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