Biogel
A biogel and blood coagulation factor technology, which can be used in drug combination, drug delivery, extracellular fluid diseases, etc., can solve the problem of toxicity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0121]Example 1: Formation of biogels or tissue adhesives using fibrinogen, and fibrinogen binding peptide immobilized on a carrier comprising human serum albumin.
[0122] Human serum albumin (HSA) was reacted with a 40-fold molar excess of the linker succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) at pH 7.4. The modified protein (HAS-SMCC) was purified and an average of one mole of HAS was determined to be modified by 18 moles of SMCC. The GPRPGGGGGGC (B10; SEQ ID NO: 24) or LVPRGPRPGGGGGGC (TC-15; SEQ ID NO: 25) peptide was added to HSA-SMCC in an amount exceeding 1.5 molar relative to the maleimide moiety. The peptide-modified HSA protein was then purified from the unreacted peptide and stored at -80°C at 7 mg / ml. Lyophilized human fibrinogen was dissolved in water at 16 mg / ml as described by the supplier (National Blood Transport Service Scotland).
[0123] Peptide-modified HSA protein (5 μl) was added to 0.3 ml of fibrinogen solution and gel was obse...
Embodiment 2-5
[0124] Example 2-5 Methods for synthesizing peptide-modified HSA
[0125] 10 mg / ml of human serum albumin (HSA) was combined with a 5, 10, 20 or 40-fold molar excess of the linker thiosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1 - Carboxylate (thio-SMCC) was reacted at pH 7.4 with a total reaction volume of 0.7 ml. by Zebra TM The x-modified protein (HSA-SMCC) was purified by desalting columns (Pierce, Rockford, IL) and an average of one mole of HSA was determined to be modified with 5, 8, 16 or 18 moles of thio-SMCC, respectively. To determine the number of maleimide groups bound per HSA molecule, 3 nanomolar HSA was incubated with a known amount of cysteine (100 nanomolar) in buffer for 30 minutes at room temperature. The remaining cysteine was then reacted with 1 mmol of 5,5-dithiobis(2-nitrobenzoate) (DTNB) for 20 minutes and measured at A 412 absorbance. Maleimide levels were calculated by comparing the absorbance of the control and protein samples. GPRPGGGGGG...
Embodiment 3
[0131] Example 3 Influence of peptide-modified HSA amount
[0132] To determine the effect of the amount of peptide-modified HSA and fibrinogen on the rheological properties of the mixture, shear strength measurements were performed with 25 or 50 μl of peptide-modified HSA with 32 and 64 mg / ml of fibrinogen. Each mole of HSA was modified with 16 moles of binding peptide, and HSA modified with this peptide was used in this assay. To ensure that the kinetic oscillation test was done within the linear viscoelastic strain limit of the gel, a strain sweep was performed. figure 2 Strain scans at 20°C and 37°C are shown for 25 μl of peptide-modified HSA mixed with 32 mg / ml of fibrinogen. Both storage modulus (G') and loss modulus (G") were shown to be stable within the stated range, and a constant strain of 1% was chosen over this linear viscoelastic strain range. Frequency sweeps demonstrated that G' values were greater than G ", which is an indicator of a cross-linked network ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More