Thrombin-Responsive Gated Silica Mesoporous Nanoparticles As

Subscriber access provided by GAZI UNIV

Letter

Thrombin-responsive gated silica mesoporous nanoparticles as coagulation regulators Ravishankar Bhat, Angela Ribes, Núria Mas, Elena Aznar, Felix Sancenón, María Dolores Marcos, Jose Ramón Murguía, A. Venkataraman, and Ramon Martinez-Mañez Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04038 • Publication Date (Web): 21 Jan 2016 Downloaded from http://pubs.acs.org on January 22, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Thrombin-responsive gated silica mesoporous nanoparticles as coagulation regulators Ravishankar Bhat,⊥, † Àngela Ribes, †, ‡ Núria Mas, †, ‡ Elena Aznar, †, ‡ Félix Sancenón, †, §, ‡ M. Dolores Marcos, †, §, ‡ Jose R. Murguía, †, ‖, ‡ Abbaraju Venkataraman,⊥ Ramón MartínezMáñez*†, §, ‡ †

Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), § Departamento de

Química and ‖Departamento de Biotecnología, Universitat Politècnica de València, Valencia, 46022, Spain ‡

⊥

CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Valencia, Spain Materials Chemistry Laboratory, Department of Materials Science and Department of

Chemistry, Gulbarga University, Gulbarga-585106, India

ABSTRACT: The possibility of achieving sophisticated actions in complex biological environments using gated nanoparticles is an exciting prospect with much potential. We herein describe new gated mesoporous silica nanoparticles (MSN) loaded with an anticoagulant drug and capped with a peptide containing a thrombin-specific cleavage site. When the coagulation cascade was triggered, active thrombin degraded the capping peptidic sequence and induced the release of anticoagulant drugs to delay the clotting process. The thrombin-dependent response

ACS Paragon Plus Environment

1

Langmuir

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 21

was assessed and a significant increase in coagulation time in plasma from 2.6 to 5 min was found. This work broadens the application of gated silica nanoparticles and demonstrates their ability to act as controllers in a complex scenario such as hemostasis.

INTRODUCTION Hemostasis can be visualized as a biological fine-tuned mechanism that controls and triggers, when appropriate, the blood coagulation cascade. In this elegant process, the body is able to transform aggressive signals, such as tissue damage or vascular leak, into biochemical information, which induces thrombin protease activation.1 Thrombin is a coagulation protein with many effects on the blood coagulation cascade, and it is known to highly and specifically recognize the Leu-Val-Pro-Arg-Gly-Ser peptide sequence. This serine protease selectively cleaves the Arg–Gly bonds of fibrinogen to form fibrin and release fibrino peptides A and B.2 Research into the discovery of new agents that are able to trigger or stop the programmed cascade has been extensively conducted3-4 given the importance of this biological process. Among other tools, particles within the nanometer range have been explored as either thrombin cascade activators or antithrombotic agents.5-6 In particular, mesoporous silica nanoparticles (MSN) with anticoagulant activity have been described by Bein and coworkers.7 In their work, the authors heparinized MSN by coating their external surface with polysaccharides via an amidation step and they obtained prolonged blood clotting times using this system. In a further step, Wu and coworkers8 used the same glycosaminoglycan to coat MSN and deliver basic fibroblast growth factor, which confirmed the possibility of developing blood-compatible MSN. However all these described MSN-based systems exhibit an anticoagulant effect from the very first time they were added, and cannot, in principle, remain latent and act as anticoagulants only


2

Page 3 of 21

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

when needed. In this context, and as far as we know, the development of nanoparticles capable of working as cascade coagulation controllers in a fine-tunable manner and of acting oncommand as antithrombotic systems has not yet been described. Therefore, we believe that designing such nanoparticles could be relevant, for example, in the blood thinning medication field for certain applications. Nowadays, the promising usefulness of gated mesoporous silica nanoparticles as delivery systems has been widely recognized.9-12 These smart nanoparticles, which are able to deliver at will selected cargoes in response to physical,13-16 chemical17-21 or biochemical stimuli,22-24 have been widely studied as potential new therapies against cancer and other current chronic diseases.25-27 Gated silica nanoparticles also have strong potential as nano-objects as they possess advanced functionalities and bio-predictable responses, and could be suitable systems for acting as controllers of biological pathways. In this context, we were interested in designing capped MSN capable of detecting the activation of clotting and controlling the blood coagulation cascade. To achieve our goal, we focused on the high proteolytic specificity of thrombin, and also on the fact that the thrombin concentration increases when the coagulation cascade is activated. Based on this concept, we designed MSN, loaded with anticoagulant drug acenocoumarol and capped with a peptide containing thrombin-specific cleavage site LVPRGS (See Scheme 1). It was expected the presence of thrombin would hydrolyze the peptidic sequence inducing the release of the antithrombotic drug that would result in a change of the cascade process course.


3

Langmuir

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 21

Scheme 1. Representation of solid S1-P performance during the coagulation process. Once the cascade is activated, the generated thrombin is able to hydrolyze the capping peptidic sequence. Then released acenocoumarol is able to stop the pathway and avoid clot formation. EXPERIMENTAL SECTION Chemicals The chemicals tetraethylorthosilicate (TEOS), n-cetyltrimethylammonium bromide (CTABr), sodium hydroxide (NaOH), 3-(triethoxysilyl)propyl isocyanate, acenocoumarol and safranin O were purchased from Sigma-Aldrich and used without further purification. 3(azidopropyl)triethoxysilane was provided by SelectLab Chemicals. Sodium ascorbate and copper (II) sulphate pentahydrate were purchased from Scharlab. Human α-thrombin was provided by Haematologic Technologies Inc. Peptide P was supplied by China Peptides. All other reagents were of general laboratory grade and were purchased from Merck unless otherwise stated.


4

Page 5 of 21

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

General Techniques Powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), elemental analysis, transmission electron microscopy (TEM), dynamic light scattering (DLS), infrared spectroscopy (IR) and N2 adsorption-desorption techniques were used to characterize the prepared materials. Fluorescence spectroscopy and HPLC were used to monitor the cargo release from solids to the solution. X-ray measurements were performed on a Bruker AXS D8 Advance diffractometer using Cu-Kα radiation. Thermogravimetric analysis were carried out on a TGA/SDTA 851e Mettler Toledo equipment, using an oxidant atmosphere (Air, 80 mL/min) with a heating program consisting on a heating ramp of 10 °C per minute from 393 K to 1273 K and an isothermal heating step at this temperature during 30 minutes. TEM images were taken with a JEOL TEM-1010 Electron microscope working at 100 kV. Elemental analysis was performed in a CE Instrument EA-1110 CHN Elemental Analyzer. N2 adsorption-desorption isotherms were recorded on a Micromeritics ASAP2010 automated sorption analyzer. The samples were degassed at 120 °C under vacuum overnight. The specific surfaces areas were calculated from the adsorption data in the low pressures range using the BET model. Pore size was determined following the BJH method. DLS measurements were performed in a Zetasizer Nano instrument from Malvern. Fluorescence measurements were carried out in a JASCO FP-8500 Spectrophotometer. HPLC measurements were performed in a Waters 2998 series instrument using a Kroma Phase C18 (5µm, 250 x 4.6 mm) column and a KromaPhase C18 (30 x 2 mm) precolumn, both from Scharlab. The mobile phase was phosphate buffer (A) and acetonitrile (B) working in the gradient mode at a flow rate of 1 ml min-1. The eluent composition varied as follows 0-9 min, 13% B; 9-28 min, 13-35% B; 28-30 min 35-80% B; 30-35min, 80-13% B. The


5

Langmuir

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 21

column temperature was 25 ºC and the injection volume was 100 µL. Acenocoumarol was detected with a UV detector at 310 nm. Synthesis of mesoporous silica nanoparticles (MSNs) MCM-41-type mesoporous silica nanoparticles were synthesized using the procedure described elsewhere. n-Cetyltrimethylammoniumbromide (CTABr, 1.00 g, 2.74 mmol) was first dissolved in 480 mL of deionized water. Then 3.5 mL of an aqueous solution of NaOH 2.00 M was added and the temperature was adjusted to 80ºC. TEOS (5 mL, 2.57. 10-2 mol) was then added dropwise to the surfactant solution and the mixture was stirred for 2 h to give a white precipitate. Finally the solid product was centrifuged, washed with deionized water and dried at 60ºC. To prepare the final porous material, the as-synthesized solid was calcined at 550 ºC using oxidant atmosphere for 5 h in order to remove the template phase. Synthesis of solid S1 To obtain the azide functionalized solid S1, 75 mg of calcined MSNs and 7 mg of acenocoumarol drug were suspended in 1 mL of DMSO and stirred for 24 h at 36 ºC with the aim of achieving maximum loading in the pores of the mesoporous scaffolding. Afterward, the suspension was centrifuged and the solid obtained was washed with 1 mL of acetonitrile and dried under vacuum. In a further step, 50 mg of the obtained solid was suspended in 5.6 mL of acetonitrile and 3-azidopropyltriethoxysilane (0.093 mL, 0.36 mmol) was added. Then, the suspension was stirred for 5.5 h. After that time, the solid was filtered and dried under vacuum to obtain the final solid S1. Synthesis of solid S1-P


6

Page 7 of 21

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

For the preparation of solid S1-P, 30 mg of the azide-functionalized nanoparticles S1 and the peptide P (30 mg) were suspended in a 50:50 mixture of DMF and H2O (30 mL) in the presence of an excess of the acenocoumarol. Then, 13.1 µL of a solution of CuSO4•5H2O 10-2 M and 130 µL of sodium ascorbate 10-3M were added and the reaction mixture was stirred at room temperature for 3 days. Finally, the nanoparticles were centrifuged and washed thoroughly with water to remove the unreacted and adsorbed molecules. The resulting capped nanoparticles S1-P were finally dried under vacuum. Synthesis of S2 The azide-functionalised solid S2 was prepared suspending 400 mg of MSNs and 112.4 mg (0.30 mmol) of safranin O in 40 mL of acetonitrile in a round-bottom flask connected to a DeanStark. The suspension was heated at 110 ºC to remove the adsorbed water. Then, the mixture was stirred during 24 hours at room temperature with the aim of achieving maximum loading in the pores of the MSN scaffolding. Afterward, an excess of 3-azidopropyltriethoxysilane (0.5 mL, 1.9 mmol) was added, and the suspension was stirred for 5.5 h. Finally, the pink solid (S2) was filtered and dried at 70 ºC for 12h. Synthesis of solid S2-P For the preparation of the solid S2-P, 30 mg of the azide-functionalized nanoparticles S2 and the peptide P (30 mg) were suspended in a 50:50 mixture of DMF and H2O (30 mL) in the presence of an excess of safranin O dye (40mg). Then, 13.1 µL of a solution of CuSO4·5H2O 102

M and 130 µL of sodium ascorbate 10-3M were added. The reaction mixture was stirred at 90

ºC for 3 days. Finally, the nanoparticles were centrifuged and washed thoroughly with water to


7

Langmuir

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 21

remove the unreacted and adsorbed molecules. The resulting capped dark pink nanoparticles S2P were finally dried under vacuum. RESULTS AND DISCUSSION As support for the gated system, we selected mesoporous silica nanoparticles (MSN) due to their unique properties, such as large load capacity, biocompatibility, large surface area and wellknown functionalization chemistries. MCM-41-type silica nanoparticles of ca. 100 nm in diameter were prepared following well-known procedures using TEOS as a hydrolytic inorganic precursor and hexadecyltrimethylammonium bromide (CTABr) as a porogen species. The solid was then calcined at 550°C to obtain mesoporous nanoparticles, which were loaded with anticoagulant drug acenocoumarol and externally functionalized with 3(azidopropyl)triethoxysilane to obtain a surface that contained azide groups capable of acting as the eye bolts of the capping peptide. This isolated solid was called S1. Peptide LVPRGSGGLVPRGSGGLVPRGSK-pentinoic acid (P) was also designed to act as a pore-capping agent and a substrate of proteolytic α-human thrombin. Peptide P contains three repeats of thrombin cleavage site LVPRGS, followed by a pentinoic acid that is able to react with the azide moiety on S1. The P attachment to solid S1 was carried out by the copper (I)catalyzed Huisgen azide/alkyne 1,3-dipolar cycloaddition “click” reaction.28 The peptidefunctionalized solid (S1-P) was isolated by centrifugation and washed thoroughly to eliminate both residual acenocoumarol and free peptide. Synthesized materials were fully characterized by powder X-ray diffraction, transmission electron microscopy (TEM), nitrogen adsorption-desorption isotherms, dynamic light scattering, thermogravimetry and elemental analysis. The structure of the nanoparticulated MCM-41


8

Page 9 of 21

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

starting material was confirmed by powder X-ray diffraction (see Figure 1, curves a) and b)). The same was also confirmed by TEM studies (see Figure 1d), in which the spherical morphology and the mesoporous structure of the material are observed. Moreover, N2 adsorption-desorption isotherms registered the typical type IV-curve of the MCM-41-type nanoparticulated materials (see Supporting Information). With the application of the BET and BJH models, a specific surface of 971.5 m2g-1 and a mesopore size of 2.40 nm were respectively calculated. Solid S1 was also characterized by the same techniques. The powder X-ray diffraction pattern of S1 is shown in Figure 1c. As seen, the characteristic diffraction peaks of (100), (110) and (200) were observed. The N2 adsorption-desorption isotherm of S1 (see Supporting Information) also displayed a significantly reduced pore volume (0.36 cm3g-1), and surface area (554.7 m2g-1), when compared with the starting material. Solid S1-P was characterized by TEM, which revealed that the mesoporous structure remained in the final capped nanoparticles. Thermogravimetric and elemental analysis studies were also carried out to quantify organic content. The acenocoumarol, azide groups and P contents in solid S1-P were 0.083, 0.035 and 0.0028 mmol/g SiO2, respectively.


9

Langmuir

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 21

Figure 1. Powder X-ray diffraction patterns of solids a) MCM-41 as synthesized, b) calcined MCM-41 and c) solid S1 containing acenocoumarol and 3-(azidopropyl)triethoxysilane. TEM images of d) solid S1 and e) solid S1-P showing the typical porosity of the MCM-41 mesoporous matrix. In another step, the gating properties of S1-P were investigated. For this purpose, in vitro acenocoumarol delivery studies were carried out in the presence and absence of thrombin. In a typical experiment, 120 µg of S1-P were suspended in 120 µL of water and the suspension was divided into two fractions. Then 500 µL of water and 250 µL of human α-thrombin (1.0 µM, 124 units/mL) were added to the first fraction, whereas the second fraction was treated with 750 µL of water. In both cases, the suspension was stirred for 2 h at 37°C. During that time, the gating behavior of solid S1-P was studied by the measurement of the fluorescence emission at 530 nm of the released acenocoumarol (λex = 370 nm). The release profiles for the described experiment are shown in Figure 2. A remarkable release of acenocoumarol was satisfactorily found in the presence of thrombin, whereas very low delivery was observed in its absence. These results are


10

Page 11 of 21

in accordance with the planned design. In the absence of thrombin, the peptide blocks the release of the entrapped acenocoumarol most likely by the formation of a dense monolayer of peptidic chains which further form a compact network governed by hydrogen bonding interactions on the external surface of MSNs. In contrast, the presence of thrombin induced the hydrolysis of the peptide P resulting in acenocoumarol delivery.

100

Released acenocumarol / %

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

b)

80

60

40

20 a) 0 0

20

40

60

80

100

120

Time / min

Figure 2. Kinetics of the release of acenocoumarol from solid S1-P a) in the absence and b) presence of human α-thrombin. To investigate selectivity in the opening protocol, acenocoumarol delivery from S1-P was also tested with α-amylase and denaturalized human thrombin following the same experimental procedure described above. In both cases, no significant release was observed, which indicates the importance of the selective hydrolysis of capping peptide P in the presence of thrombin for acenocoumarol release (data not shown). In order to further study the role played by thrombin in the opening mechanism, a safranin O-loaded solid capped with peptide P was prepared (S2-P) following the same procedure to that used to prepare S1-P. Using S2-P, dye delivery, as a


11

Langmuir

function of the thrombin concentration in water, was studied by following the fluorescence emission of the released dye. The obtained results are shown in Figure 3. As seen, the cargo release was proportional to the thrombin concentration. It can be estimated from these studies that the gated solids started to release their content when the thrombin concentration was higher than ca. 100 nM (12 units/mL).

100

80

Released Dye / %

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 21

60

40

20

0 0.00

0.05

0.10

0.15

0.20

0.25

0.30

α-thrombin concentration / µM

Figure 3. Percentage of the released concentration of safranin O as function of the thrombin concentration. α-Thrombin concentration in blood is highly variable from person to person, but typical concentrations when there is no coagulation are around 1 nM, while during coagulation values are greater than 500 nM.29 Looking to our previous experiments and keeping this information in mind, we examined the ability of the designed smart nanodevice to intervene in a real biological process, such as blood coagulation cascade. For this purpose, we designed a battery of anticoagulation assays in rabbit blood plasma. In these experiments, the thrombin time was recorded as the time required for initial clot formation.30 This time was studied in the absence of


12

Page 13 of 21

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

solid and in the presence of 1 mg of calcined MSN, solid S1-P, S2-P and acenocoumarol at the same concentration released from 1 mg of solid S1-P. For each experiment, coagulation was activated by the addition of 8 units of human α-thrombin to 100 µL of plasma (final thrombin concentration 40 units/mL, 0.333 µM). Table 1 summarizes the obtained results. Table 1. Thrombin time with no solid and in the presence of calcined MSN, solid S1-P, S2-P and free acenocoumarol. Solid

Thrombin Time (min)

Without solid

2.6 ± 0.3

Calcined MSN

2.6 ± 0.3

S1-P

5.0 ± 0.3

S2-P

2.6 ± 0.3

Acenocoumarol

5.5 ± 0.3

The coagulation time obtained in rabbit blood plasma in the presence of thrombin without nanoparticles was 2.6 ± 0.3 min (5 replicas). This value was also obtained when calcined MSN or S2-P was added, which confirms that neither silica mesoporous nanoparticles nor solid S2-P containing gating peptide P and loaded with a dye actually affected the clotting response. On the contrary, when solid S1-P was present in plasma in the presence of thrombin, the coagulation time was delayed to 5.0 ± 0.3 min. Similar results were obtained in the presence of free acenocoumarol. These results agree with the thrombin-induced hydrolysis of peptide P in S1-P which resulted in the anticoagulant release and the delay in coagulation. As a control, the effect of the presence of S2-P nanoparticles in blood plasma, but in the absence of thrombin, was also


13

Langmuir

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 21

investigated. In this case, S2-P alone induced no coagulation effect. Finally, the amount of acenocoumarol released from S1-P to plasma in the absence and presence of thrombin was studied by HPLC. In a typical assay, both samples were centrifuged after 5 min and the supernatant was collected. Presence of acenocoumarol was then analyzed by HPLC using a RPC18 column and a UV-detector (see Supporting Information for details). The sample containing S1-P, but without thrombin, showed negligible acenocoumarol delivery, whereas a clear peak for acenocoumarol was detected by HPLC in the presence of thrombin. This finding confirms that acenocoumarol delivery is strictly dependent on the presence of thrombin. CONCLUSIONS In summary, after taking advantage of the high proteolytic specificity of thrombin, we designed capped nanoparticles (i.e., S1-P) which displayed a highly specific thrombin response. Acenocoumarol drug was released from S1-P when thrombin was present, whereas negligible delivery was observed in the absence of this serine protease. Acenocoumarol delivery from S1-P was thrombin concentration-dependent and a clear delivery was detected for concentrations of thrombin over ca. 100 nM. Thrombin was also able to selectively induce acenocoumarol delivery from S1-P in a complex medium such as plasma. In particular, S1-P nanoparticles were able to interact with the coagulation cascade via the thrombin-dependent delivery of acenocoumarol. When thrombin was present, S1-P was able to delay the coagulation time in plasma from 2.6 ± 0.3 to 5.0 ± 0.3 min. This work broadens the application of gated silica nanoparticles by demonstrating their ability to respond in a complex biological pathway such as hemostasis. S1-P nanoparticles remained latent, yet were able to regulate clotting when thrombin presence was activated. Other sophisticated functionalities based on similar concepts using smart capped nanoparticles are envisioned.


14

Page 15 of 21

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

ASSOCIATED CONTENT Supporting Information. Details on characterization of materials. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Ramón Martínez-Máñez ([email protected]) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT Authors thank Spanish Government (Projects MAT2012-38429-C04, AGL2012–39597–C02– 02) and Generalitat Valenciana (Project PROMETEOII/2014/047) for support. R.B. is thankful to Svagata.Eu (Erasmus Mundus Action-II program) for his fellowship. A.R. thanks UPV for her predoctoral fellowship. N.M. thanks Spanish MINECO for her FPI fellowship. The authors also thank the Electron Microscopy Service at the UPV for support. REFERENCES (1)

Coughlin, S. R. Thrombin signalling and protease-activated receptors. Nature 2000, 407,

258-264. (2)

Church, F. C.; Pratt, C. W.; Noyes, C. M.; Kalayanamit, T.; Sherrill, G. B.; Tobin, R. B.;

Meade, J. B. Structural and functional-properties of human alpha-thrombin,


15

Langmuir

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 21

phosphopyridoxylated alpha-thrombin, and gamma-t-thrombin - identification of lysyl residues in alpha-thrombin that are critical for heparin and fibrinogen interactions. J. Biol. Chem. 1989, 264, 18419-18425. (3)

Geddings, J. E.; Mackman, N. New players in haemostasis and thrombosis. Thromb.

Haemostasis 2014, 111, 565–779. (4)

Maitz, M. F.; Freudenberg, U.; Tsurkan, M. V.; Fischer, M.; Beyrich, T.; Werner, C. Bio-

responsive polymer hydrogels homeostatically regulate blood coagulation. Nature Commun. 2013, 4, 2168. (5)

Greineder, C. F.; Howard, M. D.; Carnemolla, R.; Cines, D. B.; Muzykantov, V. R.

Advanced drug delivery systems for antithrombotic agents. Blood, 2013, 122, 1565 – 1575. (6)

Modery-Pawlowski, C. L.; Kuo, H. H.; Baldwin, W. M.; Sen Gupta, A. A platelet-

inspired paradigm for nanomedicine targeted to multiple diseases. Nanomedicine, 2013, 8, 17091727. (7)

Argyo, C.; Cauda, V.; Engelke, H.; Radler, J.; Bein, G.; Bein, T. Heparin-Coated

Colloidal Mesoporous Silica Nanoparticles Efficiently Bind to Antithrombin as an Anticoagulant Drug-Delivery System. Chem. Eur. J. 2012, 18, 428-432. (8)

Wu, Q.; Liu, C. Q.; Fan, L. N.; Shi, J. H.; Liu, Z. Q.; Li, R. F.; Sun, L. W. Heparinized

magnetic mesoporous silica nanoparticles as multifunctional growth factor delivery carriers. Nanotechnology, 2012, 23, 485703.


16

Page 17 of 21

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(9)

Aznar, E.; Oroval, M.; Pascual, LL.; Murguía, J.R.; Martínez-Máñez, R.; Sancenón, F.

Gated Materials for On-Command Release of Guest Molecules. Chem. Rev. 2016, DOI: 10.1021/acs.chemrev.5b00456. (10)

Li, Z. X.; Barnes, J. C.; Bosoy, A.; Stoddart, J. F.; Zink, J. I. Mesoporous silica

nanoparticles in biomedical applications. Chem. Soc. Rev. 2012, 41, 2590-2605. (11)

Knežević, N. Z.; Ruiz-Hernandez, E.; Hennink, W. E.; Vallet-Regi, M. Magnetic

mesoporous silica-based core/shell nanoparticles for biomedical applications. RSC Adv., 2013, 3, 9584-9593. (12)

Wu, S. H.; Mou, C. Y.; Lin, H. P. Synthesis of mesoporous silica nanoparticles. Chem.

Soc. Rev., 2013, 42, 3862-3875. (13)

Mal, N. K.; Fujiwara, M.; Tanaka, Y. Photocontrolled reversible release of guest

molecules from coumarin-modified mesoporous silica. Nature, 2003, 421, 350-353. (14)

Vivero-Escoto, J. L.; Slowing, II; Wu, C. W.; Lin, V. S. Y. Photoinduced Intracellular

Controlled Release Drug Delivery in Human Cells by Gold-Capped Mesoporous Silica Nanosphere. J. Am. Chem. Soc. 2009, 131, 3462-3463. (15)

He, D. G.; He, X. X.; Wang, K. M.; Cao, J.; Zhao, Y. X. A Photon-Fueled Gate-Like

Delivery System Using i-Motif DNA Functionalized Mesoporous Silica Nanoparticles. Adv. Funct. Mater. 2012, 22, 4704-4710.


17

Langmuir

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(16)

Page 18 of 21

Tarn, D.; Ferris, D. P.; Barnes, J. C.; Ambrogio, M. W.; Stoddart, J. F.; Zink, J. I. A

reversible light-operated nanovalve on mesoporous silica nanoparticles. Nanoscale 2014, 6, 3335-3343. (17)

Lai, C. Y.; Trewyn, B. G.; Jeftinija, D. M.; Jeftinija, K.; Xu, S.; Jeftinija, S.; Lin, V. S. Y.

A mesoporous silica nanosphere-based carrier system with chemically removable CdS nanoparticle caps for stimuli-responsive controlled release of neurotransmitters and drug molecules. J. Am. Chem. Soc. 2003, 125, 4451-4459. (18)

Park, C.; Oh, K.; Lee, S. C.; Kim, C. Controlled release of guest molecules from

mesoporous silica particles based on a pH-responsive polypseudorotaxane motif. Angew. Chem. Int. Ed. 2007, 46, 1455-1457. (19)

Diez, P.; Sanchez, A.; Gamella, M.; Martinez-Ruiz, P.; Aznar, E.; de la Torre, C.;

Murguia, J. R.; Martínez-Máñez, R.; Villalonga, R.; Pingarron, J. M. Toward the Design of Smart Delivery Systems Controlled by Integrated Enzyme-Based Biocomputing Ensembles. J. Am. Chem. Soc. 2014, 136, 9116-9123. (20)

Zheng, Z. L.; Huang, X.; Shchukin, D. A cost-effective pH-sensitive release system for

water source pH detection. Chem. Commun. 2014, 50, 13936–13939. (21)

Sancenón, F.; Pascual, LL.; Oroval, M.; Aznar, E.; Martínez-Máñez, R. ChemistryOpen

2015, 4, 418–437. (22)

Schlossbauer, A.; Warncke, S.; Gramlich, P. M. E.; Kecht, J.; Manetto, A.; Carell, T.;

Bein, T. A Programmable DNA-Based Molecular Valve for Colloidal Mesoporous Silica. Chem. Int. Ed. 2010, 49, 4734 – 4737.


18

Page 19 of 21

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(23)

Popat, A.; Jambhrunkar, S.; Zhang, J.; Yang, J.; Zhang, H. W.; Meka, A.; Yu, C. Z.

Programmable drug release using bioresponsive mesoporous silica nanoparticles for site-specific oral drug delivery. Chem. Commun. 2014, 50, 5547-5550. (24)

Zhou, S. W.; Du, X. Z.; Cui, F. B.; Zhang, X. F. Multi- Responsive and Logic Controlled

Release of DNAGated Mesoporous Silica Vehicles Functionalized with Intercalators for Multiple Delivery. Small 2014, 10, 980-988. (25)

Luo, Z.; Ding, X. W.; Hu, Y.; Wu, S. J.; Xiang, Y.; Zeng, Y. F.; Zhang, B. L.; Yan, H.;

Zhang, H. C.; Zhu, L. L.; Liu, J. J.; Li, J. H.; Cai, K. Y.; Zhao, Y. L. Engineering a Hollow Nanocontainer Platform with Multifunctional Molecular Machines for Tumor-Targeted Therapy in Vitro and in Vivo. ACS Nano 2013, 7, 10271-10284. (26)

Zhang, Q.; Neoh, K. G.; Xu, L. Q.; Lu, S. J.; Kang, E. T.; Mahendran, R.; Chiong, E.

Functionalized Mesoporous Silica Nanoparticles with Mucoadhesive and Sustained Drug Release Properties for Potential Bladder Cancer Therapy. Langmuir 2014, 30, 6151. (27)

de la Torre, C.; Casanova, I.; Acosta, G.; Coll, C.; Moreno, M. J.; Albericio, F.; Aznar,

E.; Mangues, R.; Royo, M.; Sancenón, F.; Martínez-Máñez, R. Gated Mesoporous Silica Nanoparticles Using a Double-Role Circular Peptide for the Controlled and Target-Preferential Release of Doxorubicin in CXCR4-Expresing Lymphoma Cells. Adv. Funct. Mater. 2015, 25, 687-695. (28)

Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A stepwise Huisgen

cycloaddition process: Copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angew. Chem. Int. Ed. 2002, 41, 2596 – 2599.


19

Langmuir

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(29)

Page 20 of 21

Wolberg, A.S.; Campbell. Thrombin Generation, Fibrin Clot Formation and Hemostasis.

Transfus Apher Sci. 2008, 38, 15–23. (30)

Penner, J. A. Experience with a thrombin clotting time assay for measuring heparin

activity. Am. J. Clin. Pathol. 1974, 61, 645-653.


20

Page 21 of 21

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 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Insert Table of Contents Graphic and Synopsis Here

Thrombin-responsive gated silica nanoparticles as biological clotting pathway controllers have been prepared. The system consists of silica mesoporous nanoparticles loaded with acenocoumarol and capped with a peptide containing a thrombin specific cleavage site. Nanoparticles remained latent, yet were able to control clotting when thrombin presence was activated.


21

Thrombin-Responsive Gated Silica Mesoporous Nanoparticles As

Recommend Documents