Synthesis of pegylated acrylic-based biomaterials in the shape of micro-nano particle

Tutors Prof. Giovanni Polacco

Ing. Davide Silvestri

Dr. Caterina Cristallini

Ing. Mariacristina Gagliardi

Engineering UniversityBachelor in Chemical Engineering

CandidateSimony Paolicchi da Silva Pereira

9th December 2010University of Pisa

Thesis of Bachelor

Introduction Nanotecnology: science involved in planning, synthesis, characterization and

application of materials and implants having a functional organization with almost a dimension in the range of nanometer.

Nanotechnology has lot of field of application in engineering, science and medice.In particularly in medicine it use materials and devices realized for interation with Human body in the subcellular range with high grade of specificy.

Potential biomedical applications: drug delivery systems for the treatment of cancer, diabetes, systems for repairing the human organs and biocompatibile implants.

Nanocarrier for targeted release of drug

Requirements: Ability to incorporate high doses of drugs Protect and vehicular unstable molecules, poorly soluble and

potentially toxic High persistence in the bloodstream without sedimentation and

blocking of normal blood flow. Ability to evade the capture by the immune system Recognize and bind specifically receptors of target cells

Operating Parameters:• Choice of appropriate starting materials• Development of appropriate synthesis techniques• Functional Characterizations

Aim of the Thesis

Preparatation of drug delivery system in micro-nano-particles shape.

Phases of work :1. Synthesis 2. Optimization 3. Loading of the drug 4. Functional Characterization

Requirements:

• Biocompatibility

• Hydrophilicity (to avoid removing by phagocytic cells)

• Tendency to form nanoparticle structures

• Appropriate functionality to facilitate interaction with the drug and bioactive molecules for drug targeting

Choice of materials

Operating conditions:T=65°C• 24h

• Reaction Medium ACN:H2O 75:25 v/v

SHYNTHESIS

Methacrylic acidMAA (PEG) ethyl ether

methacrylate (PEG)EEMA

Trimethylolpropane trimethacrylate

TRIM

Pentaerythritol triacrylatePETRA

Monomers

Drug

Acetylsalicylic Acid

MaterialsRatioMAA/

(PEG)EEMA

Ratiomon./Crosslinkers

Ratiomonomers/drug

PMAA 100/0

5/1 7/1Copolymers 80/20, 75/25, 70/30

P(PEG)EEMA 0/100

Polymerization by precipitation in dilute conditions

Crosslinkers:

Recovery of the solid content

• Valuation reaction yield• Morphological analysis (SEM)• Functional characterization (test of drug delivery, HPLC)

Recovery and purificationWashes in ACN for 15 minutes analysis of the washing waters with HPLC

Analysis of the final conversion of monomers

Encapsulation efficiency of the drug

Analysis HPLC

The detection of the concentrations of the monomers and of the drug present in thewashing water was carried out by the technique of High Performance Liquid Chromatography.

Chromatographic Method

Mobile phase ACN/H2O 80/20 (v/v)

Flow 1 ml/min

l 210 nm (monomers)280nm (drug)

Injection volume 100 ml

Column C18 5 mm

Final conversion of monomers

0

10

n

nnx

MAA/(PEG)EEMA

Molar ratio %

MAA (PEG)EEMA

Xmoli Xmoli

100/0 1,00

75/25 0,99 0,99

70/30 1,00 1,00

80/20 1,00 1,00

0/100 0,99

Results obtained:

MAA (PEG)EEMA

Xmoli Xmoli

0,99

0,99 1,00

0,99 1,00

0,99 1,00

0,99

MAA/(PEG)EEMA

Molar ractio%

MAA (PEG)EEMA

Xmoli Xmoli

100/0 0,97

75/25 0,99 0,99

70/30 0,99 0,99

80/20 0,98 0,99

0/100 0,99

MAA (PEG)EEMA

Xmoli Xmoli

0,99

0,99 0,99

0,99 0,99

0,99 0,99

0,99

TRIM PETRA

Withdrug

MAA/(PEG)EEMA

Molar ratio %

Drug concentration in

washing water

(mg/ml)

% Encapsulation efficiency

100/0 – TRIM 0,98 74,47%

80/20 – TRIM 1,28 75,92%

75/25 – TRIM 1,02 82,03%

70/30 – TRIM 1,19 80,12%

0/100 - TRIM 1,03 90,65%

100/0 - PETRA 0,51 86,63%

80/20 - PETRA 0,93 82,48%

75/25 - PETRA 0,89 84,15%

70/30 - PETRA 0,92 84,62%

0/100 - PETRA 1,03 91,47%

Efficiency of encapsulation

Morfological Analysis (SEM) - 1

PMAA

P(PEG)EEMA

80/20

75/25

70/30

Copolymerswith TRIM

50/50 25/75

PMAA

P(PEG)EEMA

Morfological Analysis (SEM) - 2

80/20

75/25

70/30

Copolymers with

PETRA

PMAA P(PEG)EEMA

Morfological Analysis (SEM) - 3

80/20 75/25 70/30

With drug

Conclusions of morfological analysis

• The Methacrylic acid leads to the formation of nanoparticles• Increasing amounts of MAA improves the morphology of the material• The (PEG) EEMA not gives the material a nanoparticle structure• The cross-linked systems with TRIM show more spherical particles and less aggregated• The PETRA, being more hydrophilic, leads to greater interaction with the pegylated Polymer chain• Acetylsalicylic acid has no influence on formation of particles which are more aggregated

and with smaller dimensions.

It must consider the necessity to reduce the aggregation of materials using an appropriate solvent within a system of sonication

Test of drug delivery in vitro

Preparation of samples:• A known amount of filled polymers was weighed and introduced into

suitable bags to prevent loss of material• The samples were immersed in a suitable release media (PBS buffer

solution at pH 7.4 containing 0.5% w / v sodium dodecyl sulphate)• Levies at fixed times and replacement of the release media to avoid

saturation of the solution• HPLC analysis for assessing concentrations

0 20 40 60 80 100 120 140 160 1800.00%

2.00%

4.00%

6.00%

8.00%

10.00%

12.00%

14.00%

PMAA80/2070/3075/25P(PEG)EEMA

Tempo (ore)

M(t

)/M

(0)

Materials synthesized by crosslinking TRIM

0 20 40 60 80 100 120 140 160 1800.00%

1.00%

2.00%

3.00%

4.00%

5.00%

6.00%

7.00%

PMAA80/2070/3075/25P(PEG)EEMA

Tempo (ore)

M(T

)/M

(0)

Materials synthesized by PETRA

Results

• The P (PEG) EEMA releases a greater amount of drug

• The PMAA although the nanoparticle structure release a smaller amount of drug

• The copolymers have intermediate behavior• Copolymer 75/25 release high amount due

to the small size of the nanoparticles

• P (PEG) EEMA and PMAA have behaviour similar to previous results

• The polymers with PETRA show a release kinetics low, due to a greater interaction between the polar functional groups of the drug, the crosslinking agent and the monomers.

Copolymers containing TRIM

Copolymers containing PETRA

0 20 40 60 80 100 120 140 160 1800.00%

2.00%

4.00%

6.00%

8.00%

10.00%

12.00%

14.00%

PMAA 80/20 70/30 75/25 P(PEG)EEMA

Tempo (ore)

M(t

)/M

(0)

0 20 40 60 80 100 120 140 160 1800.00%

1.00%

2.00%

3.00%

4.00%

5.00%

6.00%

7.00%

PMAA 80/20 70/30

75/25 P(PEG)EEMA

Tempo (ore)

M(T

)/M

(0)

CONCLUSIONI

In the present work have been synthesized multifunctional polymeric nanoparticles with the purpose of obtaining polymeric platforms with  controlled releasing  of drug.

It has been analyzed the influence of morphology and release of drug: polymer composition, cross-linking, effect of drug loading by optimization of these parameters has been possible to obtain the desired materials

Structure nanoparticle obtained during the reaction phase this allows to avoid additional treatments

Loading of the active principle in the reaction stage and high efficacy of encapsulation

The release profiles of the drug have shown that the polymers are able to release the drug in appreciable quantities and so prolonged over time (sustained release)

FUTURE DEVELOPMENTS

Molecular imprinting: the materials can be equipped with specific recognition sites to the drug due to the presence of carboxyl groups.

The system may be enriched in vivo in order to impart a releaseprolonged in time

The nanoparticles may be used as a carrier for the encapsulation and release of drugs having anti-tumor properties

Thanks to the presence of carboxyl groups in the material, It’ s possible use these sites for specific functionalization (eg. molecular imprinting and / or conjugation of bioactive molecules)

Evaluation of cytotoxicity, cytocompatibility and hemocompatibility