Research Article www.acsami.org
Dual-Functionalized Theranostic Nanocarriers Alexander H. Mo,∥,○ Chen Zhang,†,○ Preston B. Landon,*,‡,§,○ Woraphong Janetanakit,† Michael T. Hwang,∥ Karla Santacruz Gomez,∇ David A. Colburn,‡ Samuel M. Dossou,† Tianyi Lu,‡ Yue Cao,‡ Vrinda Sant,‡ Paul L. Sud,‡ Siddhartha Akkiraju,† Veronica I. Shubayev,⊥ Gennadi Glinsky,# and Ratnesh Lal*,‡,§,∥ †
Department. of Nanoengineering, ‡Department of Bioengineering, §Department of Mechanical and Aerospace Engineering, Materials Science and Engineering Program, ⊥Department of Anesthesiology, and #Institute of Engineering in Medicine, University of California, San Diego, La Jolla, California 92093, United States ∇ Department of Physics, Universidad de Sonora, Hermosillo, Mexico ∥
S Supporting Information *
ABSTRACT: Nanocarriers with the ability to spatially organize chemically distinct multiple bioactive moieties will have wide combinatory therapeutic and diagnostic (theranostic) applications. We have designed dual-functionalized, 100 nm to 1 μm sized scalable nanocarriers comprising a silica golf ball with amine or quaternary ammonium functional groups located in its pits and hydroxyl groups located on its nonpit surface. These functionalized golf balls selectively captured 10−40 nm charged gold nanoparticles (GNPs) into their pits. The selective capture of GNPs in the golf ball pits is visualized by scanning electron microscopy. ζ potential measurements and analytical modeling indicate that the GNP capture involves its proximity to and the electric charge on the surface of the golf balls. Potential applications of these dual-functionalized carriers include distinct attachment of multiple agents for multifunctional theranostic applications, selective scavenging, and clearance of harmful substances. KEYWORDS: Janus particle, electrostatic, adsorption, nanocarrier, golf ball, gold colloid
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INTRODUCTION
To date, dual-functionalization strategies have used internal cavities of silica nanocarriers with core−shell structures, such as mesoporous silica nanoparticles,13 silica nanocages,14 and silica nanobowls.15,16 Dual-functionalization has been achieved by attaching one kind of functional group on top of the shell and another group on the internal walls of the cavities. Without surface modification, the negatively charged particles would trigger immune response and cause cytotoxicity, while less negatively charged particles or neutral particles usually show longer circulation time.17 Another limitation with these existing systems is that the monotone surface provides limited opportunities for attaching multiple active moieties. Here we describe design, synthesis, and characterization of dual-functionalized golf ball structures which were assembled by using hierarchical core−satellite templates. The golf ball structures have dimples or pits possessing functional groups (e.g., secondary amine or quaternary ammonium) that are distinct from those on the surface of the golf balls.15,16 The core−satellite template used in this process is synthesized by modifying the silica core with an amine or quaternary
Emergence of dual-functionalized silica nanocarriers has attracted much attention in biosensing,1 adsorption,2 catalysis,3 separation,4 and clinical (drug and diagnostic) theranostic delivery.5,6 Dual-functional drug delivery vehicles with a large, multiple-therapeutic loading capacity and immune system camouflaging surface modifications would provide sensitivity and specificity.7,8 Recent advances in the hierarchical design and selective functionalization of porous silica nanocarriers allow designing more sophisticated combination theranostic delivery systems.9,10 Among existing theranostic nanocarriers, porous silica nanocarriers can be designed to possess ideal properties, including good biochemical stability, tunable carrier size, modifiable carrier surface, controllable pore sizes, ordered pore structure, augmented pore capacity, and distinctively functionalized pores and surfaces.5,10,11 The simultaneous delivery of diagnostics, bioimaging contrast molecules, and combination therapeutics then can be achieved by attaching multiple small molecules, enzymes, DNA, or RNA into the interior volume or on the surface and controlling their release rate once the carriers find their destination.5,12 This strategy can also prolong the circulation half-lives, improve the pharmacokinetics, and minimize the side effects.8 © XXXX American Chemical Society
Received: March 7, 2016 Accepted: May 4, 2016
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DOI: 10.1021/acsami.6b02761 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
nanoparticles were dispersed in 25 mL of anhydrous ethanol, respectively. Satellite Attachment. Satellite attachment was achieved by our previously published method with modification.15,16,19 Briefly, the ratio of carboxyl-functionalized 100 nm polystyrene to the amine- and quaternary-ammonium-functionalized silica nanoparticles was varied during satellite attachment process to obtain heterogeneous hierarchical templates with different polystyrene surface densities on the silica cores. A 4 mL amount (0.4%) of functionalized nanoparticles was taken out from each of the quaternary-ammonium- and aminecoated 1000 nm silica nanoparticle solutions and re-dispersed in a 500 μL 4:1 ethanol/water solution, respectively. A 132 μL amount of carboxyl-functionalized 100 nm polystyrene (PS-COOH) NPs was added inside each batch and reacted for 30 min, followed by vortex. Excessive PS-COOH was rinsed out by centrifuge at 1576 rpm for 5 min. For 100 nm templates, 25 μL of 100 nm PS-COOH was added into 500 μL of EtOH. Next, the PS-COOH solution was sonicated for 30 s to well-disperse the particles. A 6.25 μL amount (0.4%) of 100 nm amine- or quaternary-ammonium-functionalized silica nanoparticles was added into the solution, followed by adding 93.75 μL of water. The solution was tumbled for 30 min and used to prepare silica golf balls without rinsing to minimize particle aggregation. Preparation of Silica Golf Balls. Both the amine- and quaternaryammonium-functionalized 1000 nm nanoparticles were brought back in 5 mL of 4:1 IPA/water solution. A 100 μL aliquot of NH4OH was first added inside the batches. The two batches were stirred for 10 min. Then, 12 μL of TEOS was added inside the two batches, respectively. The reactions lasted for 2 h. The particles were then rinsed 3 times with IPA and resuspended in 4 mL of IPA. A 1 mL aliquot of particles from each batch was taken out and dissolved in DMF at 60 °C while solicited for 1 h to wash the PS out of the silica golf balls. The process was repeated 2 times. The silica golf balls were then left in DMF at 60 °C for 2 days. Alternatively, 1 mL of particles from each batch was dissolved in THF at 60 °C while solicited for 1 h to remove PS. Also, the same process was repeated 2 times, and the particles were left in THF at 60 °C for 2 days. Finally, the 1000 nm silica golf balls were redispersed in 1 mL ethanol and stored in the fridge at 4 °C, respectively. The amine-functionalized 100 nm templates were diluted with 3.5 mL of IPA, followed by adding 900 μL of water. A 30 μL aliquot of ammonium hydroxide was then added to the solution, before adding 8 μL of TEOS. The mixture was stirred for 3 h. Afterward, the mixture was centrifuged at 1576 rpm for 5 min, and the upper liquid was decanted and the particles were suspended in 2 mL of EtOH. The wash step was repeated 3 times. The particles were resuspended in 5 mL of DMF and stirred at 60 °C for 2 days. The particles were rinsed with 5 mL of EtOH three times. Finally, the 100 nm silica golf balls were re-dispersed in 1 mL of ethanol and stored at 4 °C. Capturing PS and GNPs with Silica Golf Balls. For the amineor quaternary-ammonium-functionalized 1000 nm golf balls, 6 × 150 μL of each kind was dispersed in 1 mL of water. Three of them were kept in water. Two of them were re-dispersed in 1 mL of 80% ethanol. The last one was re-dispersed in 1 mL of 20% ethanol. A 3 μL amount of (1) 50 nm PS and (2) 100 nm PS was added to the 1 mL water solution. A 3 μL amount of (3) 50 nm PS and (4) 100 nm PS was added to the 1 mL of 80% ethanol solution. A 3 μL amount of (5) 50 nm PS was added to the 1 mL 20% ethanol solution. A 3 μL amount of (6) 100 nm gold nanoparticles was added to the 1 mL water solution. Another 4 × 150 μL preparation of 1000 nm golf balls was made and re-dispersed in 1 mL 11.1 mM potassium biphthalate buffer (pH = 4). Three of them were kept in the buffer. The fourth one of them was re-dispersed in 1 mL 20% ethanol/potassium biphthalate buffer. A 3 μL amount of (7) 50 nm PS nanoparticles was added to the golf balls in 1 mL potassium biphthalate buffer. A 100 μL amount of (8) 40 nm gold nanoparticles was added to the second golf balls in 1 mL potassium biphthalate buffer. A 400 μL amount of (9) 20 nm gold nanoparticles was added to the third golf balls in 1 mL potassium biphthalate buffer. A 3 μL amount of (10) 100 nm gold nanoparticles
ammonium layer and adsorbing carboxylated polystyrene (PS) spheres to the surface of the core. Using the same core−satellite template, a silica-coated particle could be applied instead of gold. The PS spheres trapped in the silica shell now serve as removable nanomasks for the bottom-up construction of sophisticated silica golf balls with surface dual-functionalizing ability. Using a hierarchical template strategy to create silica golf balls, we show that selective surface functionalization of silica nanoparticles can be achieved. We demonstrate the potential utility of the silica golf balls by capturing colloidal gold nanoparticles in pH controlled buffer. The electric surface potential of the golf balls is measured and analytically modeled.
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EXPERIMENTAL SECTION
Carboxylate-modified colloidal polystyrene spheres with 100 nm diameters (2.6% (w/v); diameter, 85 nm; CV, 8%) (Catalog No. 16688), colloidal silica with 1000 nm diameters (Catalog No. 253431.5), and colloidal silica with 100 nm diameters (Catalog No. 2429810) were purchased from PolySciences. (3-Aminopropyl)trimethoxysilane (APTMS; Catalog No. A11284) was purchased from Alfa Aesar. Tetraethyl orthosilicate (TEOS; Catalog No. 131903) was purchased from Sigma-Aldrich. N-Trimethyoxysilylpropyl-N,N,Ntrimethylammonium chloride (50% in methanol) (Catalog No. SIT8415.0) was purchased from Gelest, Inc.. Ammonium hydroxide (NH4OH, 29%) (Catalog No. A669S) was purchased from Fisher Chemicals. Isopropanol (IPA; HPLC grade; Catalog No. A451-4) was purchased from Fisher Scientific. N,N-Dimethylformamide (DMF; Catalog No. 4929-08) was purchased from Macron Chemicals. Tetrahydrofuran (THF; HPLC grade) was purchased from Fisher (Catalog No. T425-1). Anhydrous ethyl alcohol (EtOH; Catalog No. 9401-06) formula 3A, 200 proof, was purchased from JT Baker. Potassium biphthalate (Catalog No. 270-4.00) was purchased from Micro Essential Lab. Sodium phosphate, dibasic, heptahydrate (Catalog No. 7914-04) was purchased from Mallinckrodt Chemicals. Citric acid monohydrate (Catalog No. C-7129) was purchased from Sigma-Aldrich. The water used in all experiments was purified using a Millipore Advantage A10 system with a resistance of 18.2 MΩ. The scanning electron microscopy (SEM) images were taken with an FEI XL30 SFEG UHR SEM instrument. Preparation of Functionalized Silica Cores. Functionalized silica cores were prepared using the published methods18 with modification. Briefly, aqueous colloidal silica was transferred to EtOH by centrifugal rinsing in EtOH at 2230 rpm for 5 min followed by decanting and re-dispersing in EtOH. The rinsing was repeated four times. A 72 mg amount of 1000 nm colloidal silica was dispersed in 12.6 mL of EtOH by rinsing a 7.4 wt % solution in water (0.973 mL of 7.4%). Also, 72 mg of 100 nm colloidal silica was dispersed in 12.6 mL of EtOH. The mixture was stirred for 10 min in a 20 mL glass scintillation vial prior to adding (1) 10.8 μL of N-trimethyoxysilylpropyl-N,N,N-trimethylammonium chloride (50% in methanol) for quaternary-ammonium-functionalized 1000 nm silica cores, (2) 5.4 μL of APTMS for amine-functionalized 1000 nm silica cores, (3) 1.08 mL of N-trimethyoxysilylpropyl-N,N,N-trimethylammonium chloride (50% in methanol) for quaternary-ammonium-functionalized 100 nm silica cores, and (4) 540 μL of APTMS for amine-functionalized 100 nm silica cores. The mixtures were left stirring for 2 h. Then, 13 mL of EtOH was added in each of the four cases, and the reaction was heated to 60 °C for 1 h while stirring. The mixtures were then centrifuged at 2230 rpm for 5 min. The liquid was decanted, and the solid was re-dispersed in 14 mL of EtOH. The centrifuge tubes containing the 14 mL solutions were then placed in a bath ultrasonic cleaner at 60 °C for 30 min. The centrifuge and disperse process was repeated 3 times. Finally, the (1) 1000 nm quaternary-ammoniumfunctionalized silica nanoparticles, (2) 1000 nm amine-functionalized silica nanoparticles, (3) 100 nm quaternary-ammonium-functionalized silica nanoparticles, and (4) 100 nm amine-functionalized silica B
DOI: 10.1021/acsami.6b02761 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces was added to the golf balls in 1 mL 20% ethanol/potassium biphthalate buffer. A 2 × 150 μL preparation of quaternary-ammonium- or aminefunctionalized 100 nm golf balls was made and re-dispersed in 1 mL 11.1 mM potassium biphthalate buffer (pH = 4). A 400 μL amount of (11) 10 nm gold NP was added to one golf ball in 1 mL potassium biphthalate buffer. (12) A set of 10 nm nanodiamonds was added to the other golf balls in 1 mL potassium biphthalate buffer. All capture reactions were left on the labquake tumbler for 1 day. The mixture underwent centrifuge under 1576 rpm, and excessive GNPs were rinsed out by 1 mL of water for 3 times. Finally, the particles were placed on the SEM grid. In addition, the reversibility of the gold capture was examined by altering the pH of the rinsing media to 7 and 10, followed by imaging with SEM. Electrophoretic Mobility and the ζ Potential Measurements. ζ potential was measured with a ζ potential analyzer (Brookhaven Instrument Corp., Zetaplus/BI-PALS) for the following five samples: (1) the quaternary-ammonium-functionalized 1000 nm silica cores; (2) PS attached 1000 nm quaternary-ammonium-functionalized silica cores; (3) 1000 nm quaternary-ammonium-functionalized silica cores with intact silica shell; (4) 1000 nm quaternary-ammonium-functionalized golf balls with PS removed by DMF; (5) 1000 nm quaternaryammonium-functionalized nano-golf balls without PS removal. For each of the six samples, ζ potential measurements were completed in five different buffers: (1) a 1 mM pH 7 sodium phosphate buffer; (2) a 1 mM pH 4 potassium biphthalate buffer; (3) a pH 3.0 citric acid− sodium phosphate dibasic buffer, which was prepared by adding Sigma’s pH 3.0 recipe20 dropwise to 50 mL of water until the pH of water was stable at 3.0; (4) a pH 2.6 citric acid−sodium phosphate dibasic buffer, which was prepared by adding Sigma’s pH 2.6 recipe dropwise to 50 mL of water until the pH of water was stable at 2.6; (5) a pH 2.2 citric acid buffer, which was prepared by adding 0.1 M citric acid solution dropwise to 50 mL of water until the pH was stable around 2.2. A 1 mL amount (1.6%) of the above six samples were centrifuged at 1576 rpm for 10 min, respectively, and EtOH was decanted. A sample was then re-dispersed in the 2 mL of buffer for the pH of choice. The ζ potential measurement result was obtained by averaging five readings. After the measurements, the designated sample was centrifuged at 1576 rpm for 10 min. The pH buffer was decanted, and particles were re-dispersed in EtOH again. The sample was centrifuged and redispersed in the next buffer for the pH of choice. The process was repeated until all measurements for all samples were finished.
Figure 1. Heterogeneous hierarchical template scheme applied in the synthesis of silica golf balls: (a) silica core (gray); (b) functionalized with a shell (red) of (3-aminopropyl)trimethoxysilane (APTMS) or Ntrimethyoxysilylpropyl-N,N,N-trimethylammonium chloride with (c) smaller polystyrene (PS) satellite spheres (purple) electrostatically attached; (d) TEOS shell growing into an interconnected silica shell outside the functionalized shell; (e) dissolution of the polystyrene satellites completes the synthesis of the silica golf ball particles. Silica golf balls capturing the gold colloidal particles (f−h): (f) silica golf ball particle with quaternary ammonium functionalized inside the pits (red); (g) silica golf ball particle capturing 20 nm or 40 nm GNPs (gold), after adding 20 nm or 40 nm GNPs to be captured; (h) silica golf ball particle capturing 20 nm GNPs with multiple GNPs in each pit (upper) or capturing 40 nm GNPs with single GNP in each pit (lower).
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RESULTS AND DISCUSSION Synthesis of Functionalized Carriers. The synthesis of functionalized carriers and the preparation of silica golf balls required three steps: (1) PS attached template synthesis; (2) TEOS plating on the template; (3) PS etching. The silica golf balls were synthesized by TEOS self-assembly on prefabricated template particles. This reaction was catalyzed by concentrated ammonia. The template particles were prepared by physically adsorbing smaller 100 nm PS particles on the amine-functionalized 1000 and 100 nm silica particles and on the quaternary-ammoniumfunctionalized 1000 and 100 nm silica particles, respectively.21 Later, the 100 nm PS particles were etched out from the silica golf ball surface by dissolving the particles in DMF heating to 60 °C in the water bath. The synthesis scheme is shown in Figure 1. The results of the amine- and quaternary-ammoniumfunctionalized NP fabrication and the silica golf ball preparations were morphologically evaluated by SEM. The SEM images of the functionalized particles are shown in Figure 2a,b and Figure 3a,b.
Figure 2. SEM images from stages of the silica golf ball synthesis process using 1000 nm silica cores: (a) pollen resembling structure created by the self-assembly of 100 nm carboxylate-modified polystyrene on the surface of quaternary-ammonium-functionalized 1000 nm silica balls; (b) 1000 nm silica golf ball after TEOS growth on the quaternary-ammonium-functionalized shell, rendered by removal of the polystyrene; (c and d) 1000 nm silica golf ball particles capturing 20 nm GNPs, with multiple GNPs in each pit (arrows indicate GNPs captured by the pores); (e) 1000 nm silica golf ball particles capturing the 40 nm GNPs with single GNP in each pit (arrows indicate GNPs captured by the pores).
The amount of APTMS and the amount of N-trimethyoxysilylpropyl-N,N,N-trimethylammonium chloride were varied C
DOI: 10.1021/acsami.6b02761 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
(Figure 2c−e). GNPs were observed inside the pits of the 1000 nm silica golf balls. For 40 nm GNPs, single GNP capture was achieved in the pits. For 20 nm GNPs, two to three GNPs can be captured by one pit. Additionally, 10 nm GNPs were captured by 100 nm functionalized golf balls (Figure 3c,d). Neither PS nanoparticles nor nanodiamonds can be captured by the synthesized silica golf balls. Potassium biphthalate buffer was the only useable capture media, while water, ethanol, or ethanol−potassium biphthalate mixture was not suitable for GNPs capture media. Thus, the capture of the GNPs by the amine- or quaternary-ammonium-functionalized silica golf balls was achieved only in acidic buffers. Adsorption of GNPs by silica golf balls was not observed when the capture reaction occurred in water or water−ethanol mixtures. The irreversibility of gold capture was examined by changing the pH of the media through rinsing the capture products with excessive pH 4, 7, and 10 media, respectively. Significantly, once the gold NPs were captured, they stayed inside the pits of the golf balls irrespective of the pH of the rinsing media. This result can be explained by the likelihood that the initial driven force of the capture reaction was ionic attraction, yet the gold NPs were held inside the pits by an interplay of covalent bond formation, van der Waals attraction, and an electrostatic complex between amine/quaternary ammonium groups and chloroaurate ions.23 Four kinds of silica golf balls were fabricated using a heterogeneous synthetic template,. The pits of the silica golf ball have an amine- or quaternary-ammonium-functionalized bottom, which may facilitate their further selective functionalization. The quaternary-ammonium-functionalized silica golf balls preferentially captured
