Nano Particle Encapsulation of Human Hemoglobin

Nano Particle Encapsulation of Human Hemoglobin

AbstractThere are many medical conditions that would greatly benefit from the ability to inject encapsulated proteins capable of maintaining their function without being scavenged by the immune system. Nano particle encapsulated proteins offer the possibility of such materials. In this study human adult hemoglobin (HbA) containing nano particles have been prepared using a new highly effective preparative platform. The research provides evidence that hemoglobin maintains functionality within the synthesized nano particles. Three different variations of the preparative methods were used to synthesize the HbA containing nano particles. In this initial study, the resulting nano particles were sized using dynamic light scattering. Additionally, there was no indication of HbA leaching from nano particles. Absorption spectra indicate that the HbA, within the nano particles, can undergo changes in both redox state and ligation state. The results suggest that this approach could lead to infusible, encapsulated, functional proteins suitable for therapeutic use.

Goals and ObjectivesSuccessfully encapsulate Met Human Hemoglobin (HbA) in sol-gel derived nano particlesHbA used as a model proteinTest functionality of encapsulated HbA

IntroductionProtein TherapyCurrently being used to treat numerous diseasesNano TechnologyNano particles: Finely ground sol gels Allow for the directed delivery of the encapsulated molecules in biological systemsApplications include cures and treatments for various ailmentsSol- gels derived from polymeric Tetra Methyl Orthosilicate(TMOS) process3 variations of TMOS sol gels were synthesized in this study

IntroductionHbA as a model ProteinHbA has been widely studiedCell- free HbA is toxic in that it dimerizes and is rapidly filtered by the kidneysEncapsulating HbA would allow HbA to exist out of the cell without dimerizing and becoming toxic

HbA has been previously been encapulated

Rapidly filtered by the kidneys5

Synthesis of nano particles40 ml Met HbA Sol gel blocks were prepared3 variations Sol Gel Blocks were converted into nano particles via Lyophilization and Ball Milling

Spectrum of nano particlesParticles were suspended in PEG 400Suspended particles were centrifuged Settle out suspended particlesWet and viscous (due to the PEG) particles were placed in between two glass slidesSlide was placed in UV- Vis for absorption spectrum

Reduction and ligation of Met HbA ParticlesPrepared diothionite/ CO buffer solution was added to Met HbA nano particlesDithionite reduced the Met HbACO liganded to the Fe(II+) Confirmed ligation and reduction via absorption spectrum

Sizing of Nano ParticlesDynapro NanoStar Dynamic Light Scattering equipment

Results

Met HbA was successfully encapsulated in the nano particles and did not leach when the particles were suspended in aq bufferMet HbA has a peak absorbance at 409 nm3 nm shift can be attributed to compenents of particles OR presence of OH- Met HbA

Ligation and reduction of HbA

Met HbA Particles were successfully reduced and liganded to CO HbA particlesCO HbA has a peak absorbance at 420 nm

Sizes of various Particle formulations

DiscussionPerformed experimentOnly CO was ligandedLack of timeCO ligands most efficiently to HbASpectra of Met HbA particles shifted about 3nmPossibly due to the other content of the particles OR the presence of some hydroxy met HbA

Future researchFunctionality of HbA nano particles will be further exploredIn vivo experimentation of the HbA particlesPotential applicationsEncapsulation of other proteins and other biological substancesProtein therapySynthetic blood substitutesDisease treatmentAdvances in biological research

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