted organoalkoxysilanes. Likewise Lin et al.[31] showed the formation of rod-like and spherically shaped particles using different hydrophilic and hydrophobic organoalkoxysilanes. The hydrogen bonding and hydrophobic interactions between the organoalkoxysilanes and surfactant molecules at the interface would appear to be strongly influencing the changes in the morphology of the MSNs. 4

ACCEPTED MANUSCRIPT Our concept is to create instead, a highly convergent pathway to MSN’s already primed for further RAFT based elaboration. The "grafting-from" approach is a more versatile method for surface functionalization allowing polymeric chains to grow directly from the surface bound initiators with less steric hindrance. It does not depend

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on diffusion of large polymeric chains to the surface thus leading to higher grafting densities, as required by “grafting-to”. In practice, we report here for the first-time this concept realized in the synthesis of MSNs by co-condensing an organoalkoxysilane

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based RAFT agent, (1) with TEOS, in the presence of the template CTAB, affording an MSN bearing an in-built RAFT agent (2) (Scheme 1). This RAFT-MSN, once isolated

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and fully characterized, could be readily elaborated further with NIPAM via a "grafting-from" surface-initiated RAFT polymerization, to give the thermally

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responsive PNIPAM-MSNs (3) (Scheme 1).

Scheme 1. Co-condensation of organoalkoxysilane based RAFT agent [1-phenylethyl(3triethoxysilyl)propyl)carbonotrithioate] (1) with TEOS and template, CTAB, leading to the synthesis of organoalkoxysilane based RAFT agent functionalized MSNs (RAFT-MSNs) (2), 5

ACCEPTED MANUSCRIPT which were functionalized in the next step with PNIPAM via controlled surface-initiated RAFT polymerization producing PNIPAM-MSNs (3).

2.1 Materials Tetraethylorthosilicate

(TEOS,

98%),

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2. Experimental Section

1-bromoethylbenzene

(98%),

3-

mercaptopropyltriethoxysilane (98%), phosphate-buffered saline (PBS) and doxorubicin were

purchased

from

TCI

India

and

were

used

as

received.

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hydrochloride

Cetyltrimethylammonium bromide (CTAB, 98%), was received from Spectrochem, India.

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Sodium hydroxide, distilled water, carbon disulphide (97%), dimethylformamide (DMF) and chloroform were purchased from Merck, India. Magnesium sulphate (97%) and ethanol was purchased from Fisher Scientific, India. N-isopropylacrylamide (NIPAM, 99%) was purchased from TCI India and was re-crystallized prior to use. Azobis(isobutyronitrile)

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(AIBN, 98%) was purchased from Sigma-Aldrich, India and was recrystallized from methanol and dried under vacuum prior to use. 2.2 Characterization

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Fourier Transform Infra-Red Spectroscopy (FTIR) Uniform size (1 mm thick) pellets were prepared from the samples: Control-MSNs, RAFT-

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MSNs, PNIPAM-MSNs; with KBr powder (1/10) using an hydraulic press. FTIR spectra were recorded on a Thermo Nicolet IR 200 spectrometer in the spectral range of 400 – 4000 cm-1.

Thermogravimetric analysis (TGA) Thermogravimetric analysis (TGA) was performed on a Perkin-Elmer Pyris-6 TGA at a heating rate of 10°C.min-1 under nitrogen atmosphere. All samples (Control-MSNs, RAFTMSNs, PNIPAM-MSNs) were dried under vacuum for more than 24 h prior to analysis. Transmission electron microscopy (TEM) 6

ACCEPTED MANUSCRIPT The mesoporous structure of the samples: Control-MSNs, RAFT-MSNs, PNIPAM-MSNs, was analyzed using transmission electron microscopy (TEM) TECHNAI series G² model using a field emission gun at an acceleration voltage of 200 keV with turbo modular pump system. Samples were prepared by dispersing in ethanol and followed by mounting on a

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copper grid. Scanning electron microscopy (SEM)

The morphological shape and size of the samples: Control-MSNs, RAFT-MSNs, PNIPAM-

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MSNs were investigated using ZEISS EVO 50 series instrument operating at 20 keV acceleration voltage with 6 mm analytical working distance under vacuum. Samples were

then gold coated prior to analysis. UV-visible Spectroscopy

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prepared by mounting them on glass cover slips on an aluminium sample stub. Samples were

UV-Visible spectra were measured with a T90+ UV Spectrometer. The absorption spectra

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were recorded from 200 to 600 nm using ethanol as a solvent. Dynamic light scattering (DLS)

DLS was measured using Malvern Zetasizer NANO ZS 90. All measurements are average of

X-ray diffraction

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at least three measurements.

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Small-angle powder X-ray diffraction (XRD) measurements were performed on a SAXSpace Anton Paar with dual beam set up. The XRD patterns were collected using a Cu Kα radiation (α=0.1542 nm) with SAXS position of 317 mm. All measurements were done with imaging plate detection system with exposure time of 10 minutes for 1 frame. Confocal Laser Scanning Microscopy The fluorescein and doxorubicin loaded control-MSNs and PNIPAM-MSNs were analyzed using TCS-SP5X Leica confocal laser scanning microscope with 60x oil objective setup.

7

ACCEPTED MANUSCRIPT Samples dispersed in ethanol were prepared by sealing the solution between a 1”×3” glass slide and a cover slide. Surface area measurement The surface area of the MSNs was measured using a Nova-e series LX version quantochrome

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2012 accelerated P0 station surface area analyzer. All samples were dried overnight at 100°C under vacuum before carrying out nitrogen adsorption experiments at 77 K. Size Exclusion Chromatography (SEC)

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The molecular weight of the polymer cleaved from the surface of PNIPAM-MSNs was determined by a Water series 1515 gel permeation chromatograph (GPC) equipped with

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Water 1515 column and Water 2414 refractive index detector system. The measurement was performed using THF as an eluent at a flow rate of 0.5 ml.min-1 at 30°C. Nuclear Magnetic Resonance Spectroscopy (NMR)

NMR spectra of soluble materials were recorded as solutions in CDCl3, with a Bruker DPX-

Solid state NMR 13

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400 spectrometer operating at 400 MHz for ¹H NMR and 75 MHz for ¹³C.

C, 29Si and 1H spectra were acquired on a Bruker Avance III 300 MHz NMR spectrometer

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(7 T) at 75, 59.4 MHz and 299.67 MHz respectively. 13C CPMAS spectra were acquired with samples packed into 4 mm zirconia rotors for use in a 4 mm H-X CPMAS probe. 1-D

13

C

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CPMAS spectra were typically acquired with the following parameters: MAS, 6.5 kHz; relaxation delay, 3 s; contact pulse, 2 ms, ramped from 70 to 100% on the 1H channel; 1H 90° pulse of 3 µs (83 kHz) decoupling with SPINAL64; 2k scans were usually acquired for satisfactory signal to noise; total time ~100 min to acquire. Inclusion of a dephasing delay of 40 µs, and with Total Suppression of Spinning sidebands (TOSS), gave sub-spectra in which the protonated 13C peaks for -C-H and –CH2- as well as spinning side bands were suppressed to aid peak assignment. 1-D

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Si CPMAS spectra were acquired on the same samples and

were typically acquired with the following parameters: MAS, 4 kHz; relaxation delay, 3 s; 8

ACCEPTED MANUSCRIPT contact pulse, 2 ms, ramped from 70 to 100% on the 1H channel; 1H 90° pulse of 3.5 µs (71 kHz) decoupling with SPINAL64; 1k scans were usually acquired for satisfactory signal to noise; total time 60 min to acquire. Direct detection of

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Si was also achieved using a spin

echo with 1H decoupling, using the following parameters: MAS, 10 kHz; relaxation delay, 60

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s; excitation 90° pulses, 5 µs; interpulse delay of ~ 100µs; approximately, 17 hr for adequate signal to noise. All spectra were acquired at ambient probe temperature. Chemical shift referencing was achieved externally with 1H to solid DSS; with

13

C to glycine carbonyl

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C

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176 ppm; with 29Si to the peak in spectra of Kaolin, 29Si -91.5 ppm.

2.3 Synthesis of control mesoporous silica nanoparticles (Control-MSNs)

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A solution of TEOS (11.2 mmol) in ethanol (11 mL) was added dropwise to a stirred solution of CTAB (4.11 mmol) and sodium hydroxide (2M, 1.75 mL) in water (120 mL), and kept for stirring at 80°C for 2 h. The resultant silica particles were washed with ethanol and water and were isolated by centrifugation. The samples were allowed to dry under vacuum for 24 h.

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Surfactant removal was performed by solvent extraction. In a typical procedure, the assynthesized MSNs (1.85 g) were treated with ethanol (150 mL) and hydrochloric acid solution (1N) for 8 h under reflux. This procedure was repeated six times in order to completely

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remove CTAB. Finally, the particles were washed with water and ethanol and were isolated by centrifugation. The samples were dried under vacuum for 24 h. Synthesis

of

organoalkoxysilane

based

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2.4

RAFT-Agent

[1-phenylethyl(3-

triethoxysilyl)propyl) carbonotrithioate] 3-Mercaptopropyltriethoxysilane (15 mmol) was added to a solution of triethylamine (TEA) (30 mmol) in chloroform (25 mL) followed by carbon disulphide (30 mmol) with continuous stirring at room temperature. After 3 h, 1-bromoethylbenzene was added and the mixture was left stirring for 16 h at room temperature. The product was extracted via washing with chloroform and water mixture. Finally, a yellow coloured viscous liquid was obtained by removing the organic solvent under reduced pressure (82% yield).¹H NMR (400 MHz, 9

ACCEPTED MANUSCRIPT CDCl₃) (as shown in Figure S1): δ (ppm) 7.27 – 7.16 (m, 5H, aromatic), 5.24 (m, 1H, SCHCH₃), 3.73 (m, 6H, (-CH₃CH O), 3.29 (t, 2H, (CH₃CH S), 1.73 (m, 2H, (CH₃CH CH₃), 1.67 (d, 3H, (-SCHCH ), 1.63 (s, 9H, (-OCH₃CH ), 0.66 (t, 2H, SiCH CH₃).¹³C NMR (75 MHz, CDCl₃) (as shown in Figure S2): δ (ppm) 226.2 (C=S),

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141.19 (tertiary carbon, aromatic), 128.66 - 127.69 (aromatic), 58.48 (-OCH₃CH₃), 50.12 (SCHCH₃), 39.46 (-CH₃CH₃S), 21.1 (-SCHCH₃) 20.9 (-CH₃CH₃CH₃), 18.47 (OCH₃CH₃)., 15.3 (-SiCH₃CH₃).

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2.5 Stability study of RAFT-Agent [1-phenylethyl(3-triethoxysilyl)propyl)carbonotrithioate] under conditions used for MSNs synthesis

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A solution of the RAFT agent [1-phenylethyl (3-triethoxysilyl)propyl)carbonotrithioate] (0.1 mmol) in ethanol (5 mL) was added dropwise to sodium hydroxide solution (2M; 1.75 mL) diluted with water( 120 mL ) and kept for stirring at 80°C for 2 h. Finally, the RAFT agent was recovered by extracting with chloroform and water mixture. ¹H NMR (400 MHz,

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CDCl₃) (as shown in Figure S3b): δ (ppm) 7.27 – 7.16 (m, 5H, aromatic), 5.24 (m, 1H, SCHCH₃), 3.73 (m, 6H, (CH₃CH O-), 3.29 (t, 2H, (-CH₃CH S), 1.73 (m, 2H, (CH₃CH CH₃), 1.67 (d, 3H, (-SCHCH ), 1.63 (s, 9H, (-OCH₃CH ), 0.66 (t, 2H, -

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SiCH CH₃). The isolated RAFT agent was then dissolved in ethanol (150 mL) and hydrochloric acid solution (1N) and refluxed for 48 h. Finally, the RAFT-Agent was

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recovered from the cooled solution by washing with chloroform and water mixture. ¹H NMR (400 MHz, CDCl₃) (as shown in Figure S3c) : δ (ppm) 7.27 – 7.16 (m, 5H, aromatic), 5.24 (m, 1H, -SCHCH₃), 3.73 (m, 6H, (CH₃CH O-), 3.29 (t, 2H, (-CH₃CH S), 1.73 (m, 2H, (-CH₃CH CH₃), 1.67 (d, 3H, (-SCHCH ), 1.63 (s, 9H, (-OCH₃CH ), 0.66 (t, 2H, SiCH CH₃). 2.6

Synthesis

of

organoalkoxysilane

triethoxysilyl)propyl)carbonotrithioate]

based

functionalized

(RAFT-MSNs) 10

RAFT

agent

mesoporous

[1-phenylethyl(3silica

nanoparticles

ACCEPTED MANUSCRIPT A

solution

of

TEOS

(11.2

mmol)

and

RAFT

agent

[1-phenylethyl(3-

triethoxysilyl)propyl)carbonotrithioate] (1.12 mmol) in ethanol (11 mL) was added dropwise to a stirred solution of CTAB (4.11 mmol) and sodium hydroxide (2M, 1.75 mL) in water (120 mL), and kept for stirring at 80°C for 2 h under reflux. The resultant silica particles were

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isolated by centrifugation, followed by washing with ethanol and water and samples were allowed to dry for 24 h. The CTAB surfactant was removed by solvent extraction method using the same procedure as used for the preparation of control-MSNs.

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2.7 RAFT polymerization of NIPAM on mesoporous silica co-condensed with RAFT-Agent (PNIPAM-MSNs)

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NIPAM (10 gm), RAFT-MSNs (500 mg), AIBN (10 mg) and DMF (30 mL) were combined in a 50-ml sample vial and sonicated for 30 min in order to uniformly disperse the RAFTMSNs. The sample vial was purged with a stream of nitrogen gas for 20 min, and then sealed and placed in an oil bath at 65°C to initiate polymerization. After 36 h, the mixture was

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washed ten times with THF in order to remove PNIPAM which was not covalently attached to the MSNs. The PNIPAM grafted MSNs were subsequently dried under vacuum for 24 h. 2.8 Procedure for aminolysis (cleaving of grafted polymer) on PNIPAM-MSNs

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50 mg of PNIPAM-MSNs, 20 mL THF and 20 mg of triphenylphosphine were taken in a round bottomed flask. The solution was then degassed with nitrogen for 30 min, followed by

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addition of 2.5 mL n-hexylamine. The mixture was allowed to stir overnight at room temperature. Finally the solution was filtered off, the THF solution was concentrated and subjected to SEC analysis.

2.9 Procedure for loading of fluorescein into control-MSNs and PNIPAM-MSNs Solution of 4 µM fluorescein was prepared in 100 mL of water at room temperature. Two samples were prepared in a 15 mL sample vial taking 10 mg of as synthesized control-MSNs and PNIPAM-MSNs with 5 mL of fluorescein solution. The samples were kept for stirring at 25°C and 35°C in the incubator shaker for 24 h. Finally, the fluorescein loaded control-MSNs 11

ACCEPTED MANUSCRIPT and PNIPAM-MSNs were washed vigorously with water (10 times) and then dried under vacuum for 24 h. 2.10 Procedure for loading of doxorubicin into PNIPAM-MSNs and determination of loading efficiency

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Solution of 18 mM doxorubicin was prepared in 50 mL of PBS at room temperature. 50 mg of PNIPAM-MSNs was added into 15 mL sample vial with 10 mL of doxorubicin solution as prepared. The samples were kept for stirring at 25°C and 35°C in incubator shaker for 24 h.

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Finally, the doxorubicin loaded PNIPAM-MSNs were washed vigorously with water (10 times) and then dried under vacuum for 24 h. The doxorubicin loaded PNIPAM-MSNs were

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characterized using confocal laser scanning microscopy. In order to determine the content of loaded doxorubicin its concentration in supernatant solutions was determined before and after addition of PNIPAM-MSNs by UV spectroscopy measurement at a wavelength of 483 nm.

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3. Results and Discussion

The first step in the synthesis of the RAFT- MSNs is to make sure that the RAFT agent bearing the triethoxysilyl group, can be formed, ready for subsequent incorporation into the

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MSN synthesis. This was achieved by reaction of 3-mercaptopropyltriethoxysilane with carbon disulphide and 1-bromoethylbenzene in the presence of trimethylamine, and affording

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the RAFT agent isolated in excellent yield. The stability of this bi-functional agent under conditions utilized for the synthesis of MSNs, that is, strongly basic (NaOH) and strongly acidic (HCl) media was established with 1H,

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C NMR and UV-visible spectroscopies

(Supporting Information, Figures S1-S4). Figure S3 and S4 shows a key comparison of 1H NMR and UV-Visible spectra of the RAFT agent, before and after its reactions with NaOH and HCl respectively. Based on 1H NMR results (Figure S3) we concluded that the RAFT agent used for co-condensation experiments was stable during the synthesis of MSNs. Additional support was deduced from UV-Visible spectroscopy shown in Figure S4. After 12

ACCEPTED MANUSCRIPT confirming the stability of organoalkoxysilane RAFT agent, it was incorporated into the organically modified MSNs by co-condensing with TEOS and the structure directing template, CTAB in aqueous base. Removal of the CTAB by extraction, gave the organoalkoxysilane RAFT functionalized mesoporous silica nanoparticles (RAFT-MSNs). Using controlled

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radical polymerization, the RAFT-MSNs were further elaborated to incorporate a temperature responsive polymer via the "grafting-from" approach. Thus, the RAFT-MSNs were reacted with NIPAM with the radical initiator in DMF to yield the PNIPAM-MSNs.

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All three materials - control-MSNs, RAFT-MSNs and PNIPAM grafted MSNs - were characterized by a number of methods, all of which strongly indicate that it is the interior

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surface of the mesoporous material that has been functionalized, and ready for further use. The FT-IR spectra of the RAFT- and NIPAM-MSNs are shown in Figure 1 a), with peaks at around 3400 cm-1, 1095 cm-1, and 806 cm-1 assigned to O-H stretching bands, asymmetric stretching vibration peak of Si-O-Si and Si-O-Si symmetric stretching vibrations respectively.

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In spectra of RAFT-MSNs and PNIPAM-MSNs, new absorption bands at 2914 cm-¹ and 2829 cm-¹ were observed, arising from the C-H stretching vibration of the organic groups. In the PNIPAM-MSN, a prominent band at 1524 cm-1 was also observed which indicates the

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secondary amide group. Therefore, FT-IR analysis shows successful co-condensation of organoalkoxysilane RAFT agent with TEOS as well functionalization of RAFT-MSNs with

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PNIPAM. TGA analysis of all samples was conducted over the range from room temperature to 850°C. Typical weight loss curves for control-MSNs, RAFT-MSNs, and PNIPAM-MSNs are shown in Figure 1 b). The control-MSNs showed a weight loss of 4.6 wt. % up to a temperature of 850°C, which is ascribed to the removal of adsorbed water, as well as dehydration of surface hydroxyl groups. In the case of RAFT-MSNs, the total weight loss was 22.6 wt. %, with a first stage at ~82°C contributing to a weight loss of 2.9 wt.%, and the second stage at ~267°C due to decomposition of the organoalkoxysilane RAFT agent contributing to the remaining weight loss of 19.7 wt.%. After NIPAM polymerization, the 13

ACCEPTED MANUSCRIPT total weight loss was 29.0 wt. %. The first weight loss was ~100°C contributing to the weight loss of 2.1 wt.%, the second weight loss at ~261°C contributing to the weight loss of 16.8 wt.% and a third loss at ~373°C of 10.1 wt.% These markedly different behaviour in the TGA analysis also confirms the success of the subsequent steps in the synthesis of the PNIPAM-

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MSN. The weight loss at different steps is summarised in Table S1 in the Supporting

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Information.

Figure 1. a) FT-IR spectra of control-MSNs, RAFT-MSNs, PNIPAM-MSNs having the characteristics signals at 1095 cm-1 (Si-O-Si asymmetric vibration), 2914 cm-1 (C-H

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stretching) and 1524 cm-1 (secondary amide group) respectively; b) TGA curves for control-

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MSNs, RAFT-MSNs, and PNIPAM-MSNs. The total weight loss for PNIPAM-MSNs was around 29.0 wt. % which is higher than weight loss for RAFT-MSNs (22.6 wt. %) and control-MSNs (4.6 wt. %).

UV-visible spectroscopy was performed in addition to other techniques to confirm cocondensation of the organoalkoxysilane RAFT agent with TEOS. Figure 2 shows a comparison of the UV spectra of the control-MSNs, the RAFT-MSNs and the PNIPAMMSNs, clearly displaying the absorption from the thiocarbonyl group at ~310-332 nm, allowing straight forward detection of this feature of the RAFT agent. The molecular weight 14

ACCEPTED MANUSCRIPT of PNIPAM grafted on MSNs was determined using size exclusion chromatography after performing aminolysis experiment. As the trithiocarbonate group is labile to aminolysis using excess of primary amine, the polymer chains were cleaved from MSNs using aminolysis. The number and weight average molecular weight obtained was 2100 g.mol-1 and 2600 g.mol-1

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respectively. The SEC trace of the polymer obtained after aminolysis is shown in the

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Supporting Information Figure S5.

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Figure 2. Comparative UV absorbance spectra for RAFT agent, control-MSNs, RAFT-MSNs,

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and PNIPAM-MSNs.

The morphology and size of the synthesized non-functionalized MSNs (control-MSNs), organoalkoxysilane RAFT co-condensed MSNs (RAFT-MSNs) and PNIPAM grafted MSNs was studied using SEM, TEM, XRD and DLS. The surface area was calculated from BET and BJH analysis. The shape and size of the MSNs is highly dependent on the reaction conditions such as solvent, temperature, pH, stirring rate and surfactant to TEOS molar ratios. With this new system described here, the organoalkoxysilane-RAFT agent (1), the interaction of its hydrophobic group, with hydrocarbon tails of CTAB, water and ethanol can plausibly leads to 15

ACCEPTED MANUSCRIPT the formation of particles featuring a distorted ellipsoid type of structure as shown in the

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SEMs, Figure 3 (and Figure S6).

Figure 3. SEM and TEM micrographs of organoalkoxysilane RAFT-MSNs and PNIPAM-

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MSNs.

As depicted in the Scheme 2, CTAB can form micellar structures in contact with water, having the hydrophilic tetra-alkylammonium groups turned outwards towards the bulk water and hydrophobic alkyl chains projected inwards. When the hydrophobic part of the organoalkoxysilane RAFT agent comes in contact with this micelle, it penetrates more towards the hydrophobic, interior part, forming intercalated layers and thus changing its shape from complete spherical particle. In this work, we have also utilized ethanol as a co-solvent 16

ACCEPTED MANUSCRIPT for the synthesis of MSNs, and because of this, the hydrolysis of TEOS is retarded, leading to the slower growth of micellar structures. In addition, the presence of the co-solvent, weakens the aggregation of the CTAB which leads to distorted ellipsoid/spherical morphology. Therefore, the presence of ethanol as a co-solvent in the reaction medium, the decreases the

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overall polarity, which in turn restricts the micellar growth. [32] Finally, the tetraethoxysilyl group and siloxy group of the RAFT agent, arranged at the Guoy-Chapman region [33] of the surface of the micelles, hydrolyze and condense with each other giving small distorted

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ellipsoid/spherical shaped nanoparticles. Thus, in this system there are two main factors which are affecting MSN morphology: one factor is organoalkoxysilane RAFT agent and the

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other factor is presence of ethanol as a co-solvent. Further studies are underway to tune the morphology of MSNs, by systematically varying these components and their relative

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concentrations.

Scheme

2.

Plausible

mechanism

of

the

interaction

of

hydrophobic

group

of

organoalkoxysilane-RAFT agent with hydrocarbon tails of CTAB, water and ethanol leading to the formation of particles featuring a distorted ellipsoid type of structure.

From the TEM micrographs as shown in Figure 3 (and Figure S7), it can be observed that the co-condensation of organoalkoxysilane RAFT agent as well polymerization of NIPAM has 17

ACCEPTED MANUSCRIPT not destroyed the mesoporous structure of the MSNs[34-36]. This is supported by the powder XRD pattern shown in Figure S8, which reveals a broad (100) peak at 2.9 (2θ) corresponding to a d-spacing of 30.5 Å as well as a broad peak at 4.58 (2θ) corresponding to a d-spacing of 19.5 Å, for all the three samples, control-MSNs, RAFT-MSNs and PNIPAM-MSNs.

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As already discussed, the structural ordering of mesoporous silica is also dependent on the loading of the surfactant as well as loading of the organoalkoxysilane. Several authors [37] have reported a decrease in the ordering of the mesopores with an increase in the amount of

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surfactant as well as organoalkoxysilane. The hydrodynamic diameter of the MSNs was determined by dynamic light scattering (DLS), using a dilute suspension of nanoparticles in

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deionized water at 25°C. The analysis was performed in triplicate and the particles size mentioned is the average for each sample: for control-MSNs, it was 97±2 nm; for RAFTMSNs was 95±3 nm; for PNIPAM was 98±3 nm. The DLS results give a clear indication that the size of MSNs has not increased after polymerization, as might be expected if the organic

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coating has accumulated on the outside surface of the nanoparticle. This leads to the conclusion that the polymerization has occurred only on the inside the pores of the MSNs. In keeping with this, it was important to investigate the remaining porosity as well as surface

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area of the MSNs after the polymerization. The surface area of MSNs was evaluated with nitrogen adsorption isotherm measurements. The surface area calculated using multi-point

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Brunauer-Emmett-Teller (BET)[38] analysis was 942.6 m2.g-1 for control-MSNs, 961.8 m2.g-1 for RAFT-MSNs and 958.3 m2.g-1 for PNIPAM-MSNs. The pore area calculated using Barrett-Joyner-Halenda (BJH) analysis was 87.8 m2.g-1 for control-MSNs, 91.1 m2.g-1 for RAFT-MSNs and 132.9 m2.g-1 for PNIPAM-MSNs. The substantial increase in pore area in the case of PNIPAM-MSNs also suggests that polymer has grafted onto the functionalized MSN, but mostly inside its pores. In order to characterize the organic functionalities on the inorganic backbone of MSNs, as well as the silica-organic interface, solid state NMR spectroscopy was utilized. Previously, 18

ACCEPTED MANUSCRIPT several authors have taken advantage of solid state NMR in order to prove organic functionalization on MSNs surface.[39-42] The ¹³C (RAFT-MSNs, PNIPAM-MSNs) and ²₃Si NMR spectra (Control-MSNs, RAFTMSNs, PNIPAM-MSNs) are shown in Figure 4 and Figure 5. The assignments of the

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observed ¹³C resonances in RAFT-MSNs and PNIPAM-MSNs are presented in Table S2 in the Supporting information. The ¹H→¹³C cross-polarization magic angle spinning (CPMAS) spectrum of RAFT-MSNs exhibits specific resonances, which can be assigned as C1(-O-Si-

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CH₃), C2(-O-Si-CH₃-CH₃), C3(-CH₃-S-), C4(-S-C=S-S), C5(-C=S-CH-CH₃), C6(SCH-CH₃), C7(tertiary carbon, aromatic) and C8 (aromatic carbon). The strong signal at

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64.10 ppm resonance can be assigned to the ethoxy group (-Si-O-CH₃-CH₃) of the RAFTagent which has not reacted during the synthesis. After comparing the intensity of the same ethoxy group for ¹³C CPMAS of PNIPAM-MSNs, this signal has disappeared which may account for the condensation of the ethoxy groups during polymerization.

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The ¹³C CPMAS of PNIPAM-MSNs shows that thorough polymerization took place which can be assigned to specific resonances such as, C1(-O-Si-CH₃), C2(-O-Si-CH₃-CH₃), C3, C5, C10(-CH2-CH2-S-), (-CH-CH₃-), (-CH-CH₃-), C6(-CH-C=O), C7(-NH-CH-), C8, C9, (-CH3-CH-),

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C12(-CH-CH₃),

C11(-CH-CH₃-),

C13(tertiary

carbon,

aromatic),

C14(aromatic carbon). The carbons site at C8 and C9 of the polymer backbone, which

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resonate at 18.26 ppm, respectively demonstrates NIPAM polymerization. The peak at ~172 ppm, which in the RAFT-MSNs was not been identified, is solely due to carbon contributions from carbonyl carbons C6 in each of the NIPAM monomer units. As the RAFT polymerization reaction progressed, attributing to the NIPAM groups, the intensities of the carbons C5–C10 increased with the decreased intensity of aromatic group carbon from the intensity of RAFT-MSNs as shown in Figure 4. The ²₃Si-CPMAS the resonances at -67.5 and -57.3 denoted as T² and T³ can be assigned to (≡SiO)₃Si(OH)-R/R' and (≡SiO)₃Si-R/R' silicon atoms respectively. The presence of T³ and 19

ACCEPTED MANUSCRIPT T² functionalities confirms the existence of the covalent linkage between the organic groups and the silica surface. The resonance representing Q (≡SiO)₃Si), Q³(≡SiO)₃SiOH) and Q²(SiO)₃Si(OH)₃) are observed in their actual spectral position denoting the network of the silica. The 29Si spectra also provides evidence that the T sites remain unaltered even after the

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polymerization indicating the RAFT functionalities remain attached to the silica network.

Figure 4. Solid state ¹³C CPMAS-TOSS spectra for a) organoalkoxysilane RAFT agent functionalized MSNs (RAFT-MSNs) and b) PNIPAM functionalized MSNs (PNIPAM-MSNs).

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Figure 5. Solid state 29Si CPMAS spectra a) control-MSNs, b) RAFT-MSNs and c) PNIPAMMSNs with the respective peak assignments for Q and T structures.

To confirm the thermoresponsive behaviour of the PNIPAM grafted inside the pores of mesoporous silica nanoparticles for drug delivery application, dye as well as drug loading experiments[43,44] were performed, for both control-MSNs as well as PNIPAM-MSNs with 21

ACCEPTED MANUSCRIPT fluorescein as a model dye and doxorubicin as drug (experimental section). The dye as well as drug loaded MSNs were analyzed using confocal laser scanning microscopy. As shown in Figure 6, green and red fluorescence corresponding to fluorescein and doxorubicin respectively was strongly retained in the PNIPAM-MSNs after 24 h of incubation at 25°C

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while lack of fluorescence after 24 h incubation at 35°C (as shown in Figure 7), demonstrates the thermoresponsive behaviour of the polymer being grafted inside the pores of mesoporous

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silica nanoparticles.

Figure 6. Confocal laser scanning micrographs of PNIPAM-MSNs loaded with fluorescein and doxorubicin at 25°C (a) dark field image, (b) in-focus bright field image, and (c) maximum intensity DIC image.

That is, when PNIPAM is below its lower critical solution temperature (LCST), all the polymer chains are swollen, facilitating the entry of dye as well as drug inside the pores, while above LCST, all the PNIPAM chains collapse, in turn restricting any entry of dye and 22

ACCEPTED MANUSCRIPT drug molecules. For comparisons, the same conditions of loading experiments were performed for control-MSNs and confocal laser scanning micrographs are shown in Figure S9 in the Supporting Information. The micrographs clearly shows green fluorescence for mesoporous silica nanoparticles loaded with dye at both 25°C and 35°C. The loading of dye at

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both 25°C and 35°C indicates no barriers for entry of the dye molecules into the

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unmodified/control MSNs.

Figure 7. Confocal laser scanning micrographs of PNIPAM-MSNs loaded with fluorescein and doxorubicin hydrochloride at 35°C (a) dark field image, (b) in-focus bright field image, and (c) maximum intensity DIC image.

The loading efficiency (LE) of doxorubicin in PNIPAM-MSNs was calculated using equation (1) discussed in the Supporting Information. The LE of doxorubicin in PNIPAM-MSNs is dependent on the initial concentration [Dox] of the doxorubicin in the buffer solution and the 23

ACCEPTED MANUSCRIPT concentration of doxorubicin after incubation for 24 h at 25°C [Dox]s. In order to determine [Dox] and [Dox]s, calibration curve of doxorubicin in PBS was prepared as shown in the Figure S10 in the Supporting Information. The loading efficiency of doxorubicin at 25°C is 46.6%. In future, these MSNs grafted with PNIPAM as well as other stimuli responsive

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polymers will be studied for loading as well release of model drugs.

4. Conclusions

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In this study, we have successfully realized for the first time the concept of directly priming MSNs for RAFT polymerization. Practically this has been achieved by co-condensing the organoalkoxysilane

RAFT

agent

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bifunctional

[1-phenylethyl(3-

triethoxysilyl)propyl)carbonotrithioate] with TEOS in the templating reaction for MSN synthesis to embed the RAFT agent directly into the mesoporous silica network. The RAFT agent functionalized MSNs were elaborated further with NIPAM via surface-initiated RAFT

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polymerization in a “grafting-from” approach. The polarity of organoalkoxysilane governs the morphology and location of organoalkoxysilane in the silica network. The appearance of carbon signals in 13C solid state NMR, T signals in 29Si solid state NMR and C-H stretching

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signals in FT-IR establishes that we have successfully obtained organic-inorganic hybrid materials. The increase in pore area of MSNs after polymerization of NIPAM confirms

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grafting of PNIPAM inside the pores of MSNs. The loading of fluorescein as well as doxorubicin at 25°C, while none at 35°C establishes thermoresponsive nature of PNIPAM grafted inside the pores of MSNs. In addition, further studies are underway, in order to tune the morphology of MSNs based on organoalkoxysilane RAFT agent. Insights from the concept executed here, into strategies for the synthesis of organoalkoxysilane RAFT agents and the controlled grafting of polymers onto the exterior and interior of the mesoporous silica surfaces will lead to the construction of sophisticated materials for targeted drug delivery, sensors and catalysis applications. 24

ACCEPTED MANUSCRIPT Supporting Information Supporting Information is available from the Online Library. Acknowledgements S.M. acknowledges a research fellowship from the Indian Institute of Technology Delhi. We

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thank the Mark Wainwright Analytical Centre, UNSW, for access to solid state NMR spectrometers supported by Australian Research Council grant, LE0989541.

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Received: Month XX, XXXX; Revised: Month XX, XXXX; Published online:

((For PPP, use “Accepted: Month XX, XXXX” instead of “Published online”)); DOI:

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10.1002/marc.((insert number)) ((or ppap., mabi., macp., mame., mren., mats.))

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Highlights

1) RAFT mesoporous silica nanoparticles (MSNs) have been synthesized for the first time agent, 1-phenylethyl(3-

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via co-condensation of an organoalkoxysilane RAFT

triethoxysilyl)propyl)carbonotrithioate, with tetraethoxysilane (TEOS) in aqueous basic cetyltrimethylammonium bromide.

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2) The success of the incorporation of the organoalkoxysilane RAFT agent into the silica network, and polymerization of PNIPAM was confirmed with TGA, FT-IR, UV-visible

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spectroscopy, 13C and 29Si solid state NMR spectroscopy.

3) The effect of organoalkoxysilane based RAFT agent and PNIPAM grafting on the morphology, size and surface area of the resulting MSNs was investigated using SEM, TEM, XRD, DLS and BET analysis.

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4) The thermoresponsive behaviour of PNIPAM grafted inside the pores of MSNs was studied by dye as well as drug loading experiments at variable temperatures, followed by

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characterization using confocal laser scanning microscopy.