final year project

Final Year ProjectSchool of Materials Science and Engineering

List of Contents

• Introduction• Motivation• Scope• Plan of action• Data• Results• Discussions• Conclusion• Recommendations• Insights into FYP• Questions and Answers

Introduction

Final Year Project

Protein loading and delivery :Comparison between mesostructured and non porous bioceramics

• Keywords: Protein, loading and delivery, mesostructured, non porous, bioceramics

Keywords

• Bioceramics- bioactive, bioinert- Hydroxyapatite (HA), Bioglass (BG)

• Mesostructured- Pore size of 2nm to 50nm- MHA, MBG

• Applications- Protein loading and delivery, scaffolds, coatings e.g.

Motivation

• Recent years, mesostructured bioceramics are used as carrier for controlled drug delivery.

• higher pore size, higher surface area and pore volume

• Initial loading concentration of protein into mesostructured biomaterials are not widely researched.

• Thus, in this project, we will like to ask ourselves whether…

Motivation

PORE SIZE

Motivation

SURFACE AREA

Motivation

of mesostructured* ; non porous* bioceramics

and

*MHA, MBG, HA, BG

Motivation

INITIAL PROTEINCONCENTRATION

Motivation

of Lysozyme and Bovine Serum Albumin will affect in

Motivation

LOADING/DELIVERYEFFICIENCIES.

Scope

• This project is divided into 2 phases.

• Phase 1 : loading of proteins into non porous and mesostructured bioceramics (completed)

• Phase 2 : releasing of proteins by the loaded non porous and mesostructured bioceramics (future studies)

Plan of action

• Synthesis of Bioceramics

• Characterization (BET)*

• Loading of different concentrations of proteins into bioceramics

• Characterizations (Uv-vis spectrometry, Thermal Gravimetric Analyzer)**

*surface area, pore diameter & volume data ** Absorption spectrum and TGA data

Data

• Bioceramics : HA, MHA, BG, MBG

Samples MBG BG MHA HA

BET Surface

Area (m2/g)

463.44 294.90 228.13 26.30

BJH Pore Volume (cm3/g)

0.61 0.02 0.96 0.22

BJH Pore Size (nm)

57.31 25.56 135.47 27.10

Data

• Proteins: Lysozyme, Bovine Serum Albumin

• Concentration of Proteins

Concentration

1mg/ml 5mg/ml 10mg/ml

Protein Dimensions Isoelectric point

Lysozyme 3.0nm x 3.0nm x 4.5nm 11.9

Bovine Serum Albumin

4.0nm x 4.0nm x 14.0nm 4.9

Theory/Concepts

• Higher initial protein concentration loaded into bioceramics will result in an enhanced efficiency of protein loading results.

-Increase protein concentration = distance between protein

decreases = increase lateral attraction force = change in conformation of protein on adsorbent net charge of

protein = strong interfacial interaction

-Higher protein concentration drives molecules to the surface

faster.

Theory/Concepts

• Mesostructured bioceramics have higher pore size, surface area, thus the efficiency of adsorption of proteins is greatly enhanced compared to non porous bioceramics.

-Ordered pore network, homogenous

-adsorb in pores larger that the hydraulic radius of protein

-Surface area of pores, the higher the surface area of higher

amount of drug being adsorbed

1.00 5.00 10.000.00

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60.00

70.00

80.00

MHA

HA

MBG

BG

Lysozyme concentration (mg/ml)

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eff

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f L

yso

zym

e (w

t%)

Results - Lysozyme

Observations

1)Increased in loading efficiency with an increased in protein concentration

2)Abnormality at 5mg/ml

3)Loading efficiencies for all bioceramics were approximately constant and high at 10mg/ml

4)Loading efficiencies for mesostructured biocermics are relatively higher compared to non porous bioceramics

Discussions - Lysozyme

1)Increased in loading efficiency with an increased in protein

concentration

• congruent to theory of higher initial protein concentration loaded into bioceramics will result in an enhanced efficiency of protein loading results.

Discussions - Lysozyme

2)Abnormality at 5mg/ml

• At a higher concentration of lysozyme solution, they may be strong repulsion forces between the lysozyme molecules since they will be positively charged when placed in deionized water of pH 6 which is below the isoelectric point of lysozyme.

Discussions - Lysozyme

3)Loading efficiencies for all bioceramics were approximately

constant and high at 10mg/ml

• adsorption kinetics of increased concentration of initial protein loading > kinetics of strong repulsion forces between the lysozyme molecules.

• May exist additional lysozyme molecules adsorbed onto the external surface of the bioceramics, being retained outside the bioceramics architecture, achieving an optimum loading efficiency.

Discussions - Lysozyme

4)Loading efficiencies for mesostructured biocermics are relatively higher to non porous bioceramics

• Pore size of MHA and MBG are bigger than HA and BG, thus a greater pore size will allow the small lysozyme molecule to enter and get adsorbed onto the bioceramics

Results - Lysozyme

Observations

1)Increased in loading efficiency with a decreased in protein concentration

2)Loading efficiencies for MHA are relatively higher compared to HA at all protein concentrations

3)Loading efficiencies for MBG are comparable or less than BG at all protein concentrations

4)Loading efficiencies for all bioceramics are relatively low at 10mg/ml

1.00 5.00 10.000.00

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MHA

HA

MBG

BG

BSA concentration (mg/ml)

Cu

mu

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f B

SA

(w

t%)

Discussions – Bovine Serum Albumin

1)Increased in loading efficiency with a decreased in protein

concentration

• Contrary to theory.

• At higher bulk concentration of BSA protein loaded = less unfolding of BSA would occur have an effect on the adsorbate-adsorbate interactions, leading to the decline in the binding affinity of protein to bioceramics

Discussions – Bovine Serum Albumin

2) Loading efficiencies for MHA are relatively higher compared to

HA at all protein concentrations

• Pore size of MHA is comparable to dimensions of Bovine Serum Albumin highest efficiency

• Surface area of MHA > Surface area of HA higher efficiency

Discussions – Bovine Serum Albumin

3)Loading efficiencies for MBG are comparable or less than BG at

all protein concentrations

• BSA is ellipsoidal in shape and might adsorb with either their long and short axes with comparable probabilities

• In mesostructured bioceramic, proteins are evenly distributed in a given mesopore; highly likely to be clustered at the pore entrance, hindering further adsorption of the BSA molecules.

Discussions – Bovine Serum Albumin

4)Loading efficiencies for all bioceramics are relatively low at

10mg/ml

• At higher bulk concentration of BSA protein loaded = less unfolding of BSA would occur have an effect on the adsorbate-adsorbate interactions, leading to the decline in the binding affinity of protein to bioceramics

• In mesostructured bioceramic, proteins are evenly distributed in a given mesopore; highly likely to be clustered at the pore entrance, hindering further adsorption of the BSA molecules.

Conclusion - Lysozyme

• Higher initial protein concentration loaded into bioceramics

will lead to an increased in loading efficiency.

• Mesostructured bioceramics have a higher loading efficiency compared to non porous bioceramics.

• Dominant protein loading factors are pore size and initial protein concentration as compared to surface area of bioceramics.

Conclusion – Bovine Serum Albumin

• Higher initial protein concentration loaded into bioceramics

will lead to an decreased in loading efficiency.

• MHA have a higher loading efficiency compared to HA, MBG have a comparable loading efficiency compared to BG.

• Dominant protein loading factors for MHA are pore size, surface area and initial protein concentration.

• Dominant protein loading factors for bioglasses are initial protein loading concentration.

Future work

• Phase 2 , a comparison of protein delivery between mesostructured and non porous bioceramics.

• Pore volume analysis by carrying out repeated protein loadings into the bioceramics.

• Different protein concentrations of lysozyme and Bovine Serum Albumin used to obtain a wider data distribution.

Future work

• Adsoprtion of protein is enhanced around isolectric point.

• Thermal gravimetric analysis could help in confirming the trends observed.

• Possible applications of this final year report study.

Insights into FYP

I feel that the final year project is the

realization of classroom education, applying to reality through practical

work.

It is a self learning process where one grew through exploration.

I learned a system, or a mindset to go about solving problems.

“Knowing is not enough; we must apply!” – Johann Wolfgang Von

Goethe

THANK YOU

Final Year ProjectQuestions and Answers

Final Year ProjectQuestions and Answers

Final Year ProjectQuestions and Answers

Data

Adsorption Mechanism• (1) Transport to the surfaces by diffusion, which could be generally

improved through mixing and shearing action.• (2) Pore diffusion, the rate-limiting step when strong interfacial

interactions occur (the protein adsorbed to the binding sites remains fixed), and hence mainly depending on the relative size of pores and protein molecules.

• (3) Adsorption/desorption at the surface, affected by the nature of surface–protein interactions, and described by an interfacial chemical reaction and its related kinetic adsorption and desorption mechanisms.

• (4) Surface diffusion, influenced mainly by the surface–protein interactions and negligible in the case of strong adsorption (no desorption occurs).

• (5) Conformational alteration of proteins in contact with the interface, and interactions with other adsorbed protein molecules

Adsorption Mechanism

Adsorption Mechanism - langmuir

• The classical Langmuir theory for gas adsorption can be applied to adsorption from solution, if the solution is sufficiently diluted [11]. The isotherm for loading studies of lysozyme and BSA exhibits a sharp initial rise in the low equilibrium concentration, which suggests there is a high affinity between proteins and the surface of the bioactive biomaterials [11]. In the high equilibrium concentration, the isotherm tends to a plateau. These isotherms are quite fit for the Langmuir monolayer adsorption equation which is expressed as above

Bioglass synthesis

Mesostructured Bioglass synthesis

TGA - Lysozyme

TGA – Bovine Serum Albumin

• inter-penetration of chains at the interface.

• contact time, temperature, molecular weight of polymers and physical form (liquid, solid)

Diffusion theory

• Adhesive penetrates into the pores, holes and crevices and other irregularities of the adhered surface of a substrate, and locks mechanically to the substrate.

• Wet the substrate, right rheological properties.

Mechanical interlocking

• Intimate intermolecular contact between two materials, and involves surface forces/chemical bonding between the atoms in the two surfaces

Adsorption theory

• Transfer of electrons across the interface creating positive and negative charges that attract one another.

Electronic theory