Subscriber access provided by CORNELL UNIVERSITY LIBRARY Characterization, Synthesis, and Modifications Ultrasmall PEGylated and Targeted Core-Shell Silica Nanoparticles Carrying Methylene Blue Photosensitizer Ferdinand F.E. Kohle, Songying Li, Melik Z. Turker, and Ulrich B. Wiesner ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b01359 • Publication Date (Web): 20 Nov 2019 Downloaded from pubs.acs.org on November 27, 2019
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1 2 3 4 5 Ultrasmall PEGylated and Targeted Core-Shell Silica 6 7 Nanoparticles Carrying Methylene Blue Photosensitizer 8 9 10 Ferdinand F. E. Kohle †, ‡,∥, Songying Li †,∥, Melik Z. Turker †, 11 12 and Ulrich B. Wiesner *, † 13 14 15 16 17 18 19 20 † 21 Materials Science and Engineering, Cornell University, Ithaca, NY 14853 22 ‡Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853 23 24 25 *Department of Materials Science and Engineering, Cornell University, 330 Bard 26 27 Hall, Ithaca, NY 14853. 28 Corresponding Author: Email: [email protected], Fax: 607-255-2365. 29 30 31 32 33 ABSTRACT 34 35 36 Photodynamic therapy (PDT) presents an alternative non-invasive therapeutic 37 38 modality for the treatment of cancer and other diseases. PDT relies on cytotoxic singlet 39 40 oxygen (reactive oxygen species, or ROS) that is locally generated through energy 41 42 43 transfer between a photosensitizer (PS) and molecularly dissolved triplet oxygen. While 44 45 a number of nanoparticle-based PS vehicles have been described, because of their 46 47 beneficial and proven biodistribution and pharmacokinetic profiles, ultrasmall 48 49 50 nanoparticles with diameters below 10 nm are particularly promising. Here, we 51 52 investigate two different particle designs deviating from ultrasmall poly(ethylene glycol) 53 54 coated (PEGylated) fluorescent core-shell silica nanoparticles referred to as Cornell 55 56 57 58 59 1 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 2 of 37
1 2 3 4 prime dots (C’ dots) by replacing the fluorescent dye with a photosensitizer (psC’ dots), 5 6 here the methylene blue derivate MB2. In the first approach (design 1), MB2 is 7 8 encapsulated into the matrix of the silica core, while in the second approach (design 2), 9 10 11 MB2 is grafted onto the silica core surface in between chains of the sterically stabilizing 12 13 PEG corona. We compare both cases with regard to their singlet oxygen quantum yields, 14 15 Φ , with the effective Φ eff per particle reaching 111 ± 3% and 161 ± 5% for design 1 16 Δ Δ 17 18 and 2, respectively, substantially exceeding single MB2 molecule performance. 19 20 Encapsulation significantly improves PS photostability, while surface conjugation 21 22 diminishes it, relatively to free MB2. Finally, we show that both particle designs allow 23 24 25 functionalization with a targeting peptide, c(RGDyC). Results suggest that psC’ dots 26 27 are a promising targeted platform for PDT applications, e.g. in oncology, that may 28 29 combine colloidal stability, efficient renal clearance limiting off-target accumulation, 30 31 targeted delivery to sites of disease, and effective ROS generation maximizing 32 33 34 therapeutic efficacy. 35 36 37 38 KEYWORDS: Ultrasmall silica nanoparticles, silica chemistry, photosensitizer, 39 40 41 photodynamic therapy (PDT), singlet oxygen quantum yield 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 2 60 ACS Paragon Plus Environment Page 3 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 5 6 1. INTRODUCTION 7 8 Photodynamic therapy (PDT) has emerged as a minimally invasive and 9 10 11 minimally toxic therapeutic modality for the treatment of cancer and other diseases.1 12 13 The principle of PDT can generally be described in four steps: A photosensitizer (PS) 14 15 is localized around diseased tissue (step 1), and activated by a light source (step 2). The 16 17 1 18 absorbed photon energy leads to the generation of highly reactive singlet oxygen, O2 19 20 (step 3), causing oxidative stress and cellular damage, eventually initiating cell death 21 22 mechanisms such as necrosis and/or apoptosis in the local environment of the PS (step 23 24 2 25 4). These steps impose chemical, photophysical, and structural requirements onto PDT 26 27 probes. 28 29 1 Photophysical requirements: Generation of O2 is caused by photoexcitation of 30 31 the PS. Figure 1A depicts a simplified Jablonski scheme illustrating the photophysical 32 33 1 34 processes leading to O2 generation. From an electronically excited singlet state the PS 35 36 undergoes a forbidden electron spin-flip (intersystem crossing, ISC) into an 37 38 energetically lower lying excited triplet state, 3PS*. From here, 3PS* relaxes into the 39 40 1 41 singlet ground state, PS, via energy transfer (ET) with dissolved molecular triplet 42 3 1 43 oxygen, O2, yielding cytotoxic reactive singlet oxygen, O2. High intersystem crossing 44 45 1 rates (kISC) and long triplet state lifetimes (τT > 1 μs) of the PS promote O2 generation, 46 47 3 48 which is reflected in high singlet oxygen quantum yields, ΦΔ. An ideal PS should have 49 50 a molar extinction coefficient of ε ≥ 50 000 M-1 cm-1 in the therapeutic window of ~600– 51 52 3,4 5 1200 nm, and a singlet oxygen quantum yield of ΦΔ ≥ 0.5. In addition, high 53 54 photostability, as well as low phototoxicity in the dark are desired.3 55 56 57 58 59 3 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 4 of 37
1 2 3 4 Chemical requirements: Besides the general requirement of a PDT probe to be 5 6 non-toxic itself, a key challenge is its localization at a specific site of interest. Singlet 7 8 oxygen is highly reactive, and locally produced by the PS. Typical diffusion lengths of 9 10 11 singlet oxygen in tissue before it reacts are on the order of only tens of nanometers.6 12 13 Therefore, to minimize damage of healthy tissue, selective targeting is crucial. Since 14 15 most PS molecules are hydrophobic and prone to aggregation in physiological 16 17 18 environments, low selectivity towards diseased tissue and adverse pharmacokinetics 19 20 have hindered their clinical translation.4 The association of PS molecules with 21 22 nanoparticles (NPs) as drug delivery systems (DDSs) can promote solubility, overcome 23 24 25 aggregation issues to improve pharmacokinetics, and protect PSs from enzymatic 26 27 degradation.7 Furthermore, NP surface functionalization with targeting moieties reduces 28 29 systemic side effects, increases the therapeutic concentration of PSs at the target site, 30 31 and gives room for multi-modality platforms simultaneously enabling diagnosis, 32 33 34 imaging, and treatment.7 Different such NP-based DDSs have been explored, including 35 36 polymeric particles, solid lipid-based materials, metal-organic framework (MOF) NPs, 37 38 metallic and inorganic particles.8-11 However, while NP-based DDSs help overcome 39 40 41 shortages associates with the PS molecules, they themselves impose additional 42 43 requirements. 44 45 Structural requirements: When PSs are loaded onto durable NPs, i.e. NP that 46 47 48 are non-degrading during administration, distribution, accumulation, and elimination, it 49 50 is essential that oxygen species can easily diffuse to and away from the PS molecule. 51 52 Second, after NPs have targeted the site of interest and PDT has been performed (or if 53 54 the NPs have failed to target the site of disease in the first place) it is necessary that NPs 55 56 57 58 59 4 60 ACS Paragon Plus Environment Page 5 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 are rapidly cleared from the body to reduce potential off-target toxicities (principle of 5 6 target-or-clear).12 Both of these requirements favor small hydrodynamic NP diameters. 7 8 Given a glomerular filtration size cutoff below 10 nm hydrodynamic diameter for rapid 9 10 11 renal clearance via the kidneys, diameters of durable NP DDSs should lie below 10 12 13 nm.13 14 15 In comparison to other pharmaceutical technologies, i.e. inhalations, 16 17 18 transdermal patches and oral sustained release preparations, NPs are characterized by a 19 20 low ratio of clinical trials to publication 14 and often fail clinical translation due to a lack 21 22 of quality control, efficacy or safety.15 A rare example for a durable NP-based DDS that 23 24 25 is able to meet above-mentioned requirements are sub-10 nm (ultrasmall) organic- 26 27 inorganic hybrid core-shell silica nanoparticles (SNPs) stabilized with polyethylene 28 29 glycol (PEGylated particles). 16-18 It has been demonstrated in first published human 30 31 clinical trials that such NPs efficiently target and clear from the human body, while no 32 33 34 toxic or adverse events were observed.19,20 This NP platform, termed Cornell prime dots 35 36 or C’ dots, is cost-effectively synthesized in aqueous solution and allows exceptional 37 38 size and structural control on the sub-10 nanometer length scale 18,21-23 with the ability 39 40 41 to introduce a library of targeting moieties to the surface, such as small peptide 42 43 molecules or antibody fragments.24,25 In addition, their small size in combination with 44 45 a reliable steric stabilization enable sterile filtration, reducing the complexity of aseptic 46 47 48 preparation of parenterals. 49 50 Here, we demonstrate the conversion of diagnostic fluorescent C’ dots into 51 52 therapeutic C’ dots by covalently binding the PS molecule MB2, a derivate of methylene 53 54 blue (MB) (Figure 1B and Figure S1) to ultrasmall C’ dots. MB was FDA-approved 26 55 56 57 58 59 5 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 6 of 37
1 2 3 4 and has been previously used in photodynamic therapy to treat cancerous and non- 5 6 cancerous diseases.27 MB2 has a high singlet oxygen quantum yield and maximum 7 8 extinction coefficient in the near infrared (Φ ≈ 0.5, ε = 105 M-1 cm-1 at about 670 nm, 9 Δ 10 11 see Figure S1).28 Furthermore, the optical transparency of silica and high silica matrix 12 13 microporosity render SNP an ideal DDS candidate for PS molecules to be used in PDT. 14 15 There have been attempts to incorporate MB into SNPs and other particle platforms.29- 16 17 31 18 However, in the past many attempts relied on physical incorporation of MB into large 19 20 (>10 nm) SNPs and mesoporous SNPs increasing the risk of dye leakage during 21 22 systemic circulation. In the following we will refer to sub-10 nm PEGylated SNP 23 24 25 covalently binding PS molecules as photosensitizing C’ dots, or simply psC’ dots. 26 27 In contrast to previous studies where we encapsulated MB2 in C’ dots to study 28 29 the effects of spin-orbit coupling to heavier atoms like iodide co-condensed into the 30 31 amorphous silica particle matrix (referred to as iodide containing C’ dots, or simply iC’ 32 33 34 dots),32 here we compare two different psC’ dot designs (Figure 1C). In design 1, MB2 35 36 is encapsulated within the silica network similar to a regular dye in a typical fluorescent 37 38 C’ dot; in design 2, MB2 is grafted onto the SNP surface, inserted between the PEG 39 40 41 corona chains. In both cases the PS molecules are covalently bound to the SNPs via a 42 43 thiol-Michael addition click reaction between maleimide functionalized MB2 and (3- 44 45 mercaptopropyl)trimethoxysilane (MPTMS) co-condensed into the silica matrix. We 46 47 48 show that both particle types can be further functionalized with αvβ3 integrin-targeting 49 50 cyclic(arginine-glycine-aspartic acid-D-tyrosine-cysteine) peptide (cRGDyC, Figure 51 52 1D and 1E) using established protocols.33,34 Photosensitizing action is successfully 53 54 demonstrated using the singlet oxygen sensor 1,3-diphenylisobenzofuran (DPBF). 55 56 57 58 59 6 60 ACS Paragon Plus Environment Page 7 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 Relative to free MB2, we demonstrate effective per particle singlet oxygen quantum 5 6 yields of 111 ± 3% (design 1) and 161 ± 5% (design 2), respectively. These designs 7 8 therefore suggest targeted and highly efficient probes for PDT which meet demonstrated 9 10 11 physicochemical particle criteria necessary for clinical translation.20 12 13 14 15 2. MATERIALS AND METHODS 16 17 18 19 20 2.1 Materials 21 22 Aluminum-tri-sec-butoxide (ASB), (3-aminopropyl)triethoxysilane (APTES), 23 24 25 ammonium hydroxide (28 wt% in H2O), ammonia solution (2.0 M in ethanol), dimethyl 26 27 sulfoxide (DMSO), 1,3-diphenylisobenzofuran (DPBF, 97%), hydrochloric acid (HCl, 28 29 0.5018 N in H2O), (3-iodopropyl)trimethoxysilane (IPTMS), methylene blue (MB), (3- 30 31 mercaptopropyl) trimethoxysilane (MPTMS), 2-propanol (anhydrous 99.5%), and 32 33 34 tetramethyl orthosilicate (TMOS) were purchased from Sigma Aldrich. (3- 35 36 aminopropyl)trimethoxysilane (APTMS) and methoxy-terminated poly(ethylene glycol) 37 38 (PEG-silane, molar mass of ~0.5 kg/mol) were purchased from Gelest. 39 40 41 Heterobifunctional PEG (NHS-PEG-mal, molar mass of ∼960 g/mol) was purchased 42 43 from Quanta BioDesign. ATTO MB2-maleimide was purchased from ATTO-Tec. 44 45 46 Tetramethylrhodamine-5-maleimide (TMR) was purchased from AnaSpec. Ethanol 47 48 (absolute anhydrous 99.5%) was purchased from Pharmco-Aaper. c(RGDyC) was 49 50 purchased from Peptide International. Deionized (DI) water (18.2 MΩ·cm) was 51 52 generated using a Millipore Milli-Q system. All chemicals were used as received. 53 54 55 56 57 58 59 7 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 8 of 37
1 2 3 4 2.2 Synthesis of photosensitizing C’ dots according to design 1 5 6 First, 3.67x10-7 moles MB2 with a maleimide group were reacted with MPTMS 7 8 in DMSO at a molar ratio of 1:25 (fluorophore:MPTMS) to generate a MB2-silane 9 10 11 conjugate. To synthesize sub-10 nm PEGylated SNPs with MB2 inside the silica core, 12 13 2 mL of 0.02 M ammonia aqueous solution was first added into 8 mL of DI water 14 15 yielding a pH of ~9. The solution was then stirred at room temperature for 5 min. As 16 17 18 the silica precursor, 0.43 mmol of TMOS were added under vigorous stirring, followed 19 20 by the addition of all MB2-silane. The molar ratio of MB2-silane to TMOS was about 21 22 1:1000. The solution was left stirring at room temperature overnight. Then, 0.21 mmol 23 24 25 of PEG-silane were added and the solution was kept stirring at room temperature 26 27 overnight. Finally, to promote covalent bond formation between PEG-silane and 28 29 particles, stirring was stopped and the particle dispersion was heated to 80 C for 8 hours 30 31 35 32 (also see Figure 1E). To remove any unreacted precursors, aggregates, or dust from 33 34 the particle dispersion, particles were transferred into a dialysis membrane tube 35 36 (molecular weight cutoff, MWCO = 10,000), and dialyzed in 2 L of DI water with three 37 38 39 water exchanges every 8 hours. After dialysis, the dispersion was subject to syringe 40 41 filtration (0.2 µm, Fisherbrand) and finally up-concentrated for gel permeation 42 43 chromatography (GPC) using a membrane spin filter (GE Healthcare, molecular weight 44 45 cutoff = 30,000) and a centrifuge at 2300 rpm. 46 47 48 49 50 2.3 Synthesis of photosensitizing C’ dots according to design 2 51 52 Particles binding MB2 to the particle surface were synthesized according to the 53 54 55 synthesis described for design 1, but excluding the addition of MB2-silane, or replacing 56 57 58 59 8 60 ACS Paragon Plus Environment Page 9 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 MB2 with TMR-maleimide (see main text). MB2 was added to the final silica particles 5 6 by using the method of post-PEGylation surface modification by insertion (PPSMI).24 7 8 To that end, MPTMS was added to the PEGylated particle dispersion under vigorous 9 10 11 stirring at a concentration of 2.3 mM. The particle/MPTMS mixture was stirred at room 12 13 temperature overnight, followed by the addition of 3.67x10-7 moles MB2-maleimide at 14 15 a concentration of 37 µM. The solution was vigorously stirred at room temperature for 16 17 18 24 hours for the dye to react with the thiol group on the silica core surface of the particles 19 20 (see Figure 1E). Afterwards, the particle dispersion was subjected to the same cleaning 21 22 process as described under design 1 (dialysis, syringe filtration, GPC). Particles 23 24 25 containing TMR on the surface were synthesized in the same way by replacing MB2- 26 27 maleimide with TMR-maleimide. 28 29 30 31 2.4 Targeting peptide c(RGDyC) functionalization 32 33 34 Particles were peptide-functionalized with c(RGDyC)-PEG-silane 35 36 (Supplementary Figure S2 in the Supporting Information). c(RGDyC)-PEG-silane was 37 38 prepared by exploiting the mercapto group of cysteine of c(RGDyC) (Figure 1D) to 39 40 41 click to the maleimide group of a heterobifunctional mal-PEG-NHS first, and then 42 43 clicking the NHS to the amine group of (3-aminopropyl)trimethoxysilane (APTES). The 44 45 concentration of NHS ester-PEG-maleimide in DMSO was 0.23 M. The mixed solution 46 47 48 was left at room temperature in the glovebox for 3 hours to form silane-PEG-maleimide. 49 50 After that, c(RGDyC) was added and the solution left at room temperature in the 51 52 glovebox overnight to produce c(RGDyC)-PEG-silane. The molar ratio 53 54 c(RGDyC):NHS-PEG-mal:APTES was 1.0:1.0:0.9. In order to functionalize particles 55 56 57 58 59 9 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 10 of 37
1 2 3 4 with c(RGDyC) peptide ligands, previously prepared c(RGDyC)-PEG-silane was added 5 6 to the particle dispersion immediately before the addition of PEG-silane.33,34 The 7 8 remainder of the synthesis and purification protocol was as described before (also see 9 10 11 Figure 1E). 12 13 14 15 2.5 Gel permeation chromatography 16 17 18 To remove unreacted precursors and particle aggregates from the native particle 19 20 dispersion, samples were purified using gel permeation chromatography (GPC) using 21 22 established protocols.18 A BioLogic LP system with 275 nm UV detector and cross- 23 24 25 linked copolymer of allyl dextran and N,N'-methylene bisacrylamide (Sephacryl S-300 26 27 HR, GE Healthcare) as solid phase was used. Before GPC purification each sample was 28 29 up-concentrated with centrifuge spin-filters (Vivaspin with MWCO 30k, GE Healthcare) 30 31 to an approximate sample volume of 600 μL, run through the column with a 0.9 wt% 32 33 34 NaCl solution, and fraction-collected by a BioFrac fraction collector. Sample fractions 35 36 were transferred to DI water by washing samples five times with centrifuge spin-filters. 37 38 The resulting particles could be subjected to long-term storage in nitrogen bubbled DI 39 40 41 water in the dark at 4 °C. 42 43 44 45 2.6 Steady-state absorption spectroscopy 46 47 48 Absorbance spectra were measured on a Varian Cary 5000 spectrophotometer. 49 50 Spectra were measured in DI water using a quartz cuvette (HellmaAnalytics) with a 10 51 52 mm light path, and baseline corrected using a second cuvette with pure DI water as a 53 54 55 56 57 58 59 10 60 ACS Paragon Plus Environment Page 11 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 reference cell. All spectra were measured in 1 nm increments and peak intensities were 5 6 kept between 0.01 and 0.06. 7 8 9 10 11 2.7 Fluorescence correlation spectroscopy 12 13 A homebuilt confocal fluorescence correlation spectroscopy (FCS) setup was 14 15 used to determine particle hydrodynamic diameter, solution concentration, and number 16 17 36,37 18 of dye molecules per particle as described previously. Particles containing TMR dye 19 20 were excited with a 543 nm HeNe laser, that was focused by a water immersion 21 22 microscope objective (Zeiss Plan-Neofluar 63x NA 1.2). The fluorescence signal passed 23 24 25 through a 50 μm pinhole and a long pass filter (ET560lp, Chroma) before being detected 26 27 by an avalanche photo diode (APD) detector (SPCM-AQR-14, PerkinElmer) and auto- 28 29 correlated with a digital correlator (Flex03LQ, Correlator.com). Data were fitted using 30 31 a non-linear least-squares Levenberg-Marquardt algorithm and a triplet corrected 32 33 34 correlation function, G(), shown in equation (1): 35 36 1 1 1 1 2 1 37 � �(�) = 1 + (1 ― � + �exp ( ��)) (1) 38 ��(1 + � � )(1 + � (� �2)) (1 ― �) 39 � � 40 41 42 43 Where � is the lag time, �� the time- and spaced-averaged number of TMR labeled 44 45 ω 46 particles in the FCS observation volume, that is defined by a structure factor κ = � ω�� 47 48 with radial (ω�� ) and axial (ω� ) radii. �� is the time that a particle takes to diffuse 49 50 51 through the observation volume. � is the fraction of TMR molecules being in the triplet 52 53 state, with a triplet relaxation time, �� . FCS correlation curves were normalized using 54 55 equation (2): 56 57 58 59 11 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 12 of 37
1 2 3 4 �(�) = (�(�) ― 1) �� (2) 5 6 7 8 9 All samples were measured in 35 mm glass bottom dishes (P35G-1.5-10-C, Mattek 10 11 Corporation) at nanomolar concentration in DI water at 20 °C, 5 kW cm-2 laser power, 12 13 and in triplets with five 30 s long collection intervals. The observation volume was 14 15 calibrated before each FCS measurement. Particle diameters, � , were calculated using 16 17 18 the Stokes-Einstein equation (3) with the diffusion constant, � , obtained from equation 19 20 (4): 21 22 ��� 23 � = 2 (3) 24 6��� 25 2 26 ω�� 27 � = (4) 28 4�� 29 30 The number of TMR or MB2 molecules per particle, �� , was determined by comparing 31 32 33 the dye concentration from steady state absorption spectroscopy, CAbs, and the particle 34 35 concentration measured in FCS,
1 2 3 4 absorption band at 410 nm. Measurements were carried out at sample optical densities 5 6 of 0.15 – 0.50 in a 100 µL quartz cuvette (Starna). Samples were evenly exposed to a 7 8 635 nm, expanded, and collimated laser beam of a solid-state laser (Power Technology 9 10 11 Inc.) at 3 mW/cm2 with a spot size of about 1 cm in the same cell. To acquire a 0.5 - 0.6 12 13 absorption, DPBF was added at a concentration of approximately 18.75 µM. All 14 15 absorption spectra were measured in 1 nm steps and baseline-corrected against a second 16 17 18 cuvette with ethanol as a reference cell. The sample absorption was recorded at defined 19 20 time intervals and corrected for the sample absorption spectrum in the absence of DPBF. 21 22 Φ∆ was calculated by comparing all samples to the standard methylene blue (MB) dye 23 24 38 25 with known singlet oxygen quantum yield of Φ∆ = 0.52 (in ethanol) by plotting the 26 27 natural logarithm of the reduction of the 410 nm DBPF band against the exposure time 28 29 and using equation (6), where � represents the slope of a linear fit through the data 30 31 32 points: 33 34 �(������) Φ (������) = Φ (��) (6) 35 ∆ ∆ �(��) 36 37 To determine the effective singlet oxygen quantum yield, Φ eff, the particle 38 ∆ 39 40 concentration as determined by FCS and the MB concentration were matched. Errors 41 42 were calculated by determining Φ∆ three separate times for one sample. 43 44 45 46 47 48 49 2.9 Transmission Electron Microscopy (TEM) 50 51 1 µL of a ~15 µM concentrated sample solution (design 1, PEG-MB2-psC’ dots, 52 53 54 and design 2, MB2-PEG-psC’ dots) was diluted into 100 µL DI water. ~10 µL of the 55 56 57 58 59 13 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 14 of 37
1 2 3 4 diluted solution was then dropped onto carbon-film-coated TEM copper grids (Electron 5 6 Microscopy Sciences), and allowed to air dry. TEM imaging was performed using a FEI 7 8 Tecnai T12 Spirit microscope operated at 120 kV. 9 10 11 12 13 2.10 Photostability Measurements 14 15 Measurements were carried out in DI water using a 100 μL quartz cuvette 16 17 18 (Starna). All samples (design 1, PEG-MB2-psC’ dots, and design 2, MB2-PEG-psC’ 19 20 dots) were measured at optical densities of ~0.01 to ~0.02 with respect to the monomer 21 22 peak, and were evenly exposed to a Diode Red 640 nm Laser (Opto Engine LLC) 23 24 2 25 expanded and collimated solid-state laser (Inc.) at 50 mW/cm with a spot diameter of 26 27 about 1 cm in the same cell. All absorption spectra were measured in 1 nm steps and 28 29 baseline-corrected against a reference cell containing DI water. The absorption of each 30 31 sample was recorded at exposure times of 10 s, 20 s, 30 s, 30 s, 60 s, 120 s, 240 s, 480 32 33 34 s, 900 s, 1800 s and 3600 s. Spectra were qualitatively interpreted by fitting the 35 36 absorption spectrum with two Gaussians representing the monomer and dimer peaks. 37 38 39 40 41 42 3. RESULTS AND DISCUSSION 43 44 3.1 Synthesis of Photosensitizing C’ dots 45 46 47 48 49 Photosensitizing C’ dots (psC’ dots) covalently encapsulating the methylene 50 51 blue derivate MB2 inside the particle (design 1, Figure 1C and 1E) were synthesized by 52 53 54 combining tetramethylorthosilicate (TMOS) and MB2-silane (Figure 1B) in basic 55 56 57 58 59 14 60 ACS Paragon Plus Environment Page 15 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 aqueous solution. After particle formation, further particle growth was quenched by the 5 6 addition of PEG-silane (Figure 1B) to the reaction mixture, which was subsequently 7 8 heated to promote covalent bond formation between PEG-silane and surface silanol 9 10 11 groups.18,35 Particles containing MB2 on the particle surface (design 2, Figure 1C) were 12 13 synthesized in the same way, however, MB2 was attached using a grafting method 14 15 referred to as post-PEGylation surface modification by insertion (PPSMI).24 This 16 17 18 method employs sulfhydryl-reactive click chemistry, by adding thiol-silanes below the 19 20 nucleation threshold into an aqueous dispersion of PEGylated SNPs. The small molar 21 22 mass silane precursors diffuse through the PEG corona chains and react with the silica 23 24 25 core particle surface. The pending amine or thiol groups can further be reacted with 26 27 maleimide functional groups.24 For design 2, we used (3-mercaptopropyl) 28 29 trimethoxysilane (MPTMS) to functionalize the particle surface with thiol groups to 30 31 click MB2-maleimide to the particle (Figure 1E). Finally, all particles were cleaned 32 33 34 from unreacted precursors and particle aggregates via gel permeation chromatography 35 36 (GPC) prior to further characterization (see Materials and Methods section). 37 38 3.2 Particle Characterization 39 40 41 Fluorescent correlation spectroscopy (FCS) is a powerful tool for the 42 43 development and characterization of ultrasmall fluorescent silica nanoparticles.36,37 FCS 44 45 probes particle behavior with high signal-to-noise, and enables to assess a number of 46 47 48 sample properties simultaneously including particle concentration, hydrodynamic size, 49 50 and particle brightness. Particle diameter and concentration are determined by 51 52 measuring the fluorescence fluctuations of particles diffusing through a well-defined 53 54 observation volume of a laser beam and subsequently auto-correlating the fluorescence 55 56 57 58 59 15 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 16 of 37
1 2 3 4 time signals. In combination with steady-state absorption measurements of the same 5 6 sample, the number of fluorescent molecules can then be determined. 7 8 However, due to the weakly-emissive nature of MB2 (Supplementary Figure S3 9 10 11 in the Supporting Information), fluorescence-based size determination of MB2 12 13 functionalized particles by FCS was not possible. To render MB2-containing particles 14 15 accessible to FCS characterization, particles were co-functionalized with the fluorescent 16 17 18 dye conjugate tatramethylrhodamine-silane (TMR-silane) (Figure 1B). For design 1 19 20 with encapsulated MB2 we grafted TMR onto the particle surface using PPSMI. For 21 22 design 2 we turned the situation around: We synthesized SNPs encapsulating TMR dye 23 24 25 before MB2 was grafted on the particle surface (Figure 1E). A combination of FCS and 26 27 steady state absorption spectroscopy was then used to determine the number of MB2 28 29 and TMR molecules per particle. The resulting FCS correlation curves were fitted with 30 31 a correlation function (see equation (1), Materials and Methods) from which the time 32 33 34 averaged number of particles and the diffusion constant were extracted. To determine 35 36 the number of dyes per particle, the dye concentration as determined by steady-state 37 38 absorption spectroscopy was compared to the concentration of the particles as 39 40 41 determined by FCS (equation (5), Materials and Methods), yielding the average number 42 43 of dyes per particle. For accurately determination of the number of MB2 dyes per 44 45 particle, it is necessary that every MB2 containing particle carries at least one TMR dye, 46 47 48 a requirement that is not necessarily met. We accounted for that to the best possible 49 50 degree by working with high TMR concentrations in the synthesis. Dye molecules that 51 52 were not covalently bound during synthesis were washed away by dialysis and separated 53 54 from the particles by GPC. Supplementary Figure S4A and S4B in the Supporting 55 56 57 58 59 16 60 ACS Paragon Plus Environment Page 17 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 Information show the GPC elugrams before and after TMR and MB2 surface 5 6 functionalization of particles, respectively. Both elugram-pairs show a single peak and 7 8 were congruent to each other, indicating that TMR dye molecules (design 1), or MB2 9 10 11 dye molecules (design 2) were grafted onto the respective SNP surface. 12 13 Figure 2A and 2B show the FCS correlation curves of the fluorescent particles 14 15 containing the photosensitizer MB2. The curves were fitted using a triplet corrected 16 17 18 translational diffusion correlation function (equation (1), Materials and Methods). 19 20 Particle hydrodynamic diameters of 5.9 nm for MB2 encapsulating C’ dots with TMR 21 22 surface functionalization (TMR-PEG-MB2-psC’ dots), 5.2 nm for TMR encapsulating 23 24 25 C’ dots (PEG-TMR-C’ dots), and 5.2 nm for TMR encapsulating C’ dots with MB2 26 27 surface functionalization (MB2-PEG-TMR-psC’ dots) were obtained (a comprehensive 28 29 nomenclature to describe C’ dots is presented in the supporting information of reference 30 31 24). For comparison, TEM images of particles of design 1 and 2 were taken. As expected, 32 33 34 due to the low contrast of the PEG corona and hydration layer slightly smaller particle 35 36 diameters of about 4.0 nm were determined for both designs, (Supplementary Figure 37 38 S5A and S5B). 39 40 41 Figure 2C and 2D show the UV-vis absorption spectra of PEG-MB2-psC’ dots, 42 43 TMR-PEG-MB2-psC’ dots, PEG-TMR-C’ dots, and MB2-PEG-TMR-C’ dots in water, 44 45 respectively. For comparison, the absorption spectra of TMR-maleimide and MB2- 46 47 48 maleimide are superimposed onto the particle spectra. For TMR-PEG-MB2-psC’ dots 49 50 and MB2-PEG-TMR-psC’ dots, a TMR absorption peak can be observed indicating 51 52 successful functionalization of particles with TMR and MB2, respectively. 53 54 55 56 57 58 59 17 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 18 of 37
1 2 3 4 Comparing the absorption profiles of MB2 for the two different designs, a 5 6 relative hypsochromic shift (blue-shift) from 668 nm to 644 nm of the main peak for 7 8 design 1 relative to free MB2 is observed that is absent in design 2. This hypsochromic 9 10 11 shift likely originates from dimethylation of the auxochrome groups of MB2, from - 12 39,40 13 N(CH3)2 to -NH(CH3) and/or -NH2, which is promoted in basic media. In addition, 14 15 both designs display a heightened left shoulder in the absorption peak as compared to 16 17 18 free MB2 dye that is more pronounced in design 1 than it is in design 2. The heightened 19 20 shoulders around 620 nm and 605 nm for design 2 and one, respectively, are a result of 21 22 dimerization of MB2 at high concentrations (1×10−6 to 4×10−4 M) in aqueous media 23 24 -5 41 25 (MB2 concentration during synthesis is 3.67x10 M). MB monomers and dimers are 26 27 known to have distinct absorption peaks located at 664 nm and 590 nm, respectively, 28 29 with an equilibrium constant of 3.8x103 M-1 in water.42 However, the formation of 30 31 dimers is not only dependent on concentration but is additionally promoted by the 32 33 34 presence of oppositely charged surfaces.43 For design 1, the cationic MB2 sensitizer was 35 36 added to the synthesis during the silica particles formation and hence was exposed to 37 38 negatively charged silica nucleation seeds/clusters (at pH 9). For design 2, MB2 was 39 40 41 grafted onto the PEGylated silica particle surface at neutral conditions (pH 7), 42 43 consequently showing no peak shift and relatively fewer MB2 dimers, despite the same 44 45 MB2 concentration during the synthesis as for design 1. 46 47 48 To determine the number of MB2 molecules per particle we compared the 49 50 particle concentrations estimated by FCS and the MB2 concentrations from steady-state 51 52 absorption measurements. For practical reasons, we assumed that the extinction 53 54 coefficient remained unaffected in the particle synthesis. This is not necessarily true due 55 56 57 58 59 18 60 ACS Paragon Plus Environment Page 19 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 to the metachromatic nature of methylene blue and demethylation. Nevertheless, based 5 6 on this assumption we estimated the average number of dyes per particle (equation (5), 7 8 Materials and Methods) to be 2.4/3.3 for MB2/TMR for design 1 and 3.4/2.3 for 9 10 11 MB2/TMR for design 2. 12 13 3.3 Determination of Singlet Oxygen Quantum Yields 14 15 Next, we measured the singlet oxygen quantum yield, Φ , for both particle 16 ∆ 17 18 designs using the singlet oxygen sensor 1,3-diphenylisobenzofuran (DPBF). For these 19 20 measurements, we matched the particle concentrations as determined by FCS to yield 21 22 eff an effective singlet oxygen quantum yield per psC’ dot (ΦΔ (psC’ dot)). Figure 3A 23 24 25 demonstrates the principle of oxygen sensing using DPBF and the particle TMR-PEG- 26 27 MB2-psC’ dots (design 1) in ethanol. The mixture is evenly exposed to an expanded 28 29 and collimated 635 nm laser beam for defined time intervals. The singlet oxygen that is 30 31 generated by the psC’ dots reacts with DPBF molecules, yielding 1,2-dibenzoylbenzene 32 33 34 (Figure 3A inset).44 The formation of 1,2-dibenzoylbenzene was monitored via a 35 36 reduction of the absorption band at 410 nm (Figure 3B). By comparing samples to a 37 38 methylene blue standard (Φ (MB) = 0.52), Φ eff(psC’ dot) was determined (see equation 39 Δ Δ 40 41 (6), Materials and Methods), resulting in values of 111 ± 3% for design 1 (TMR-PEG- 42 43 MB2-psC’ dots) and 161 ± 5% for design 2 (MB2-PEG-TMR-psC’ dots). This 44 45 translates to an estimated per dye singlet oxygen quantum yield of 46% and 47%, 46 47 48 respectively, based on the estimated number of MB2 dyes per particle. The slightly 49 50 lower values for ΦΔ of the dyes associated with the particles versus free methylene blue 51 52 dye can be rationalized by the steric shielding effects of encapsulation or grafting within 53 54 the PEGylation corona. The silica network and/or the PEG molecules shield diffusing 55 56 57 58 59 19 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 20 of 37
1 2 3 4 oxygen, first, from MB2, and then, from DPBF resulting in a reduced singlet oxygen 5 6 quantum yield. Although for both designs the per dye ΦΔ values are similar, surface 7 8 grafted MB2 molecules are likely less shielded than dyes fully encapsulated in the silica 9 10 11 network. In addition, it is known that methylene blue dimers and monomers engage in 12 13 different photochemical processes. While monomers undergo energy transfer reaction 14 15 with triplet oxygen, dimers engage in electron transfer reactions with other methylene 16 17 43 18 blue molecules. These different energy dissipation pathways of dimers correlate 19 20 negatively with the singlet oxygen quantum yield possibly also contributing to the 21 22 slightly reduced singlet oxygen quantum yield per dye molecule of design 1 and design 23 24 45 25 2. However, relative to a single MB2 molecule, the multiplicity effect stemming from 26 27 multiple MB2 molecules colocalized on one particle far overcompensates for a reduced 28 29 per dye singlet oxygen quantum yield by steric shielding and/or dimerization. 30 31 To exclude the possibility of 1O formation in the absence of irradiation with 32 2 33 34 light (dark toxicity) for the different particle designs, we repeated singlet oxygen 35 36 quantum yield measurements, but did not expose the samples to the laser beam. 37 38 Supplementary Figure S6 in the Supporting Information shows results of the same 39 40 41 experiment as shown in Figure 3A for design 1 (PEG-MB2-psC’ dots). This time, the 42 43 DPBF peak at 410 nm remains unchanged, however, indicating no formation of 1,2- 44 45 dibenzoylbenzene and hence no generation of singlet oxygen. This is the case for both 46 47 48 designs. 49 50 To qualitatively investigate how MB2-particle encapsulation influences MB2 51 52 photostability we performed photobleaching experiments comparing free MB2 to 53 54 particles of design 1 and design 2 in aqueous solution. Samples were exposed to an 55 56 57 58 59 20 60 ACS Paragon Plus Environment Page 21 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 expanded and collimated 640 nm laser for defined time intervals and bleaching 5 6 progression was recorded via steady state absorption spectroscopy (Figure S7A to S7C). 7 8 To distinguish the MB2 monomer bleaching from the dimer peaks we fitted the spectra 9 10 11 with two Gaussians (Figure S7D to S7F). We found that MB2 monomer dye 12 13 incorporated in the silica matrix bleaches substantially slower compared to free MB2 14 15 dye in solution (Figure S7G and S7H). This agrees with previous findings where it was 16 17 46 18 shown that incorporated organic dyes are shielded from dissolved oxygen. On the 19 20 other hand, MB2 dyes grafted onto the particle surface had a tendency to bleach faster 21 22 as compared to free MB2 dye. This could possibly be understood by the presence of a 23 24 25 higher per particle dye average of MB2 molecules on the particle surface, leading to an 26 27 effectively increased local concentration of MB2 dyes around the particle. 28 29 3.4 Functionalization of psC’ dots with c(RGDyC) 30 31 Specific targeting of photosensitizers to diseased tissue increases the efficacy of 32 33 34 PDT and minimizes collateral damage to healthy tissue. Here, we functionalized psC’ 35 36 dots with the targeting moiety cyclo(arginine-glycine-aspartic acid-D-tyrosine-cysteine) 37 38 (c(RGDyC)) (Figure 1C), which targets α β integrins overexpressed, e.g. on various 39 v 3 40 20 41 cancer cells including melanoma. It has been shown that the endocytosis-mediated 42 43 cellular uptake of c(RGDyC)-functionalized particles correlates with the αvβ3- 44 45 expression levels of cells,47,48 and increases the intracellular particle concentration, 46 47 48 rendering c(RGDyC) a specific targeting moiety with high affinity for the treatment of 49 50 melanoma.12,34 In previous studies, it could be shown that ultrasmall c(RGDyC)- 51 52 functionalized SNP (Cornell dots) demonstrated selective disease targeting in 53 54 55 56 57 58 59 21 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 22 of 37
1 2 3 4 combination with bulk renal clearance in human clinical trials.20 We therefore believe 5 6 that c(RGDyC)-functionalized psC’ dots are a relevant first model system. 7 8 Particles were functionalized by adding c(RGDyC)-PEG-silane (Figure 1E and 9 10 11 Supplementary Figure S2 in the Supporting Information) during the PEGylation step 12 13 (for details see Materials and Methods).18 To allow more steric freedom for ligand 14 15 binding to integrins, the c(RGDyC)-PEG-silane was chosen to be three ethylene oxide 16 17 18 (EO) units longer than the PEG-silane (twelve versus six to nine units). Due to the 19 20 weakly-fluorescent nature of MB2, a FCS analysis could not be conducted. Instead we 21 22 compared the GPC elugrams before and after peptide functionalization for particle 23 24 25 design 1 (PEG-MB2-psC’ dots and c(RGDyC)-PEG-MB2-psC’ dots), and design 2 26 27 (MB2-PEG-psC’ dots and MB2-c(RGDyC)-PEG-psC’ dots) (Supplementary Figures 28 29 S8A and S8B in the Supporting Information). In both cases, we observed single peaks 30 31 that were congruent to each other. All particles were then characterized using steady- 32 33 34 state absorption spectroscopy. Figures 4A and 4D show the absorption spectra of design 35 36 1 and design 2, respectively, with and without c(RGDyC)-functionalization in water. In 37 38 both cases, increased absorption between 200 and 300 nm was noticeable. Due to strong 39 40 41 absorption features in that region it is difficult to clearly identify the peptide absorption 42 43 by qualitative comparison. For that reason, we subtracted the two spectra from each 44 45 other and displayed the difference spectra in Figures 4B and 4E. In both cases a band at 46 47 48 ~260 to 270 nm can clearly be identified, which coincides with the absorption band of 49 50 the c(RGDyC) spectrum (Supplementary Figure S8C in the Supporting Information).24 51 52 Using the relative absorption peaks of tyrosine in c(RGDyC) and of MB2, we estimated 53 54 55 56 57 58 59 22 60 ACS Paragon Plus Environment Page 23 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 17 and 14 c(RGDyC) units per MB2 molecule for design 1 and design 2, respectively, 5 6 which is close to the desired number based on earlier studies.34 7 8 Finally, we tested the effect of c(RGDyC)-functionalization on the relative 9 10 11 singlet oxygen quantum yield performance. We compared particles with and without 12 13 c(RGDyC) for absorption matched samples of the same design. For both designs we 14 15 measured a reduction of singlet oxygen quantum yield by a relative 25% for design 1 16 17 18 and by a relative 12% for design 2 (Figure 4C and 4F). This finding is surprising. Given 19 20 the spatial proximity of surface MB2 and c(RGDyC), one would expect a stronger effect 21 22 of c(RGDyC) in design 2. Results suggest, however, that c(RGDyC) increases the steric 23 24 25 shielding more significantly for the encapsulated MB2 than for the surface grafted MB2. 26 27 28 29 30 31 4. CONCLUSIONS 32 33 34 In this study, we have presented two different designs for the covalent loading 35 36 of ultrasmall organic-inorganic hybrid silica nanoparticles with photosensitizer 37 38 molecules (psC’ dots) in the form of methylene blue derivate MB2. MB2 was either 39 40 41 covalently encapsulated in the silica core or attached to the silica core surface. Both 42 43 designs yielded sub-10 nm size particles that could be further functionalized with 44 45 c(RGDyC) as a targeting moiety. We found that the properties of MB2 strongly depend 46 47 48 on its position in the particle. While design 1 protected MB2 from photobleaching, 49 50 design 2 showed increased photobleaching as compared to free MB2 dye. Despite 51 52 slightly reduced per molecule singlet oxygen quantum yields of MB2 upon particle 53 54 association, the effective per particle singlet oxygen quantum yields far exceeded the 55 56 57 58 59 23 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 24 of 37
1 2 3 4 quantum yield of a single MB2 dye as a result of multiple MB2 molecules per particle. 5 6 The advantages established for fluorescent dye encapsulating C’ dots and expected for 7 8 ultrasmall organic-inorganic hybrid psC’ dots as a delivery and protective system for 9 10 11 photosensitizers make such probes interesting candidates for applications in PDT: This 12 13 includes surface PEGylation providing particle stability, e.g. in blood serum, surface 14 15 targeting moieties enabling enhanced accumulation at desired sites of disease, and 16 17 18 ultrasmall particle size enabling rapid renal clearance thereby minimizing possible side 19 20 effects.20 21 22 Although this study focuses on the photosensitizer MB2, described design 23 24 25 principles and synthesis methods are in principle applicable to other photosensitizers. 26 27 This might be of special interest for hydrophobic NIR and IR photosensitizers with large 28 29 singlet oxygen quantum yields, i.e. porphyrins, chlorins, phthalocyanines, 30 31 naphthalocyanines, bacteriochlorins, and BODIPY dyes. PEGylated silica can provide 32 33 34 a water-soluble carrier for these cargos, to allow specific targeting and achieve high 35 36 local concentrations at targeted sites, while avoiding aggregation in aqueous media. 37 38 While more work is necessary to fine-tune such synthesis protocols to fully harvest the 39 40 41 potential of ultrasmall organic-inorganic hybrid silica nanophotosensitizers (psC’ dots), 42 43 we believe that psC’ dots carry high potential for clinical translation. 44 45 46 47 48 49 50 51 52 53 FIGURES 54 55 56 57 58 59 24 60 ACS Paragon Plus Environment Page 25 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Figure 1. (A) Simplified Jablonski diagram illustrating the creation of reactive singlet 1 1 1 42 oxygen, O2. PS denotes the singlet ground state, PS* the electronically excited singlet 43 state, and 3PS* the electronically excited triplet state of a photosensitizer. 3O denotes 44 2 45 the triplet ground state of molecularly dissolved oxygen. (B) Precursor molecules for 46 the synthesis of sub-10 nm silica nanoparticles, showing the methylene blue derivate 47 MB2-silane, the rhodamine dye TMR-silane, tetramethyl orthosilicate (TMOS), and 48 49 polyethylene glycol-silane (PEG-silane). (C) Schematic representation of two different 50 designs of functionalized photosensitizing sub-10 nm silica nanoparticles (psC’ dots). 51 Design 1: Covalent encapsulation of one or more MB2 molecules in the silica matrix of 52 53 the particle core (PEG-MB2-psC’ dots). Design 2: Particle surface functionalization 54 with one or more MB2 molecules (MB2-PEG-psC’dot). (D) Molecular structure of 55 targeting moiety cyclo(Arg-Gly-Asp-D-Tyr-Cys) (cRGDyC). (E) Synthesis scheme for 56 57 58 59 25 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 26 of 37
1 2 3 4 functionalized sub-10 nm silica photosensitizers of design 1 and 2 showing the three 5 general synthesis steps. A comprehensive nomenclature to describe C’ dots is presented 6 7 in the supporting information of reference 24. 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 26 60 ACS Paragon Plus Environment Page 27 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 Figure 2. (A) and (C) FCS autocorrelation curve of MB2-PEG-TMR-psC’dot (design 5 1) and absorption spectra before and after TMR surface functionalization as compared 6 7 to free TMR dye and MB2 photosensitizer. (B) and (D) FCS autocorrelation curves of 8 PEG-TMR-C’ dots and TMR-PEG-MB2-psC’ dots (design 2) and absorption spectra 9 before and after MB2 surface functionalization as compared to free TMR dye and MB2 10 11 photosensitizer. 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 27 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 28 of 37
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Figure 3. (A) Schematic representation of a photosensitizing measurement using 1,3- 21 1 diphenylisobenzofuran (DPBF) as a singlet oxygen, O2, sensor. Absorption of a 22 23 solution containing TMR-PEG-MB2-psC’ dots and DPBF irradiated at 635 nm for 60 s 1 24 in intervals of 5 and 10 s (see legend). (B) Comparative O2 generation of methylene 25 blue, TMR-PEG-MB2-psC’ dots, and MB2-PEG-TMR-psC’ dots. 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 28 60 ACS Paragon Plus Environment Page 29 of 37 ACS Biomaterials Science & Engineering
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Figure 4. (A) Intensity matched absorption spectra of PEG-MB2-psC’ dots and 27 28 c(RGDyC)-PEG-MB2-psC’ dots. (B) Difference spectrum of the two spectra in (A) 29 revealing the absorption band of c(RGDyC). (C) Photosensitizing measurement of 30 intensity matched PEG-MB2-psC’ dots and c(RGDyC)-PEG-MB2-psC’ dots. (D) 31 32 Intensity matched absorption spectra of MB2-PEG-psC’ dots and MB2-c(RGDyC)- 33 PEG-PsC’ dots. (E) Difference spectrum of the two spectra in (D) revealing the 34 absorption band of c(RGDyC). (F) Photosensitizing measurement of intensity matched 35 36 MB2-PEG-psC’ dots and MB2-c(RGDyC)-PEG-psC’ dots. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 29 60 ACS Paragon Plus Environment ACS Biomaterials Science & Engineering Page 30 of 37
1 2 3 4 ASSOCIATED CONTENT 5 6 Supporting Information 7 8 Absorption spectra of MB and MB2, chemical structures of (cRGDyC)-PEG(12)-silane 9 10 11 and MPTMS, comparative fluorescence emission spectra of free dye, C’ dots and psC’ 12 13 dots, GPC elugrams before and after surface functionalization of psC’ dots, TEM 14 15 images of psC’ dots, photobleaching experiments of MB2 and psC’ dots. 16 17 18 19 20 AUTHOR INFORMATION 21 22 ORCID 23 24 25 Ferdinand F. E. Kohle: 0000-0002-7351-5940 26 Songying Li: 0000-0002-7977-9293 27 28 Melik Z. Turker: 0000-0001-7801-4275 29 30 Ulrich B. Wiesner: 0000-0001-6934-3755 31 32 33 34 Author Contributions 35 36 ∥F.F.E.K. and S.L. contributed equally to this paper. 37 38 39 40 41 Notes 42 43 The authors declare the following competing financial interest: F.F.E.K. and U.B.W. 44 45 46 have filed for a patent based on these findings. Other authors declare no competing 47 48 interests. 49 50 51 52 53 ACKNOWLEDGEMENTS 54 55 This work was funded by the National Institutes of Health (NIH) under Award 56 57 58 59 30 60 ACS Paragon Plus Environment Page 31 of 37 ACS Biomaterials Science & Engineering
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1 2 3 4 TABLE OF CONTENTS GRAPHIC 5 6 For Table of Contents Use Only. 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Title: Ultrasmall PEGylated and Targeted Core-Shell Silica Nanoparticles Carrying 29 30 Methylene Blue Photosensitizer 31 32 33 34 Authors: Ferdinand F. E. Kohle, Songying Li, Melik Z. Turker, and Ulrich B. 35 36 Wiesner 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 37 60 ACS Paragon Plus Environment