Development of photo-responsive hydrogels for controlled release of encapsulated molecules

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Thesis

DEVELOPMENT OF PHOTO-RESPONSIVE HYDROGELS

FOR CONTROLLED RELEASE OF ENCAPSULATED MOLECULES

UPPSALA UNIVERSITY

Final Report

Johan

Verhoeven

ATGM

Avans University of applied science

‘s Hertogenbosch

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Graduation Project

Development of photo-responsive hydrogels for

controlled release of encapsulated molecules

Document data

Graduation report Chemistry

Version 1.1

Deadline: 15 January 2015

Semester 1

Implementation Details

Avans University of Applied Sciences ’s_Hertogenbosch ATGM Chemistry Study year 2014-2015 Author Johan Verhoeven (2041242) jmga.verhoeven@student.avans.nl

Supervisor Avans University:

Dr. W.H.A. Kuijpers

wha.kuijpers@avans.nl

Supervisor Uppsala University

Associate professor Dmitri Ossipov

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A

BSTRACT

Hydrogels are becoming more used within the field of tissue engineering and drug delivery. A hydrogel with the property of controlled release of encapsulated drug or biomacromolecule is one of many possible applications. Encapsulated material within these hydrogels will not interact with its surrounding enviroment until it will be released. One of these stimuli used, used to trigger the release of the encapsulated material is UV light. the goal was to synthesize the photo sensitive macromolecules and to form hydrogels from this material. Once obtained, these hydrogels would be used in degradation studies by measuring the release of photo-degraded material from the hydrogel network and the release of a drug model so called “SAMSA fluorescein”.

The synthesis of the starting material, bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) has been started with 4-(4-(1-(hydroxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid. From here on several synthesis pathways were introduced to obtain the best yield of the required material for hydrogel formation.

Once formed, these hydrogels were used in the degradation study by measuring the release of photo degraded material and the release of fluorescein. This study has been performed by comparing the observed release from hydrogels which are exposed to UV light to the release from hydrogels kept in dark conditions.

Off all the synthesis pathways, the synthesis using PEG diamine and 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid-N-hydroxysuccinimide ester showed the best results. The degradation studies showed that the release of photosensitive material or fluorescein from hydrogels could be triggered by UV light exposure. The studies also showed that the hydrogels made out of this photo sensitive macromolecules are stable in dark conditions. However, constant release of fluorescein observed from hydrogels kept in dark conditions, proves that the covalent bond used for encapsulation of fluorescein is not stable.

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T

ABLE

OF

1 I

NTRODUCTION

Use of hydrogels and their utility rises every day. Hydrogels are polymer networks made for different applications. The biggest interest has been observed in using hydrogels as drug (protein) delivery systems and in tissue engineering [1, 2]. Hydrogels are made from a synthetic polymer polyethylene glycol (PEG), or from natural biomacromolecules for example hyaluronic acid. A network can be formed from one or more types of polymers and the resulting networks forming a hydrogel [3]. In drug/macromolecule delivery applications, a drug can be encapsulated in hydrogel either in situ (that is during the network formation from soluble polymer precursors) or post-synthetically (that is by incubation of hydrogel in drug solution)[3]. When the gel, loaded with the cargo, is injected in human body, the drug molecules cannot come in contact with the surrounding cells immediately. One advantage of this system is that toxic medicine cannot destroy any cells except for the residue surrounding the hydrogel. By linking a drug to hydrogel, it will bring more control over the release of the drug. In this case, drug can be released in response to some stimulus. A chemical linkage has been designed to brake when exposed to such stimulus, thus enabling the release of toxic compound. Some examples of these stimuli are temperature, pH, biomolecules, or UV light [1,2,3,]. For the release mechanism triggered by UV light, a photo-labile group should be built into the hydrogel’s structure. When the hydrogel absorbs UV light, a photo-labile chemical bond between the network and the drug molecule will be cleaved. This will slowly release the drug molecule from the hydrogel [4,5,6]. The goal of this work was to synthesize precursor molecules containing photo-labile groups and use them for hydrogel formation. Stability and degradation of these hydrogels would be studied by measuring and comparing the absorbance of the released photo-degraded compounds and the absorbance of the released drug mimic (SAMSA fluorescein) in UV-light and dark conditions. If the absorbance recorded from hydrogels exposed to UV light rises faster than the absorbance recorded from hydrogels kept in dark conditions, then it can be concluded that UV exposure on hydrogels triggers the degradation of the gel. However, if there is an linear absorbance rate observed from hydrogels kept in dark conditions, then it can be concluded that the hydrogel is not stable and slowly degrades.

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2 T

HEORETICAL

B

ACKGROUND

In this chapter, a theoretical background for the study is given. Information on hydrogels is presented at the beginning of this chapter. It is followed up by the theoretical aspects of synthesis and utilized purification procedures. This chapter ends with the information about analytical methods used to detect this degradation process.

2.1 H

YDROGELS

Every day hydrogels are becoming more important within the medical healthcare. These hydrogels are made out of polymers like polyethylene glycol (PEG) or hyaluronic acid (HA). Hydrogels are widely used for their biocompatible properties. Hydrogels may have mechanical and structural similarities with the extracellular matrix (ECM) [1] and have the ability to hold large quantities of water up to 99% [5]. Some types of hydrogels are designed to carry specific kinds of medicines or bio-macromolecules. These encapsulated medicines will not interact with the human body for as long as they remain encapsulated [7,8].

There are three different ways to encapsulate different kinds of biomacromolecules: by physical entrapment, affinity-based sequestration or covalent tethering. Physical entrapment is used with large molecules like proteins or nucleic acids. these will be trapped within the mesh of the hydrogel, which impedes the diffusion of the trapped molecules. Covalent tethering is basically chemical bonding the desired material to the hydrogel network. This way of encapsulation is mostly used for small molecules. Affinity-based sequestration is an alternative method for the covalent tethering and this technique uses the same principle [3].

All kind of encapsulated materials are released from each kind of hydrogel in a different way. For example, physically entrapped molecules will diffuse out of a hydrogel in a slow rate, while material what has been encapsulated by covalent tethering has to be released by some kind of trigger (see figure 1) [3]. Therefore the release of the encapsulated material depends on the developed goal of the hydrogel, the structure of the hydrogel and the structure of the encapsulated molecule [9].

Figure 1 A schematic figure of a hydrogel undergoing a degradation process. The degradation proces will release the encapsulated material (in this case protein) from the hyrogel [6].

2.2 P

HOTOSENSITIVEHYDROGELS

Some hydrogels are developed for controlled release of encapsulated molecules. These hydrogels will not release any encapsulated material until the degradation is triggered by U-light. When

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molecule, called 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid, belongs to the o-nitrobenzene ether based photo-labile groups (see figure 2) [1]. The advantage of this system is that the encapsulated materials can be released from the hydrogels at any place and time within the human body [3]

C H₃ O N+ O -O O O O OH CH3 CH2 O

Figure 2 Molecular structure of 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid.

These photo sensitive hydrogels can be prepared out of a material containing poly ethylene glycol (PEG) or PEG diamine connected with the photosensitive molecules. PEG is a polymer with bio-compatibility and non-toxic properties [4]. Therefore PEG is widely used for drug delivery vehicles [11]. For this design of hydrogels, two photo-sensitive molecules are bound to a PEG molecule. This material, called bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000), forms the starting material for gel formation. The structure is shown in figure 3. The difference between PEG and PEG diamine is indicated by an O or NH in figure 3.

C H₃ O N+ O -O O O O O CH3 CH₂ O O O C H₃ O N+ O -O O O CH₂ O C H₃ O (NH) (NH)

Figure 3 Molecular structure of bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) (or

PEG diamine).

2.3 H

YDROGELFORMATION

The formation of hydrogels can take place because of the presence of two acryl groups on each end on the photo sensitive PEG molecule (see figure 3). This makes it possible for this photo sensitive molecule to participate in radical polymerization [1,2]. This reaction will form a network on both acryl groups. Ammonium persulfate has been used as a source of radicals which attack the acryl group causing a chain propagation reaction. Tetramethylethylenediamine (TEMED) has been used as a catalyst for this reaction [2]. This reaction forming a network is shown in figure 4.

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Figure 4 Radical polymerization of bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000)

forming a hydrogel network. This is shown in the circle on the right side. The long chains represent PEG. The dots represent the connection point of cross-linked photo sensitive molecules.

2.4 E

NCAPSULATIONOFADRUGMODEL

The mostly used technique off encapsulation of the drugs, biomacromolecules or other material in photo sensitive hydrogels is covalent tethering [3]. This way of binding has to take place before the hydrogel formation would be triggered. Once the hydrogel is formed, it is not possible anymore to connect something to the hydrogel. Therefore the material has to be connected to bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000). One way to connect a molecule is by Michael addition [12]. Therefore the material requires a thiol group to react with the acryl group present on the starting material. When this molecule is connected to the starting material, hydrogel formation may be triggered. A hydrogel network with encapsulated material is shown in figure 5.

Figure 5 Hydrogel network with encapsulated material. The purple dots within this figure represents the encapsulated material.

A drug model will be used to study the encapsulation progress and other properties of the hydrogel. This molecule is called SAMSA fluorescein or 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino) fluorescein (see figure 6) [13]. This is a fluorescent molecule which will be used to measure any release from the hydrogel in the degradation studies. This molecule can be connected to an acryl group on bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000), but first the acetyl protecting group has to be removed from SAMSA fluorescein. This is done by adding a basic phase. When the protecting group is removed, the solution will be neutralized with an acidic phase. The available thiol group can be used for the Michael addition reaction [12].

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Figure 6 Structure of a SAMSA fluorescein molecule [12].

2.5 D

EGRADATIONPROCESS

When the hydrogel is exposed to UV light, the degradation process well be triggered. The photo sensitive molecule build into the network of the hydrogel will absorb the UV light. The absorption will affect the network and break the ester bond between the photosensitive group and the cross-linked connection point. The polymerized part of the molecule leaves with an acid. The photosensitive part leaves with a ketone and the nitrate group is changed to a nitroso group. This change is due to the properties of the photo sensitive material [1,2,3]. The disconnected particles and encapsulated material will dissolve and diffuse out of the gel until the whole gel is degraded and dissolved within the human body. The degradation process is shown in figure 7.

Figure 7 Degradation process of a photo-sensitive hydrogel triggered by UV absorption.

2.6 S

YNTHESISWALKTHROUGH OFPHOTOSENSITIVE

PEG

MACROMOLECULES

The synthesis of the photosensitive PEG macromolecules, necessary for hydrogel formation, starts with the commercially available photosensitive 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid. The synthesis has been proceeded step by step to obtain the material bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) or PEG diamine (Mn = 6000). All the synthesis pathways present in this process are stated in figure 8.

The first step was the synthesis of 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid. This reaction has been performed by esterification of an acryloyl group and the photo sensitive material. Triethylamine has been added as an organic base [1,15]. The second step was the synthesis of bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000). This could be done in multiple ways. The first way of synthesis has been done by esterification of the photosensitive material and the PEG (Mn = 6000) molecules. Oxalyl chloride is added as a condensing agent and potassium bicarbonate is added

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as a base [2,14]. The other way to obtain bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) is by using dicyclohexylcarbodiimide (DCC) as condensing agent [15,16]. This condensing agent is eliminated as dicyclohexyl urea (DCU) [17]. 4-dimethylaminopyridine (DMAP) had been used as a catalyst in this synthesis [15, 16].

Another way to obtain the required material for hydrogel formation is by obtaining the molecule bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG diamine (Mn = 6000). Therefore the photosensitive material, obtained after step one, had to be used in another synthesis path, step three.

In the synthesis of step three, a hydroxysuccinimide molecule had to be connected to the photosensitive molecule to form 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid-N-hydroxysuccinimide. The hydroxysuccinimide molecule will be connected to the carboxylic acid by esterification. N-(3-dimethylaminopropyl-N’-ethyl-carbodiimide hydrochloride (EDC)is used as a catalyst in this synthesis [3, 18].

This obtained material can be used in the synthesis of step four. In this synthesis the obtained material from step three is used to form bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG diamine (Mn = 6000). This will be done by dissolving the photosensitive material from step three with PEG diamine (Mn = 6000). No extra catalysts are necessary for this reaction, an amide bond will be formed after the esterification [1, 3, 19].

C H3 OH N+ O -O O C H3 O O OH Cl CH2 O C H3 O N+ O -O O C H3 O O OH CH2 O C H3 O N+ O -O O O O O CH3 CH2 O O O C H3 O N+ O -O O O CH2 O C H3 O N N Or O O Cl Cl P E G (6 0 0 0) , K2C O3 0 o_{C , A r g o n} P E G (6 0 0 0) , D M A P A r g o n Radical polymerisation A P S T E M E D C H3 O N+ O -O O C H3 O O O CH2 O N O O O H N O O C H3 O N+ O -O O O NH O CH3 CH2 O O NH C H3 O N+ O -O O O CH2 O C H3 O D C M A r g o n Hydrogel formation P E G d i a m i n e (6 0 0 0) S t e p 1 S t e p 2 . 1 s t e p 2 . 2 D C M , E D C 0 o_C S t e p 3 S t e p 4 T r i e t h y l a m i n e 0 o_{C , A r g o n}

Figure 8 Synthesis walkthrough from step 1 to step 4. Step 1: synthesis by attaching a acryloyl group. Step 2.1 esterification of PEG and the photosensitive material using oxalyl chloride. Step 2.2: other approach for this synthesis by using DCC. Step 3: connection of hydroxy succinimide to the photo sensitive material after step 1. Step 4 esterification using the photosensitive obtained from step 3.

2.7 P

URIFICATION

After each synthesis, a reaction mixture contains unwanted compounds such as side products, unreacted starting material and other materials. Purification techniques like liquid-liquid extraction, column chromatography and precipitation were used to purify the product from the crude reaction mixtures.

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2.7.1

LIQUID

-

LIQUIDEXTRACTION

One technique to purify the product is by liquid-liquid extraction. This technique uses two immiscible liquid phases, an organic and an aqueous layer, to separate impurities from the product. The product should be concentrated in one of the two phases, while impurities would transfer to the other one. Addition of bases or acids may cause protonation or deprotonation of the product or impurities. This change may activate the transfer of some material to the other layer.

Liquid-liquid extraction has been used in the purification of 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid. DCM has been used a solvent of organic layer. The product is dissolved in the organic layer. This layer has been washed with 10% sodium bicarbonate to remove any unreacted starting material 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid. 0.1 M HCl has been used to wash the organic layer to remove triethylamine. Concentrated sodium chloride has been used to remove any water remaining in the organic layer [1].

2.7.2

COLUMNCHROMATOGRAPHY

Column chromatography is a purification technique which separates the product from its impurities basing on interaction with the material of stationary phase and the solvent of the mobile phase. One of such stationary phase materials is silica (SiO2), while solvent is used as a mobile phase flowing through the column. When the product is loaded to the column and eluted, it interacts with both stationary and mobile phases. The polarity of the components and polarity of the eluent and stationary phase determines the movement for each different component present in the column. More polar molecules will interact strongly and retain in the column, while less polar molecules will move faster.

When the mixture has been loaded to a column, DCM is used as a mobile phase to remove any non-polar impurities. A mixture of DCM and methanol (40:1) is used for the separation. If the product moves slowly, a mobile phase of 20 : 1 of DCM and methanol is used.

2.7.3 P

RECIPITATION

Precipitation is a purification technique which removes the impurities from the product basing on solubility of different compounds in different solvents. Normally, in re-crystallization procedure, a product is insoluble in the cooled solvent, while impurities are. By adding the crude mixture in cold solvent, the product will preticipate while the impurities dissolve [15].

Purification by precipitation has been used for bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) and bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEGdiamine (Mn = 6000) after the synthesis using cooled diethyl ether [2, 15, 17]

2.8 C

HARACTERIZATION

2.8.1 T

HINLAYER CHROMATOGRAPHY

Thin layer chromatography (TLC) is a fast and easy type of analytic assay. TLC can be used to determine the composition of a mixture. By applying a mixture on a TLC plate, the mixture will be separated upon moving the solvent through the plate by capillary forces. Different spots belonging to different compounds can be then observed on the TLC plate. UV light can be used to see UV absorbing substances. The separation is based on interactions between the mobile (mostly non polar) and solid (mostly polar) phases. Solid phase consists of silica (SiO2) attached on a glass or aluminum plate. Mobile phase is chosen basing on the expected composition of the mixture. The mixture is applied on solid phase at the bottom of the plate and placed into liquid

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mobile phase. Polar compounds interact more strongly and moves more slowly than non-polar components [20].

TLC has been mainly used as a control analytical tool after each synthesis. TLC plates were used for examination for formed impurities, which will be shown with another spot besides the starting material. It can give the information about how much of starting material is left, how much of other unwanted products are formed and what is the polarity of the end product. The mobile phase that has been used consisted of non-polar dichloromethane and polar methanol (or ethanol) at different ratios, such as 20:1 for synthesized low molecular weight compounds and 40:1 and 20:1 for modified polymers.

2.8.2 NMR

Nuclear magnetic resonance spectroscopy (NMR spectroscopy) is a mostly used technique for determining structures of organic materials. It is a non-destructive method with high reproducibility and a high sensitivity. With NMR, it is possible to determine the structure of a compound. By applying a magnetic force, it is able to excite magnetic dipole moments of magnetic nuclei (1H, 13C, 19F and 31P) and measure the relaxation time to the ground state [21, 22]. This relaxation process is detected as signals with certain intensities and a chemical shifts generating an NMR spectrum. Intensity of a signal is directly linked to the amount of corresponding nuclei, while its chemical shift is determined by electronic shell of the nuclei. The last is a “chemical” fingerprint of the nuclei [21, 23].

NMR has been used to determine if the synthesis has been successful, and has been used to confirm the chemical structures of 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid, 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid-N-hydroxy succinimide ester, bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) and bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000).

2.9 UV-

VISSPECTROSCOPY

UV-vis spectroscopy is a technique that measures the amount of absorbed light. UV-vis spectroscopy is a suitable technique to measure concentration of a compound in solution. In UV-vis spectrophotometer, a source of UV-UV-vis light projects the light through a sample solution at different wavelengths. After absorption by the sample, the intensity of this light is measured by the detector. It is possible to measure the concentration by using Lambert Beer law [24].

The UV-vis spectroscopy has been used to measure the process of gels degradation study. In this study, absorbance of photo-labile o-nitrobenzene ether group has been measured [1, 2]. The release of fluorescein from SAMSA fluorescein-linked hydrogels has been studied by measuring the absorbance of at 495 nm [13].

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3 E

XPERIMENTAL

PART

In this section, all the experiments are revealed together with the used equipment and materials.

3.1 M

ATERIALS

4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid, TRC, CAS nr. H825255; Triethylamine (TEA) extra dry, Sigma Aldrich, CAS nr. 121-44-8;

Dichloromethane (DCM) extra dry, Acros Organic, Cas nr. 814-68-6; Acryloyl chloride, Sigma Aldrich, CAS nr. 814-68-6;

Hydrochloric Acid (HCl), Scientific Fischer CAS nr. -; Dichloromethane (DCM) Scientific Fischer CAS nr 75-09-2; Methanol (MeOH), Scientific Fischer CAS nr. 67-56-1; Diethyl Ether, Scientific Fischer CAS nr. 60-29-7;

Sodium Bicarbonate (NaHCO3), Sigma Aldrich, CAS nr. 144-55-8; Sodium Chloride (NaCl), Scientific Fischer, CAS nr. 7647-14-5; Sodium Hydroxide (NaOH), VWR, CAS nr. 28 245.298;

Magnesium Sulfate, Sigma Aldrich, CAS nr. 7487-88-9; Oxalyl Chloride 98%, Acros Organics, CAS nr. 79-37-8; Polyethylene glycol (PEG Mn =6000), Merck, CAS nr. 70217-1;

potassium bicarbonate (K2CO3) 99%, Sigma Aldrich CAS nr. 584-08-7; N-hydroxysuccinimide (NHS) 97%, Sigma Aldrich, CAS nr. 6066-82-6; 4-dimethylaminopyridine (DMAP) Acros Organics, CAS nr. 1122-58-3;

N,N'dicyclohexylcarbodiimide (DCC) 99%, Sigma Aldrich, CAS nr. 538-75-0;

Polyethylene glycol diamine (PEGNH2 Mn =6000), Sigma Aldrich, CAS nr. 24991-53-5; Ammonium Per sulfate (APS) 98%, Sigma Aldrich, CAS nr. 7727-54-0;

Tetramethylethylenediamine (TEMED) 99%, Sigma Aldrich, CAS nr. 110-18-9; Deuterated Chloroform (CDCl3) 99, 8%, Cambridge Isotope, CAS nr. -;

Deuterated Dimethyl sulfoxide (DMSO) 99,9%, Cambridge Isotope, CAS nr. -.

N-(3-Dimethylaminopropyl)-N’-ethyl-carbodiimidehydrochloride (EDC), Fluka, CAS nr. 25952-53-8;

5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein (SAMSA fluorescein), Molecular Probes, CAS nr.-;

3.2 D

EVICES

UV-vis spectrophotometer: Perkin Elmer Instruments, Cambda 35. Centrifuge: Hettich Zentrifugen, EBA 30.

NMR: Oxford NMR AS 4000, 400 MHz.

Magnetic stirrer: Heidolph MR Hei- standard. Vortex: VWR international.

Analytical laboratory scale: Metler Toledo AX 504 Deltarange. Vacuum pump: Labinet CVC 3000.

Rotary evaporator: Laborota 4000.

3.3 S

YNTHESISOF

4-(4-(1-(

ACRYLOYLOXY

)

ETHYL

)-2-

METHOXY

-5-NITROPHENOXY

)

BUTANOICACID

4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (488 mg, 1.63 mmol) and 3 mL of anhydrous DCM were added to a 25 mL round bottom flask, and the flask was purged with argon. TEA (661 mg, 6.53 mmol) was added to the round-bottom flask and a funnel was attached on top of the RB flask. The flask was placed into an ice bath and 1 mL of anhydrous DCM and AC (470 µL, 5.77 mmol) were added to the funnel. The AC solution was added drop-wise (1 drop every 5 sec) into the solution of starting material at 0°C and the combined mixture was set up to

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react overnight. On the next day the reaction turned yellow/brown containing white TEA•HCl crystals. The reaction mixture was filtrated and the solid phase was washed with sodium bicarbonate, 0,1M aqueous HCl, and finally with concentrated brine. After washing, the DCM phase was removed by rotary evaporation. 50 mL of acetone-water 1:1 v/v mixture was added to the residue and the resulting mixture was stirred overnight. The next day, acetone was removed by rotary evaporation. The remaining water layer was washed four times with DCM. The DCM phase was thereafter washed with 0.1 M HCL and brine. DCM was finally evaporated and the product was dried under vacuum overnight. NMR: 6-8 mg in 0.6 mL of CDCl3. Yield 430 mg (1.22 mmol, 75%).

3.4 S

YNTHESISOFBIS

(4-(4-(1-(

ACRYLOYLOXY

)

ETHYL

)-2-

METHOXY

-5-NITROPHENOXY

)-

BUTANATE

)PEG (MN = 6000)

USINGOXALYLCHLORIDE

4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (431 mg, 1.22 mmol) and 3 mL of anhydrous DCM were added to a 25 ml round bottom flask and the flask was purged with argon. The solution was cooled on an ice bath. Oxalyl chloride (200 µl, 2.33 mmol) was added to the cooled solution and the reaction mixture was stirred for five hours. Solvent was removed by rotary evaporation. The residue was dissolved in 10 mL of anhydrous DCM. This mixture was slowly added to a mixture of PEG (963 mg, 0,16 mmol, Mn = 6000) and K2CO3 (171 mg, 1,24 mmol) under argon atmosphere. After stirring overnight, the product was precipitated by slowly adding to cold diethyl ether. The precipitate was filtrated from the solvent and dried in a vacuum oven overnight at room temperature. NMR: 6-8 mg in 0.6 mL of CDCl3.. Additional purification by column chromatography was performed using DCM : MeOH at ratio of 40:1 followed by 20:1 as an eluent. Yield 237 mg (0,035 mmol 22%).

3.5 S

YNTHESISOFBIS

(4-(4-(1-(

ACRYLOYLOXY

)

ETHYL

)-2-

METHOXY

-5-NITROPHENOXY

)-

BUTANATE

)PEG (MN = 6000)

USING

DCC

4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (64 mg, 0.181 mmol), PEG (435 mg, 0,073 mmol, Mn = 6000) and 5 ml of anhydrous DCM were added to a 25 mL round bottom flask and it was purged with argon. DCC (37 mg, 0,181 mmol) and DMAP (5 mg, 0,029 mmol) were dissolved in 5 mL of anhydrous DCM. This solution was added to the solution of starting materials in DCM. The reaction mixture was stirred overnight under argon at room temperature. The product was precipitated by adding the reaction mixture to cold diethyl ether. The precipitate was dried in a vacuum oven at room temperature. After drying, NMR analysis was performed on 6 mg of the product after dissolving in 0.6 mL of CDCl3. NMR: 6-8 mg in 0.6 mL of CDCl3. Yield 400 mg (0.06 mmol 82%).

3.6 S

YNTHESISOF

4-(4-(1-(

ACRYLOYLOXY

)

ETHYL

)-2-

METHOXY

-5-NITROPHENOXY

)

BUTANOICACID

-N-H

YDROXYSUCCINIMIDEESTER

4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (79.6 mg, 0.225 mmol), N-hydroxysuccinimide (29.4 mg, 0.27 mmol) and EDC (43.4mg, 0,28 mmol) were added to a 25 mL round bottom flask. This flask was purged with argon for 10 min. 4 mL of anhydrous DCM was then added. The reaction mixture was stirred overnight at room temperature. It was then washed three times with concentrated NaCl, and dried with magnesium sulfate. Magnesium sulfate was filtered out, the solvent was evaporated and the obtained residue was dried under vacuum overnight. NMR: 6-8 mg in 0.6 mL of DMSO-d6. Yield 69 mg (0.153 mmol 68%).

3.7 C

OUPLINGOF

4-(4-(1-(

ACRYLOYLOXY

)

ETHYL

)-2-

METHOXY

-5-NITROPHENOXY

)

BUTANOICACID

-N-

HYDROXYSUCCINIMIDETO

PEG

DIAMINE

(MN =

6000)

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10 mL round bottom flask. The flask was purged with argon and 4 mL of anhydrous DCM was added to the flask. The reaction mixture was stirred overnight. The reaction mixture was precipitated in cooled diethyl ether. The precipitate was filtered of and dried in a vacuum oven. After drying, NMR analysis was performed on 6 mg of the product after dissolving in 0.6 mL of CDCl3. NMR: 6-8 mg in 0.6 mL of CDCl3. Yield 133 mg (0,02 mmol66%).

3.8 G

ELFORMATIONUSINGBIS

(4-(4-(1-(

ACRYLOYLOXY

)

ETHYL

)-2-

METHOXY

-5-NITROPHENOXY

)-

BUTANATE

)PEG (MN = 6000)

Gels were prepared between two glass coverslips covered with hydrophobic Parafilm. Two pieces of plastic (≈1 mm thick) were placed on the edges of one of the two cover slips. 50 L of gel mixture was placed on the slip with plastic spacers and another slip was placed over exactly matching the geometries of the cover slips. Two clamps were attached to hold coverslips firmly. For each gel formulation, 6 mg of bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) was added to an Eppendorf tube and dissolved in 51 µL of degassed water. 6 µL of 2M APS solution was added to the Eppendorf tube and the mixture was mixed by vortexing. 3 µL of TEMED was added to the Eppendorf tube and the mixture was mixed by quick vortexing. 50 µl of the obtained mixture was quickly transferred onto a coverslip. Other coverslip was placed on top of the first one. The coverslips were locked with two clamps placed over the two edges of the coverslips. After 30 minutes of setting, the gels were removed from the set up and stored in vials which were kept in a fridge.

3.9 E

NCAPSULATIONOF

SAMSA

FLUORESCEIN

For preparation of gels with covalently linked fluorescein, the same set up was used. Specifically, 2 mg of SAMSA fluorescein was dissolved in 200 µL of 0.1 M NaOH. After 15 minutes of reaction of deprotection, 3.4 µL of 5 M HCl was added to neutralize the mixture. Another 40 µL of prepared 0.5 M sodium phosphate buffer (pH 7) was added. Meanwhile, 6 mg of bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) was dissolved in 40 µL of degassed water. After PEG was dissolved, 11.4 µL of the prepared fluorescein solution was added. After 30 minutes of Michael addition reaction, 6 µL of APS and 3 µL of TEMED were added and the mixture was quickly vortexed. Shortly after addition of TEMED, 50 µL of the gel solution was quickly transferred to a coverslip. Setting time was 30 minutes, after which the gels were removed from the set up and stored in vials which were kept in a fridge.

3.10 S

TUDYOFDEGRADATIONPROCESSANDRELEASEOFTHEPHOTO

-

DEGRADED

COMPOUNDSFROMHYDROGELSWITH ANDWITHOUT

UV

EXPOSURE

For this experiment, 3 mL of 10 mmol PBS was added to a vial containing a 50 L gel sample made from bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000). This translucent vial was then placed under a UV lamp. After 30 seconds of exposure to UV light, the vial was removed and stored protected from light. The gel was incubated in PBS for another 9.5 minutes from the moment it was removed from the UV lamp. When a total of 10 minutes of incubation has passed, the PBS phase was transferred from the vial to a quartz cuvette. A UV-vis spectrum was recorded for wavelengths between 250 nm and 700 nm. After the measurement, the PBS phase was transferred back to the vial containing the gel sample for further UV light treatment. The procedure was repeated 10 times. This means that each gel sample had an incubation time of 100 minutes and was exposed to UV light for a total of 300 seconds. The absorbance reading was taken at a wavelength of 276 nm. Number of repeats – 3. The same experiment was performed for 3 gels without exposure to UV light. Fresh 10 mM of PBS was used as blank for UV-vis measurements.

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3.11 S

TUDYOFDEGRADATIONPROCESSANDRELEASEOFFLUORESCEINFROMTHEGELS WITHANDWITHOUT

UV

EXPOSURE

For this experiment, hydrogels were made from bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) and SAMSA fluorescein. First, the gels were washed with 3 mL of 10 mM PBS for one hour to remove any non-reacted SAMSA fluorescein or other components which were not bonded to the hydrogels. After washing, 3 mL of PBS was added to the vials containing the gel. The vial is placed under the UV lamp for 30 seconds. After exposure to UV light, the gel was incubated for another 9.5 minutes in the same PBS (3 ml). Thereafter the PBS was transferred to a quartz cuvette and UV-vis spectrum was recorded for wavelengths between 250 nm and 700 nm. After the measurement, the PBS phase was transferred back to the vial containing the gel sample for further UV light treatment. The procedure was repeated 10 times. The absorbance reading was taken at a wavelength of 493 nm. Number of repeats – 3. The same experiment was performed for 3 hydrogels without exposure to UV light. Fresh 10 mM of PBS was used as blank for UV-vis measurements.

3.12 S

TUDYOFDEGRADATIONPROCESSANDRELEASEOFFLUORESCEINFROMTHEGELS

WITHANDWITHOUT

UV

EXPOSUREINCUBATEDUNDERCONSTANTFLOW CONDITIONS

For this experiment, gels were made from bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) and SAMSA fluorescein. After washing the gels with 3 mL of 10 mM PBS for one hour, 3 mL of PBS was added to a vial containing a gel sample. The vial was placed under a UV light for 30 seconds and the gel was incubated another 19.5 minutes in the same PBS. After the incubation, the PBS was collected and fresh 3 ml PBS was added to the vial, thus creating ‘constant flow’ conditions. The procedure was repeated 10 times. A gel was incubated for a total of 200 minutes and was exposed to UV light for 300 seconds in total. Number of repeats – 3. The same experiment was performed for 3 hydrogels without exposure to UV light. The collected PBS samples were measured using a UV-spectrophotometer. The absorbance reading was taken at a wavelength of 493 nm.

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4 R

ESULTS

AND

DISCUSSION

The obtained results are described in this chapter. First the results of the performed syntheses and corresponding NMR spectra are shown. It is followed by presentation of the results on gel formation and finally the results obtained from the degradation studies are described.

4.1 NMR

SPECTRUMOF

4-(4-(1-(

ACRYLOYLOXY

)

ETHYL

)-2-

METHOXY

-5-NITROPHENOXY

)

BUTANOICACID

After the synthesis of 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid, a NMR spectrum had been recorded. This spectrum is shown in Figure 9. This synthesis is noted as step one in the synthesis walktrough scheme which is shown in figure 8.

Figure 9 NMR spectrum of 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid.

The molecular structure is included in the spectra and each proton has been marked with a letter. Signal in the spectra corresponding to particular protons is marked with the letter assigned to this type of protons. The integrals are shown besides the letter corresponding to each proton. Protons G and F attached to aromatic ring appeared around 7.5 ppm in the spectra because of pi bonds present in the aromatic ring. A, B and C protons are shown between 6 and 6.5 ppm in the spectrum and belong to the acryloyl group of the molecule. D is also shown in the same area. I and H protons are shown around 4 ppm; these two protons are positioned near an ether bond. The protons K, J and E are shown between 1.5 and 2 ppm.

After this synthesis, an acryloyl group should be attached to the photosensitive material. This material is marked with the letters a, b and c. The ratio of integrals between these protons and the proton marked with f should be 1:1. The observed average ratio is 1:0.96. This proves that 96% of the total starting material had been synthesized with the acryloyl group, with an average yield of 46%. This low yield was expected and did not differ much from the reported in the literature of 53% [1]. The peak marked with j showed a total integral of 5.16. This peak should contain an integral of 2 accroding to the protons present in the molecule. This difference is due to the present of acetone, which overlaps with the peak from J at 2.17ppm.

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4.2 NMR

SPECTRUMOF

BIS

(4-(4-(1-(

ACRYLOYLOXY

)

ETHYL

)-2-

METHOXY

-5-NITROPHENOXY

)-

BUTANATE

)PEG (MN = 6000)

After the synthesis of bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000), a NMR spectrum had been recorded. This spectrum is shown in Figure 10. This synthesis is noted as step two in the synthesis walktrough scheme which is shown in figure 8.

Figure 10 NMR spectrum of bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000).

Spectra of bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) contains the same peaks and integrals and corresponds to the spectrum shown in figure 9. The new peaks recorded in this spectrum, is the big peak that belongs to the protons from the PEG molecule. The protons related to this peak are marked with as M.

After the synthesis by using oxalyl chloride or DCC, the photo-labile groups should be connected to both terminals of the PEG macromolecules. This should give a ratio of 2 : 1 between the photo-labile group and the PEG polymer. The used PEG molecules has a Mn of 6000 mol/g. This molecular weight corresponds to 136 ethylene glycol repeat units per macromolecule. Each repeating unit of PEG contains four hydrogen atoms. This means that one PEG macromolecule should contain 544 protons versus the protons from two photo-labile groups, giving an integral ratio of 2 : 544 .

After step 2.1 in the synthesis walkthrough using oxalyl chloride, a ratio of 2:740 had been observed in the spectrum. This means that only 74% of the terminals of PEG macromolecules have been linked to the photo-labile group. This conversion of the terminals is higher than 50%, which is the approximately minimum required for hydrogel formation [1,2] The synthesis had a yield of 22%. A lot of the product is lost during column chromatography purification. In other trials using this synthesis, amount of linked photo-labile groups to PEG macromolecules terminals were always below 50% (between 20 and 40%). Therefore, new synthesis routs were introduced to synthesize this compound.

The new synthesis route is marked as step 2.2 in the synthesis walkthrough. DCC has been used as new condensing agent in this synthesis to improve the conversion PEG terminals with DMAP as catalyst [15,16]. After this synthesis, a NMR spectrum has been recorded to observe the ratio.

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desired 50%. Therefore no hydrogel can be formed out of this material theoretically. This result could be due to low concentration of reagents when the reaction had been performed. Thus, the applied concentration for the PEG was at 0.365 mM while the suggested concentration should be 6.6 mM [17]. On the other hand, the yield of this synthesis had been improved to 82%, which was close to the expected yield in the literature (89%) [16, 17].

4.3 NMR

SPECTRUMOF

4-(4-(1-(

ACRYLOYLOXY

)

ETHYL

)-2-

METHOXY

-5-NITROPHENOXY

)

BUTANOICACID

-N-

HYDROXY

-

SUCCINIMIDEESTER

After the synthesis of 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid-N-hydroxy-succinimide ester, a NMR spectrum had been recorded. This spectrum is shown in Figure 11. This synthesis is noted as step three in the synthesis walktrough scheme which is shown in figure 8.

Figure 11 NMR spectrum of 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid-N-hydroxysuccinimide ester.

The spectrum of 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid-N-hydroxy succinimide ester should be similar to the spectrum of 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid. The only difference in the spectrum is that there should be an extra peak corresponding to N-hydroxysuccinimide. This peak should be shown in the spectra at 2.75-2.95 ppm with an integral of 4. However, the peak observed at 2.75-2.95 ppm contains an integral of 5.64. This is due to the overlap of the peak k with the peak belonging to N-hydroxysuccinimide. Peak k should contain an integral of 2. The total integral of the overlapped peaks should be 6. The observed integral 5.64 is close to the expected integral of 6. This concludes that all present carboxylic acids are modified to N-hydroxysuccinimide ester. An average yield of 68% was obtained, which is close to the reported one (69%) [3].

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4.4 NMR

SPECTRUMOF

BIS

(4-(4-(1-(

ACRYLOYLOXY

)

ETHYL

)-2-

METHOXY

-5-NITROPHENOXY

)-

BUTANATE

)PEG-

DIAMINE

(MN = 6000)

After the synthesis of bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG-diamine (Mn = 6000), a NMR spectrum had been recorded. This spectrum is shown in Figure 12. This synthesis is noted as step four in the synthesis walktrough scheme which is shown in figure 8.

Figure 12 NMR spectrum of Bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG-diamine (Mn =

6000).

Besides the peaks referring to the photo-labile group and PEG macromolecules, a new peak was observed at 7.9 ppm. This peak belongs to the amide proton. This means that the photo-labile group is linked to the amine of the PEG diamine molecule via an amide bond. The observed ratio between protons of the photo-labile group and the PEG protons was 2:768. This means that at least 71% of PEG terminals were conjugated to the photo-labile group. This conversion was therefore good enough for use of the product in gel formation The obtained yield of this synthesis was 66%. These results showed that this synthetic pathway is the most suitable for preparation of photo-labile PEG precursor with highest yield observed.

4.5 G

ELFORMATION

It was able to form a hydrogel out of the polymers bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) and PEG diamine variant. Once TEMED had been added, a gel was formed within a minute. It was possible to quickly transfer the final gel solution to the made set up before a hydrogel had been formed. If it had been done not quickly enough, the gel could be formed already in Eppendorf tube or in a pipette tip. After 30 minutes of setting, the gel could be removed from the set-up and stored. The gel formed had a cylinder shape and a

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formation with fluorescein encapsulation, some yellow residue was left behind on the setup. This liquid may belong to unreacted and non-cross-linked SAMSA fluorescein After washing the gel and measuring the absorbance of SAMSA fluorescein, it was observed that only 28 % of the total amount of SAMSA fluorescein in the hydrogel was actually linked to the gel network. Despite of the low concentration of present SAMSA fluorescein, the remaining amount was still high enough to use for the release studies.

Figure 13 Picture of a photo-labile hydrogel.

4.6 D

EGRADATION STUDY BY MEASURING THE RELEASE OF THE PHOTO

-

DEGRADED

MOLECULES

The study of UV light-sensitive properties of photo-labile hydrogels was performed by measuring the amount of released photo-degraded material from a hydrogel. This study had been done on hydrogels which were exposed to UV-light for 30 seconds and on hydrogels which were kept in dark conditions. This measurement had been done ten times. A recorded spectrum from the 10th measurement is shown in figure 14.

Figure 14 Recorded UV-vis spectrum of the 10th measurement of the degradation study. The blue curve corresponds to the hydrogel which is exposed to UV light. The red curve corresponds to the hydrogel kept in dark conditions.

After the 10th_{measurements, all the observed absorbances form every measurement at a} wavelength of 276 nm was recorded. The green line in figure 14 is stationed at this wavelength . A graph was made from this data, comparing the obtained results from hydrogels kept in dark conditions and from hydrogels exposed to UV-light. This graph is shown in figure 15.

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0 2 4 6 8 10 12 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 f(x) = 0.05 x + 0.06 R² = 0.99 average ab-sorbance exposed gels Linear (average absorbance exposed gels) amount of measurements ab so rb an ce a t w v: 2 7 6 n m

Figure 15 Graph of the measured absorbance of photo-degraded compounds released from a group of gels which had been exposed to UV light (blue rhombs) and from a group of gels which as kept in the dark (red squares).

In this figure, the amount of photo-degraded compounds released from the gels can be observed from both curves. The absorbance from blue curve, representing the exposed hydrogels, increases linearly. The observed absorbance rose with an average increment of 0,054 each time the gel was exposed to the UV light for 30 seconds. A release of photo degraded molecules can be observed from the red curve, which represents the hydrogels kept in dark conditions. However, this release rises slowly eventually the level of the released PEG macromolecules reached equilibrium. The fact of observed release might be probably due to the release of some not cross-linked photo-labile PEG diacrylate macromolecules. This free material diffused out of the gel and was measured The observed equilibrium indicates that these hydrogels are stable in dark conditions..The high difference between these profiles indicates that photo-labile bonds were cleaved under UV light leading to release of some free photo sensitive PEG macromolecules [1].

4.7 D

EGRADATIONSTUDYBY MEASURINGTHERELEASEOFFLUORESCEINDERIVATIVES

Hydrogels containing linked SAMSA fluorescein were studied in a similar manner in order to observe the ability of these gels to release a model fluorescent molecule under light-controlled conditions. Before the experiment was performed, the gels were washed multiple times with fresh PBS to remove any unreacted SAMSA fluorescein and other materials. A recorded spectrum from the 10th_{measurement is shown in figure 16.}

Figure 16 Recorded UV-vis spectrum of the 10th measurement in study of released fluorescein. The green curve corresponds to the hydrogel which is exposed to UV light. The blue curve corresponds to the hydrogel kept in dark

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all the observed absorbances form every measurement were recorded at a wavelength of 493 nm. The green line in figure 16 is stationed at this wavelength. A graph was made from this data, comparing the obtained results from hydrogels kept in dark conditions and from hydrogels exposed to UV-light. This graph is shown in figure 17.

0 2 4 6 8 10 12 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 f(x) = 0 x + 0.03 R² = 0.89 f(x) = 0.01 x + 0.02

R² = 1 average absorbance with _{UV exposure} Linear (average ab-sorbance with UV ex-posure)

average absorbance without UV exposure Linear (average ab-sorbance without UV exposure) amount of measurements ab so rb an ce a t w v: 4 9 3 n m

Figure 17 Graph of the absorbance of fluorescein released from a group of gels which was exposed to UV light (blue curve) and from a group of gels which had been kept in dark conditions (red curve).

The observed absorbance of fluorescein released from hydrogels which were exposed to UV light and hydrogels which were kept in the dark increased linearly for the two groups of gels. It was expected that the release of fluorescein from hydrogels which were exposed to UV light would rise linearly. However, this was not expected to occur from hydrogels kept in dark conditions. The difference between the release rate from exposed hydrogels is 0,012 which is three times higher as 0,003 observed from the hydrogels kept in dark conditions. This proves that the UV light triggers the degradation, releasing the fluorescein from the gel.

It was unexpected that fluorescein-linked gels kept in dark conditions showed a linear release rate of fluorescein from the hydrogels with time. Examination of absorbence proves that only 2/3 of the total released amount had been released by the trigger of UV exposure, while the other 1/3 was released by some other mechanism. (it was assumed that after washing the hydrogels, only linked fluorescein was left in the gels). In other words, even without UV light some bonds linked fluorescein to the matrix are not stable. For further analysis, this experiment was done again under constant flow conditions.

4.8 D

EGRADATION STUDY BY MEASURING THE RELEASE OF FLUORESCEIN FROM GELS

INCUBATEDUNDERCONSTANTFLOWCONDITIONS

.

The difference from the above experiment was that fluorescein-linked gels were incubated always in fresh PBS which provided so-called flow conditions. In the previous set of experiments, PBS was placed back in the vial containing the gel sample after UV-vis measurement at each time point. In this experiment, new fresh PBS was added to the vial containing the gel sample and the old PBS phase had been stored separately for further UV-vis measurement. a recorded spectrum of the 10th_{measurement is shown in figure 18.}

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Figure 18 Recorded UV-vis spectrum of the 10th measurement in study of released fluorescein in constant flow conditions. The green curve corresponds to the hydrogel which is exposed to UV light. The blue curve corresponds to the hydrogel kept in dark conditions.

This spectrum shows the 10th measurement of the degradation study by measuring the amount of released fluorescein. Because of the constant flow conditions and no accumulated absorbances, a low absorbance is observed. Still at the 10th measurement some release can be observed form the hydrogels kept in dark conditions. The absorbances off all measurements have been recorded at wavelength 493 nm. This is stationed with a green line in figure 18. All collected data is plotted in graph shown in figure 19.

0 2 4 6 8 10 12 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 f(x) = 0 x + 0.02 R² = 0.96 f(x) = 0.01 x + 0.03 R² = 0.99 Average ab-sorbance with UV exposure Linear (Average absorbance with UV exposure) Average ab-sorbance with-out UV exposure amount of measurements ab so rb an ce a t w v: 4 9 3 n m

Figure 19 Graph of the amount of released SAMSA fluorescein from a group of gels which were exposed to UV light and from a group of gels which were kept in the dark. This was performed in constant flow conditions.

The absorbance of released fluorescein from the gels treated under different conditions increased linearly as it was in the previous experiment. The increasing rate of the absorbance was constant. It should be noted that it was the same 10 times of UV exposure as in the previous experiment. However, the incubation time was increased to 20 min per measurement. Overall, the amounts of released fluorescein was the same as in the previous experiment, i.e. ~0.15 and ~0.05 for the gels exposed to UV and the gels kept in dark conditions.

The fact that UV light exposure turned into a higher amount of the released fluorescein indicates that this release is still triggered by UV exposure and the hydrogels do show UV light sensitive properties [1]. However, the fact that without UV light the absorbance was still increasing constantly proved that there is some other release mechanism. This mechanism could be the

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5 C

ONCLUSION

It was possible to synthesize the required material for hydrogel formation. It had been done by first synthesizing bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000) with oxalyl chloride as a condensing agent. In the first trial, 74% of PEG hydroxyls were converted. However, the yield was only 22% and it was not possible to increase the conversion of PEG terminals in other trials. By implementing condensation of PEG-diamine (Mn = 6000) with 4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid-N-hydroxysuccinimide ester, a yield of 66% and a conversion of 71% were achieved.

The gels were successfully formed from the obtained modified photo sensitive PEG molecules by radical polymerization cross-linking reaction. After setting, nice round yellow cylindrical gels were obtained with an average thickness of 1 mm. It also was possible to form a gel containing SAMSA fluorescein.

The formed hydrogels exposed to UV light showed a linear degradation rate. This means that the hydrogel degrades when exposed to UV light and it is releasing the degraded products. However, the formed gels kept in the dark conditions also showed some release of non-crosslinked photo sensitive PEG molecules. It was observed that the release of photo sensitive molecules from hydrogels kept in dark conditions was much less than from UV exposed hydrogels and the release stopped after some time. This means that the formed gels are stable under dark conditions and the release can be triggered at any time by the gel exposure to UV light.

Encapsulation of thiol containing molecules through Michael addition to hydrogels containing acrylate groups was shown not optimal. This is due to hydrolysis of this bond, which makes it this bond not stable. Despite that the gels containing the SAMSA fluorescein also showed release under UV conditions, encapsulation of drug/molecules by thiol to acrylate is not very efficient due to the unstable bond. This proves that encapsulation of thiol containing molecules does not work properly as expected for controlled release conditions.

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6 R

ECOMMENDATION

’

S

Because of the low stability of ester bond located at 1,3-position to thioester group, alternative ways of encapsulation of drug molecules within hydrogels should be introduced. One possibility is to synthesize a drug molecule containing an acryl group. The encapsulation off the molecules then takes place while the hydrogel formation is initiated by radical polymerization.

the procedure for hydrogel formation relies on radical polymerization and therefore can be harmful for in situ cell encapsulation. Using other non-toxic mechanisms, for example Michael addition reaction for cross-linking can be introduced. For example crosslinking by so called “click chemistry” with hyaluronic acid derivatives.

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B

IBLIOGRAPHY

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[3] Griffin DR, Schlosser JL, Lam SF, Nguyen TH, Maynard HD, Kasko AM: Synthesis of photodegradable macromers for conjugation and release of bioactive molecules. Biomacromolecules, ACS publications. 2013; 14: 1199-1207

[4] Mosiewicz KA, Kolb L, Vlies van der AJ, Martino MM, Lienemann PS, Hubbell JA, Ehrbar M, Lutolf MP: In situ cell manipulation through enzymatic hydrogel photo patterning. Nature materials. 2013; 12: 1072-1078

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[6] Tabata Y, Yamada K, Miyamoto S et all: Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels. Biomaterials 1998;19:807-815. [7] Banerjee SS, Aher N, Patil R, Khandare J: Poly(ethylene glycol) – prodrug conjugates:

concept, design, and applications. Journal of drug delivery. 2012; 2012: 1-17

[8] Greenwald RB, Conover CD, Pendri A, Choe Y H, Martinez A, Wu D, Guan S, Yao Z, Shum K L: Drug delivery of anticancer agents: water soluble 4-poly (ethylene glycol) derivatives of the lignin, podophyllotoxin. Journal of controlled release 1999; 61: 281-294

[9] Kim MS, Diamond S:. Photocleavage of o-nitrobenzyl ether derivatives of rapid

biomedical release applications. Bioorganic & Medicinal Chemistry Letters. 2006; 16(15): 4007-4010

[10] Kolate A, Baradia D, Patil S, Vhora I, Kore G, Misra A: PEG – a versatile conjugating ligand for drug delivery systems. Journal of Controlled Release. 2014; 192: 67-81.

[11] Poly(ethylene glycol) and Poly(ethylene oxide) [internet] cited from 14-8-2014

http://www.sigmaaldrich.com/materials-science/material-science-products.html? TablePage=20204110

[12] Serban MA, Yang G, Prestwich GD: Synthesis, characterization and

chondroprotectiveproperties of a hyaluronanthioethylether derivative. Biomaterial. 2007;29(10): 1388-1399.

[13] SAMSA Fluorescein [package insert]. Leiden; Molecular Probes Europe BV; 2001.

[14] Hickmott PW: Reaction of αβ Unsaturated aid chlorides with alcohols in the presence of tertiary amines. Royal society of chemistry. 1964; 171: 883-887.

[15] Li Y, Tong R, Xia H, Zhang H, Xuan J: High intensity focused ultrasound and redox dual responsive polymer micelles. The royal society of chemistry, 2010

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[16] Nayak A, Jain A: In vitro and In vivo study of poly(ethylene glycol) conjugated ibuprofen to extend the duration of action. Scientia Pharmaceutica. 2011; 79: 359-373

[17] Lele B, Gore M, Kulkarni M: Direct esterification of poly (ethylene glycol) with amino acid hydrochlorides. Synthetic communications. 1999; 29(10): 1727-1739

[18] Hayworth D, Ph.D, Carbodiimide Crosslinker Chemistry [Internet] 2014 [Updated: unknown, Cited: 3-12-2014] available from:

http://www.piercenet.com/method/carbodiimide-crosslinker-chemistry

[19] Cline GW, Hanna SB: Kinetics and mechanics of the Aminolysis of N-hydroxysuccinimide ester in aqueous buffers. The journal of organic chemistry. 1988; 53: 3583-3586.

[20] Santiago M, Strobel S: Thin layer chromatography. Methods in enzymology. 2013; 533: 303-323.

[21] Reusch W. Nuclear magnetic resonance spectroscopy. [internet] 05/05/2013 [cited: 22-8-14] available from:

http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/nmr/nmr1.htm

[22] Gemmecker G. Basic principles of FT NMR. [internet]1999 [cited 22-8-14]available form:

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[23] Wong KC: Review of NMR spectroscopy: basic principles, concepts and applications in chemistry. Journal of chemical education 2014; 91: 1103-1104

[24] Clarck J. The Lambert-Beer law [internet] 2007 [cited: 9-12-2014] available from:

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A

PPENDIX

NMR

SPECTRA

’

S

Figure 20 NMR spectrum of bis(4-(4-(1-(acryloyloxy)ethyl)-2-methoxy-5-nitrophenoxy)-butanate)PEG (Mn = 6000)

obtained using DCC as a condensing agent.

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Figure 22 NMR spectrum of poly ethylene glycol (PEG) Mn =6000.

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R

ESULTSOBSERVEDRELEASEOFPHOTOLABILEGROUPS time incubation (min) A Gel 3.2 (27,2 mg) B Gel 4.1 (22,5mg) C gel 4.2 (25,2 mg) average exposed gels ST.DEV. 10 0,0601 0,085 0,087 0,077 0,015 20 0,151 0,168 0,17 0,163 0,010 30 0,22 0,232 0,249 0,234 0,015 40 0,279 0,287 0,302 0,289 0,012 50 0,334 0,337 0,364 0,345 0,017 60 0,39 0,384 0,416 0,397 0,017 70 0,444 0,428 0,467 0,446 0,02 80 0,494 0,466 0,519 0,493 0,027 90 0,544 0,506 0,551 0,534 0,024 100 0,583 0,543 0,598 0,575 0,028

Table 1: Measured absorbance of the photo labile group released from gels which were exposed to UV light, measured at Wv 276 nm. time incubation (min) Gel1.1 (27,4mg) Gel 2.1 (24,7mg) Gel3.1 (25,4mg) average none exposed gels ST.DEV. 10 0,083 0,06 0,058 0,067 0,014 20 0,114 0,099 0,12 0,111 0,010 30 0,129 0,14 0,14 0,136 0,006 40 0,14 0,132 0,157 0,143 0,013 50 0,147 0,144 0,172 0,154 0,015 60 0,156 0,169 0,179 0,168 0,012 70 0,164 0,161 0,185 0,17 0,013 80 0,17 0,173 0,171 0,171 0,002 90 0,172 0,172 0,175 0,173 0,002 100 0,174 0,175 0,181 0,177 0,004

Table 2: Measured absorbance of the photo labile group released from gels which were kept in the dark, measured at Wv 276 nm.

R

ESULTSOBSERVEDRELEASEOF

SAMSA

FLUORESCEIN

Time incubation (min)

G4 absorbance G5 Absorbance G6 absorbance average

exposed gels STDEV 10 0,037 0,023 0,023 0,028 0,008 20 0,052 0,042 0,037 0,044 0,008 30 0,074 0,055 0,05 0,06 0,013 40 0,088 0,064 0,061 0,071 0,015 50 0,1 0,075 0,072 0,082 0,015 60 0,117 0,083 0,085 0,095 0,019 70 0,132 0,089 0,097 0,106 0,023 80 0,143 0,097 0,108 0,116 0,024 90 0,158 0,106 0,118 0,127 0,027 100 0,176 0,124 0,13 0,143 0,028

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Table 3: Measured absorbance of SAMSA fluorescein released from gels which were exposed to UV light, measured at WV 493 nm.

G1 absorbance G2 Absorbance G3 absorbance average non

exposed gels STDEV 10 0,021 0,022 0,022 0,022 0,0006 20 0,03 0,034 0,034 0,033 0,0023 30 0,034 0,038 0,044 0,039 0,0050 40 0,038 0,042 0,042 0,041 0,0023 50 0,043 0,05 0,044 0,046 0,0038 60 0,044 0,046 0,05 0,047 0,0031 70 0,045 0,048 0,048 0,047 0,0017 80 0,048 0,05 0,05 0,049 0,0012 90 0,052 0,05 0,054 0,052 0,002 100 0,056 0,058 0,054 0,056 0,002

Table 4: Measured absorbance of SAMSA fluorescein released from gels which were kept in the dark, measured at WV 493 nm.

gel 8

absorbance gel 11 absorbance gel 12 absorbance average exposed gels

STDEV 20 0,0323 0,0356 0,0298 0,033 0,0029 40 0,0221 0,0276 0,0204 0,023 0,0038 60 0,0131 0,0143 0,0126 0,013 0,0009 80 0,0111 0,0116 0,0108 0,011 0,0004 100 0,0104 0,0113 0,0103 0,011 0,0006 120 0,0116 0,0118 0,0101 0,011 0,0009 140 0,0105 0,0115 0,0103 0,011 0,0006 160 0,0102 0,0114 0,01 0,011 0,0008 180 0,0116 0,0116 0,0104 0,011 0,0007 200 0,0125 0,0115 0,0103 0,011 0,0011

Table 5: Measured absorbance of SAMSA fluorescein released from gels which were exposed to UV light, measured at WV 493 nm in constant flow conditions.

gel 7 absorbance gel 9

absorbance gel 10 absorbanc

e average non exposed gels STDEV 20 0,022 0,025 0,02 0,022 0,0025 40 0,007 0,0071 0,0061 0,0066 0,0005 60 0,0041 0,0045 0,0038 0,0041 0,0004 80 0,0029 0,0031 0,0028 0,0029 0,0002 100 0,0025 0,0026 0,0023 0,0025 0,0002 120 0,0031 0,0025 0,0021 0,0026 0,0005

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180 0,0016 0,0019 0,0025 0,002 0,0005

200 0,0019 0,002 0,0023 0,0021 0,0002

Table 6: Measured absorbance of SAMSA fluorescein released from gels which were kept in the dark, measured at WV 493 nm in constant flow conditions.

UV-

VISSPECTRA

Figure 24 Degradation study by measuring the amount of released photo-sensitive degraded molecules from UV exposed hydrogels gel 1.

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Figure 27 Degradation study by measuring the amount of released photo-sensitive degraded molecules from hydrogels kept in dark conditions gel 4.

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Figure 30 Degradation study by measuring the amount of released fluorescein from hydrogels from hydrogels exposed to UV light. gel 1.

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Figure 33 degradation study by measuring the amount of released fluorescein from hydrogels from hydrogels kept in dark conditions. gel 4.

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Development of photo-responsive hydrogels for controlled release of encapsulated molecules

Thesis

DEVELOPMENT OF PHOTO-RESPONSIVE HYDROGELS

FOR CONTROLLED RELEASE OF ENCAPSULATED MOLECULES

Final Report

Johan

Verhoeven

ATGM

Avans University of applied science

‘s Hertogenbosch

Graduation Project

Development of photo-responsive hydrogels for

controlled release of encapsulated molecules

A

BSTRACT

T

ABLE

OF

CONTENTS

1 I

NTRODUCTION

2 T

HEORETICAL

B

ACKGROUND

2.1 H

2.2 P

2.3 H

2.4 E

2.5 D

2.6 S

PEG

2.7 P

2.7.1

-

2.7.2

2.7.3 P

2.8 C

2.8.1 T

2.8.2 NMR

2.9 UV-

3 E

XPERIMENTAL

PART

3.1 M

3.2 D

3.3 S

4-(4-(1-(

)

)-2-

)

3.4 S

(4-(4-(1-(

)

)-2-

)-

)PEG (MN = 6000)

3.5 S

(4-(4-(1-(

)

)-2-

)-

)PEG (MN = 6000)

DCC

3.6 S

4-(4-(1-(

)

)-2-

)

-N-H

3.7 C

4-(4-(1-(

)

)-2-

)

-N-

PEG

(MN =

6000)

3.8 G