SYNTHESIS OF PAPAIN NANOPARTICLES USING E- BEAM AND GAMMA IRRADIATION: A RADIATION CHEMISTRY APPROACH Gustavo H. C. Varca 2014 Nuclear and Energy Research Institute IPEN- CNEN/SP BRAZIL
Protein- Based Nanoparticles Overview
Protein in Pharmaceutics ü Drug delivery systems ü Tissue engineering (scaffolds) ü Therapeutical peptides ü Protein pharmaceutics
Protein Based Drug Carrier Protein NPs cell recognition v Specific receptors in the cell membrane Site specific delivery v Specific orientation to interact with receptor Similar conformation v Nanoparticle- bound protein/free protein competition for the receptor Affinity Modifications Lynch et al., Nature Nanotechnology 4, 546-547 (2009)
Radiation Synthesized Protein- Based Nanoparticles Technique Overview
Particle Synthesis Technique Overview Step 1 Step 2 Step 3 - Concentration - ph - Cosolvents [ ] - Temperature - Dosis - Irradiation temp. Varca et al. Radiat. Phys. Chem. (2014a); BR Patent 1020130050342 01/03/2013
Controlled Nanostructure Drug Carrier Covalent Attachment More stable structure Caused Structural rearrangement Biological properties Drug carrier - potential - Bioactive - Controllable size distr. - No use of monomers Varca et al. Radiat. Phys. Chem. 2014(a); BR Patent 1020130050342 01/03/2013
Radiation Synthesized Protein- Based Nanoparticles The case of Papain
Papain Figure 1. Papain structure (frontal view). Structural properties - 212 amino acids (2 domains) - Active site - Asn, Cis and His Ø Cysteine protease Broad specificity Carica Papaya L. (EC 3.4.22.2) Thiol protease Ø Applications Biotechnology esterase amidase Industrial level endopeptidase Biomedical (Rosenberg, et al., 2004; Kamphuis et al., 1984)
Particle Size Increment γ irradiation Size (d.nm) 14 12.5mg.ml-1 20mg.ml-1 50mg.ml-1 12 10 8 6 4 0% 10% 20% 30% 35% Ethanol% Figure 1. Scale up experiments involving different papain (12.5, 20 and 50 mg.ml -1 ) and ethanol (0-35% v/v) concentrations irradiated at 10 kgy. Size (d.nm) 10,5 20% EtOH 10,0 0% EtOH 9,5 9,0 8,5 8,0 7,5 7,0 6,5 6,0 5,5 5,0 4,5 0,0 2,5 5,0 7,5 10,0 Radiation dose (kgy) Figure 2. Papain particle size increment as a function of irradiation dose in presence (20% v/v) and absence of ethanol. Controlled Particle Size increase Varca et al. Radiat. Phys. Chem. (2014B);
Effect of γ- irradiation Non irradiated samples Agglomeration Irradiated Samples Agglomeration Size increase Van der Waals/Electrostatic forces/ dipole- dipole Size increase Crosslinking (intermolecular) Covalent attachment 0% EtOH 30% EtOH 40% EtOH Dilution 30% EtOH 40% EtOH
Mechanism of NP Formation Ø Direct effects - Minimum Indirect effects Irradiation in presence of L- Cysteine No particle formation Oxidizing radical species 0% EtOH 30% EtOH
Mechanism Approach - Bityrosine A B Figure 3. Bityrosine monitoring in papain (12.5 mg.ml -1 ) non-irradiated (a) and irradiated (b) at 10kGy under different ethanol concentrations (0-35%). The samples were properly diluted in buffer to reach equivalent absorbance. The readings were performed at λ=280nm using a Cary 1E Uv-Vis Varian spectrophotometer. The samples were then checked for bityrosine emission on a F4500 Hitachi Fluorescence spectrophotometer using λex=325nm λem=340-500nm, scan speed of 240nm/min, Exslit of 5nm and Emslit of 10nm. Controlled Bityrosine Crosslinking Induced by Radiation Varca et al. Radiat. Phys. Chem. (2014B);
E- Beam - Radiation Chemistry Overview Absorbance units (a.u.) 0,010 0,009 0,008 0,007 0,006 0,005 0,004 0,003 0,002 300 350 400 450 500 Wavelenght (nm) Figure 4. UV-Vis absorption spectra of OH -papain adduct determined by pulse radiolysis. N 2 O saturated aqueous papain solution (4.2 10-5 mol dm -3 ) irradiated with 7 ns electron pulses. Dose per pulse 20 Gy. UV-Vis absorbance recorded at wavelength ranging from λ = 290 to 500 nm. k' OH [1/s] 7E6 6E6 5E6 4E6 3E6 2E6 1,5E-5 2,0E-5 2,5E-5 3,0E-5 3,5E-5 4,0E-5 4,5E-5 Papain concentration [mol dm -3 ] Figure 5. Determination of papain and OH radical reaction rate constant. Pulse radiolysis of papain (concentrations ranging from 1.9 10-5 to 4.2 10-5 mol dm -3 ) in N 2 O saturated aqueous solution. Pulse duration 7 ns, dose per pulse 20Gy. UV-Vis absorbance recorded at 330 nm. OH 1.3 10 11 (dm - 3 mol - 1 s - 1 )
E- Beam - Radiation Chemistry Overview 4,5E6 k'e [1/s] 4,0E6 e - 1.44 10 6 (dm - 3 mol - 1 s - 1 ) 3,5E6 1,0E-5 1,5E-5 2,0E-5 2,5E-5 3,0E-5 3,5E-5 4,0E-5 4,5E-5 Papain Concentration (mol dm -3 ) Figure 6. UV-Vis absorption spectra of OH -papain adduct determined by pulse radiolysis. N 2 O saturated aqueous papain solution (4.2 10-5 mol dm -3 ) irradiated with 7 ns electron pulses. Dose per pulse 20 Gy. UV-Vis absorbance recorded at wavelength ranging from λ = 290 to 500 nm.
E- Beam - Radiation Chemistry Overview 7,0E-4 6,0E-4 t 1/2 5,0E-4 4,0E-4 3,0E-4 2,0E-4 This indicates that even for 10% (w/w) ethanol solution OH radicals are not scavenged by alcohol and are still able to interact with papain 1,0E-4 0 1E-5 2E-5 3E-5 4E-5 5E-5 Papain Concentration (mol dm -3 ) Figure 7. Competitive kinetics of papain and ethanol with hydroxyl radicals. Pulse radiolysis of papain (concentrations ranging from 0 up to 4.5 10-5 mol dm -3 ) in N 2 O saturated aqueous solution with addition of ethanol 10% (w/w). Pulse duration 7 ns, dose per pulse 20Gy. UV-Vis absorbance recorded at 330 nm.
Conclusions Ø The synthesis of protein NP s with controllable size distribution was achieved by gamma and electron; Ø The OH. is more likely to be involved in the process of NP Formation / Bityrosine formation; Ø There are still papain molecules interacting with OH. At higher ethanol concentrations;
Acknowledgments ü Piotr Ulansky (IMTR- Lodz, PL) ü Janus Rosiak (IMTR- Lodz, PL) ü Slawomir Kadlubousky (IMTR- Lodz, PL) ü Mariano Grasselli (UNQ Buenos Aires, AR) ü Ademar B. Lugão (IPEN- CNEN/SP, Brazill) Process number 2010/10935-9
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