Polymeric Nanoconstructs as Polymeric Nanoconstructs as Oxygen CarriersOxygen Carriers
Anna Maria Piras, Federica Chiellini, Alberto Dessy
BIOlab UdR INSTM, Department of Chemistry and Industrial Chemistry, University of Pisa, Italy
VI Convegno Nazionale sulla Scienza e Tecnologia dei Materiali
12-15 June 2007, Perugia, Italy
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Oxygen CarriersOxygen Carriers
Free Hb: not feasibleFree Hb: not feasibleShort circulation timesAbnormally high O2 affinityBreakdown into dimers
Renal Toxicity Haemoglobinuria Malaise Abdominal pain
Acellular Hb-based OAcellular Hb-based O22 Carriers CarriersCross-linked HbPolymerised HbPolymer Conjugated Hb
Low O2 affinity Plasma expanders Hypertension
Cellular Hb-based OCellular Hb-based O22 Carriers Carriers
Encapsulated HbCo-encapsulation of enzymes and reductants
Enhancement of Hb half-lifeNo osmotic effect
To be administered for the immediate re-establishment of O2
homeostasis in case of urgent need No need of cross-matching Prolonged storage conditions
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Aim of the WorkAim of the Work
Development of Polymeric Bioerodible Nanoparticles as Hemoglobin Based Oxygen Carriers
PEG moieties
Hemoglobin
Bioerodible Polymer
Cofactors
Oxygen Release
Particle Erosion
Removal from Blood Circulation
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
MaterialsMaterials
Stabilizer GiG-CD:1–O–glycidyl–2,3–O–isopropylidenglyceryl--cyclodextrin
PolymersVAM41-PEG:2-methoxyethanol hemiesters of poly(maleic anhydride-co-butylvinylether)5% PEG2000
Active AgentHb: Human Hemoglobin
OH
O
O
OH
O
HO
O OH
O
HO
HO
O
HO
OH
OH
OHO
HO
OH
OOH
HO
OH
OO
OH
O
HO
OH
O
OH
OHO
HO
O
GLYx-HPMAy: poly(methacryloylglycylglycine-OHx
-co-hydroxypropylmethacrylamidey)
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Bioerodible Polymers: VAM41Bioerodible Polymers: VAM41
C6H6AIBN60 °C
THF75°C
DCMDCM
DMAPDMAP
Polymer Mw(kDa)
VAM41-1 320
VAM41-2 325
VAM41-3 315
VAM41-A 223
VAM41-B 150
VAM41-C 130
VAM41-D 105
VAM41-E 97
VAM41-F 40
VAM41-G 38
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
VAM41-5%PEGVAM41-5%PEG20002000
Purified by ion exchange chromatography
Process yield: 92%
5% PEG grafting5% PEG grafting
To achieve stealth effect on nanoparticles
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
The influence of nanoparticle components on Hb-Oxygen interaction was evaluated through UV-VIS spectroscopy under oxygenated and deoxygenated conditions. Mixtures of nanoparticle components and Hb were tested.
Materials Compatibility with HbMaterials Compatibility with Hb
No major difference on Hb capability of reversibly bind oxygen
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Nanoparticles PreparationNanoparticles Preparation
Co-precipitation TechniqueCo-precipitation TechniqueUnder controlled atmosphere and Temperature
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Particle Characterisation MethodsParticle Characterisation Methods Granulometry in Suspension: Dynamic Light Scattering Scanning Electron Microscopy (SEM) Surface Features: Zeta Potential
Protein Detection: Drabkin’s Assay─ Encapsulation Efficiency (EE%)
─ Loading (%)
Particles Erosion Rate: Dialysis Diffusion technique Citotoxicity: LDH Assay on Mouse Embryo Fibroblasts
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Formulation Set - UpFormulation Set - Up
Run Polymer Diameter Distribution (nm ± SD) V% N%
3T-2 VAM41-C 814 ± 803 84 ± 46 5T-2 VAM41-C 398 ± 667 61 ± 17 11T-2 VAM41-C 990 ± 831 87 ± 56 12T-2 VAM41-C 2740 ± 5100 88 ± 51 V3T-2 PLVAM41-5%PEG 131 ± 18 124 ± 17 V5T-2 PLVAM41-5%PEG 136 ± 42 114 ± 24 V11T-2 PLVAM41-5%PEG 149 ± 23 139 ± 21 V13T-2 PLVAM41-5%PEG 70 ± 42 55 ± 11
Formulation 12T-2 Formulation 5T-2 Formulation V11T-2
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Nanoparticles: Controlled ConditionsNanoparticles: Controlled ConditionsHomogeneous colloidal suspensions
SEM Micrograph
Drabkin’s reagent
Hb loading: (57 ± 1.7)% w/w
Hb Encapsulation Efficiency: 88%
Diameter (139 ± 21)nm
Zeta Potential (-5.38 ± 0.22) mV
Light Scattering
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Erosion KineticsErosion Kinetics
Nanoparticles displayed slow and progressive bioerosion that reached the 77% of complete dissolution after 55 hours.
Diffusion through dialysis membrane (EC, MWCO:100 kDa)
Hb loaded nanoparticles
Conditions: PBS pH 7.4, RT.
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Citotoxicity EvaluationCitotoxicity Evaluation
Cell line: Balb/3T3 Clone A31 balb/c murine embryo fibroblastLDH assay: lactate dehydrogenase (LDH) release assay
Hb loaded Nanoparticles suspensions show Hb loaded Nanoparticles suspensions show Reduced Cell Mortality in comparison to free HbReduced Cell Mortality in comparison to free Hb
Hb displays a high cytotoxicity in vitro due to its active role in oxidative reactions and for strong interactions with cellular membranes.
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Anti-oxidantsAnti-oxidantsThe encapsulated Hb displays a predominant fraction of metHb
No major benefit on Hb preservation was evidenced
Citric Acid Ascorbic Acid/Leucomethylene
Antioxidant agents were employed in nanoparticles preparation
Good nanoparticles morphology and diameter distribution
Leuco/Hb=75(103 93)nm
2mM Citric Acid(80 51)nm
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
New StrategiesNew Strategies
Alternative Materials
GLY25-HPMA75
VAM41-PEG modified with antioxidant moieties, reduced number of free COOH
M. Gizdavic-Nikolaidis et al. Curr. Appl. Phys 4:347-350 (2004)
M. Li et al., Biomaterials 27:2705-2715 (2006)
Modulators of Hemoglobin Affinity to O2
2,3-diphosphoglycerate (2,3 DPG)
Pyridoxal-5’-Phosphate (PLP)
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
GLYGLY2525-HPMA-HPMA7575 Nanoparticles Nanoparticles
Optimized with Human Albumin as Model Protein
(12717)nm
Applied to Hb, under N2 and at 4°C
(7842)nm
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
ConclusionsConclusions
Core-Corona Hb loaded Bioerodible Nanoparticles were prepared.
Nanoparticles display an homogeneous diameter distribution (14020)nm, good surface properties, high EE% and Loading.
Preliminary toxicity investigation evidenced a reduced cytotoxicity compared to free Hb.
GLYx-HPMAy polymers appear also suitable for the development of Hb based Oxygen carriers.
New co-factors and polymeric matrices will be investigated for preserving Hb functionality.
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
• The presented results have been attained within the framework of the “Sviluppo Nuovi Derivati del Sangue” financed project by MIUR.
• Polymer Laboratories (UK) for supplying polymeric materials, prepared within the EC-project “TATLYS” G5RD-CT-2000-00294
• Mr. Piero Narducci in recording SEM images of Nanoparticles
AcknowledgmentsAcknowledgments
A.M. Piras INSTM conference. 12-15 June 2007, Perugia, Italy
Thanks for Your AttentionThanks for Your Attention
Top Related