Plasminogen activator entrapped within injectable alumina: A novel approach to thrombolysis treatment

Vasiliy V. Vinogradov,aAlexander V.Vinogradov,a Vladimir E. Sobolev,aIvan P. Dudanov,b,c Vladimir V. Vinogradov*a

aITMO University, St. Petersburg, 197101, Russian Federation

bPetrozavodsk State University, Petrozavodsk,185910, Russian Federation

cRegional Vascular Center, St. Petersburg, Russian Federation

E-mail:

Electronic Supplementary Information

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1. Materials and methods

Chemicals: Aluminum isopropoxide, plasminogen from bovine plasma (PG, cat. No. P9156), fibrinogen from bovine plasma (FG, cat. No. F8630), thrombin from bovine plasma (TB, cat. No. T7513), tissue plasminogen activator (TPA, cat. No. T5451) were all obtained from Sigma-Aldrich. Glycine buffer (pH = 7.4) was prepared from glycine solutions (0.05 M; from Sigma-Aldrich) with desired volumes of 1.0 M NaOH.

Sol-gel synthesis of alumina: Alumina sol was prepared as described in Ref. [1]. In detail, 2.2 g of Al(C3H7O)3 was added to 50 mL of deionized water at 90°C and a white precipitate was formed immediately. Before US treatment the precipitate was kept at 90°C under vigorous stirring for 15 min to complete the production of boehmite nanoparticles and to complete the evaporation of the isopropanol formed during hydrolysis. The final suspension was ultrasonically treated for 2 h. In 2 h a viscous sol was formed. The resulting sol was cooled to room temperature. The dried matrix had a surface area of 153 m2/g, pore volume of 0.097 cm3/g and an average pore size of ~ 2.5 nm.

Entrapment procedure of the TPA within alumina: For the entrapment of TPA, a mixture of 50 µl glycine-NaOH buffer solution (pH 7.4) and 150 µl freshly prepared alumina sol was transferred to a cuvette and then 20 µL of TPA (500 U/ml) was added. Ten minutes later the sol was left in a vacuum desiccator at room temperature for 24 h. The TPA@alumina was rinsed inside the final polystyrene cuvette with 1.0 ml of glycine solution (pH 7.4) to assure removal of any adsorbed protein. The entrapment of the enzyme was complete, as indicated by the lack of activity in the supernatant solutions and washings (see below).

Enzymatic activity:

After rinsing with 1.0 ml glycine solution (pH 7.4), the bioactive hybrid was left for incubation at 37°C for 30 min. Then, the rinsing solution was replaced with 1.0 ml5 mg/ml bovine fibrinogen solution and 0.5 ml bovine plasminogen solution (1.2 U/ml) with subsequent addition of 0.5 ml solution of thrombin (50 U/ml) in glycine buffer (pH 7.4), and the enzymatic activity was measured by following the formation of a clot spectroscopically through the absorption at 340 nm, at a temperature of 37°C. The representative curve of clot formation and lysis (CloFAL), demonstratingprincipal CloFAL parameters is shown in Fig.1. The rising part of the curve corresponds to the formation of a fibrin clot, and the descending one to its lysis. The rinsing solution was also tested for enzymatic activity by the transfer of 500 µl of the solutions into cuvettes and adding clot-forming solution. To compensate for the slower reactivity of the entrapped enzymes, 10x lower concentrations of the free enzymes were taken. The kinetic curves were interpreted according to [2] with an activity of free TPA as a standard.

Figure 1S. Curve of Clot Formation and Lysis (CloFAL) assay, demonstrating principal CloFAL parameters. Buffered thrombin solution is added to solution of TPA, fibrinogen and plasminogen. Clot formation and lysis are monitored as continuous changes in absorbance. Measurements include maximum amplitude (MA), time to maximum absorbance (T1) and completion of the first phase of decline in absorbance (T2), and area under the curve (AUC), from which a coagulation index (CI) and various fibrinolytic indices (FI) can be calculated.

The curvesare generated overthe course of the assay reactions, showing aninitial baseline absorbance (Fig.1S), followed by a progressive rise in absorbance to a point of maximum absorbance (MA) (achieved at T1), then an earlyphase of decline in absorbance (ending at T2, thetime point at which the slope of decline inabsorbance changes by +10 mOD/min), andcompleted by a late phase of decline in absorbance to baseline.MA, T1 and T2 are directly obtained from theabsorbance data. Using the area under the curve(AUC) over the course of the initial period of theassay until T2, a coagulation index (CI) is then calculatedthat relates the AUC of the sample to that ofthe reference (activity of free TPA was taken as a reference),as follows:

Various fibrinolytic indices (FI) were also measured.FI can be calculated byrelating the ratio of the time to completion of thefirst phase of decline in absorbance (T2) to thetime to maximum absorbance (T1) for the sampleas compared to the reference, with a correctionfactor for differences in maximum absorbanceas follows:

In the present work, the clot formation and lysis (CloFAL) assay curves of entrapped TPA were analyzed for MA, T1, T2, CI,and FI using the aforementioned calculation.

Stability of TPA@alumina composite in the model blood system:

To test the stability of synthesized composite in a real blood system, tenfold Ringer's solution was used. Ringer’s solution has a similar ratio of salt concentrations to that typically found in the blood. For this aim, a plastic cuvette with TPA@alumina was run with Ringer’s solution and kept under stirring for 30 days. The activity of TPA@alumina after the test was compared with the initial activity. Only 2,5% decrease in activity was observed.

Characterization techniques:

Specific surface areas, pore volumes and pore size distributions were determined using the nitrogen adsorption-desorption method at 77 K (Quantachrome Nova 1200 series e). Surface areas were calculated using the BET equation. Pore volumes and pore size distributions were calculated using the BJH method.Micropore size distribution was calculated using the Dubinin–Astakhov method. Prior to analysis the sample was degassed for 24 hours at room temperature. The crystal phase and crystallinity of the samples were studied by X-ray diffraction method (Bruker D8 Advance) using Cu-Kα irradiation (λ = 1,54 Å), samples being scanned along 2θ in the range of 4–75° at a speed of 0.5 degrees per minute. The spectral analysis of enzymatic activity was carried out using PG Instruments T80. For scanning electron microscopy (SEM, ultrahigh resolution Magellan 400L electron microscope), the final suspension of the entrapped enzyme was coated on silicon wafer and fully dried under vacuum. The samples for transmission electron microscopy (TEM) were obtained by dispersing a small probe in ethanol to form a homogeneous suspension. Then, a suspension drop was coated on a copper mesh covered with carbon for TEM analysis (FEI TECNAI G2 F20, at an operating voltage of 200 kV). DSC curves were obtained with 204 F1 Phoenix NETZSCH apparatus and a heating rate of 10 °C min–1 was used from 30 °C to 150 °C under nitrogen.

2. SEM images of TPA@alumina

Figure 2S.SEM images of TPA@alumina. Presence of pores more 4 nm is shown here.

3. Thrombolytic activity

Figure 3S. The rate of fibrinolysis of free and rinsing solutions (a) compared to entrapped TPA (b). 1- afterand 2- before the test in tenfold Ringer’s solution.

References for ESI:

1. A. Rutenberg, V. Vinogradov and D. Avnir, Chem. Commun.,2013, 49, 5636.

2. N.A. Goldenberg,W. E. Hathaway,L. Jacobson, M. J. Manco-Johnson, Thrombosis Research, 2005, 116, 345.