Preparation of hollow nano-capsules via RAFT polymerization by vesicle-template method

Department of Chemical Engineering and Chemistry

Laboratory of Polymer Materials

Den Dolech 2, 5612 AZ Eindhoven P.O. Box 513, 5600 MB Eindhoven The Netherlands

www.tue.nl

Date June 13, 2013

Preparation of hollow nano-capsules

via RAFT polymerization by

vesicle-template method

Supervisor TU/e: MSc. M.A. Moradi

Abstract

This report is about the synthesis of polymer nanocapsules, to be used in targeted drug delivery. The ultimate goal is to synthesize temperature responsive polymer nanocapsules which can be administrated intravenous by human tissue and thus have to be smaller than 200 nm. To synthesize these nanocapsules a simple RAFT-based vesicle templating approach is used. In the first step the vesicles are prepared by extruding a dioctadecyldimethylammonium bromide (DODAB) solution through filters with 100 nm or 200 nm pores. During the polymerization the premade RAFT-oligomer was attached to the vesicles after which the monomers were added. The gained capsules were analyzed by dynamic light scattering (DLS) to measure the size, by gel permeation chromatography (GPC) to measure the molecular weight, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to give information on shape and morphology of the capsules.

The parameters varied to optimize the results was the ratio of the monomers methyl methacrylate (MMA), tert-butyl acrylate (t-BA) and ethylene glycol dimethyl acrylate (EGDMA) and the ratio of the monomers

N-isopropylacrylamide (NIPAm), N, N-methylenebisacrylamide (MBA) and

acrylic acid (AA). Both vesicles gained with the 200 nm filters and the 100 nm filters were used during this research. The average diameter found with DLS was 190 nm for the capsules made with the vesicles that were extruded through the 200 nm filters. An average of 133 nm was found for the capsules that were made with vesicles that were extruded through the 100 nm filters. The synthesized polymer capsules are reproducible. The capsules made with NIPAm, MBA and AA show size change due to temperature differences. This means that temperature responsive capsules can be made. Unfortunately the size change caused by the temperature difference between healthy and diseased human tissue is only 0.3 nm, which not enough for targeted drug delivery. In future research different copolymers could be tested to change the large peak to the biologically critical temperature of diseased tissue. At last a spherical shape of the capsules was found with TEM analysis as expected.

List of abbreviations

AIBN Azobisisobutyronitrile BA Butyl acrylate

DLS Dynamic Light Scattering

DODAB Dioctadecyldimethylammonium bromide EGDMA Ethylene glycol dimethyl acrylate GPC Gel permeation chromatography NaOH Sodium hydroxide

NIPAm N-isopropylacrylamide

NMR Nuclear Magnetic Resonance MBA N, N-methylenebisacrylamide

MMA Methyl methacrylate

RAFT Reversible Addition-Fragmentation chain-Transfer t-BA tert-Butyl Acrylate

SEM Scanning Electron Microscope TEM Transmission Electron Microscope TGA Thermo Gravimetric Analysis THF Tetrahydrofuran

Table of contents

3 Materials and methods 12

3.1 Materials 12 3.2 Methods 12 3.2.1 Vesicle preparation 12 3.2.2 Preparation RAFT-oligomer 12 3.2.3 Synthesis nanocapsules 13 4 Results 15 4.1 DLS 15 4.2 GPC 19 4.3 SEM 20 4.4 TEM 22 5 Discussion 24 5.1 Preparation 24 5.2 Size 24 5.3 Shape 25 5.4 Temperature responsiveness 25 6 Conclusions 26 7 Recommendations 27 8 Literature 28 Appendices

1

Introduction

In current drug delivery systems medicine is distributed throughout the body through blood circulation. Due to this system only a small portion of the medication reaches the organ to be affected. Because of this the medicine must be administered in high doses, which makes the chance of overdoses and side effects larger. This can be avoided by using targeted drug delivery systems. With these relatively new techniques the medication can be concentrated in the targeted tissues, while reducing the relative concentration of the medication in the remaining tissues. A targeted delivery system improves the efficacy of the medication while reducing the side effects. Examples of targeted drug delivery vehicles are polymeric micelles, liposomes, lipoprotein-based drug carriers, nano-particle drug carriers and dendrimers.

The targeted delivery system discussed in this report is based on the encapsulation of the drug in a hollow nanoparticle, specifically by the layer-by-layer method. In this method a vesicle template is prepared on which a negatively charged RAFT (Reversible Addition-Fragmentation chain Transfer) oligomer is attached, and finally, a RAFT-polymerization is used to make a more sustainable shell around the vesicle.

Nanoparticles are only a good option for targeted drug delivery if they are temperature or pH responsive. This is achieved by adding temperature or pH responsive materials to the shell. Since in previous reports the temperature and pH responsiveness wasn’t as good as hoped for, other monomers will be tested during this research to achieve this goal.

Another requirement for intravenous administration is a mean particle diameter of <200 nm and a narrow size distribution, to avoid the risk of embolism [1]. These factors will also be analyzed in this report.

The research described in this report is based on the findings of Ali, S. [2,3] and de Roos, R. [4]. Ali has reported the synthesis of stable nanoparticles produced by the layer-by-layer method and Roos tried to verify and optimize the same method.

In this research the preparation method of Ali [3] is repeated and monitored carefully. Based on the research of Ali and Roos the influence of the amount of monomer, the amount of cross linker in the monomer solution and the type of monomer is tested. The monomers used are different mixtures of MMA:t-BA:EGDMA or NIPAm:MBA:AA. Both vesicles gained with extrusion through 100 nm filters and vesicles gained with extrusion through 200 nm filters are used during these tests. During this research it is tried to produce temperature or pH responsive hollow nanocapsules, which can be filled with medicine for targeted drug delivery.

Since size is an important parameter for drug delivery systems the size of the prepared capsules is analyzed with two different Dynamic Light Scattering (DLS) devices [1]. The particles are also analyzed with Gel Permeation Chromatography (GPC) to measure the molecular weight of the polymer chains, Scanning Electron Microscopy (SEM) to analyze the morphology of the nanocapsules and Transmission Electron Microscopy (TEM) to analyze the shape of the nanocapsules.

In this report the theoretical background information is described in chapter 2. In chapter 3 the materials and methods used are described. The results are described in chapter 4. The discussion of the results is described in chapter 5. In chapter 6 the conclusions are given. The recommendations are given in chapter 7. The used literature is listed in chapter 8.

2

Theoretical background

2.1

Nanocapsules

Nanocapsules are expected to be good drug carriers, which is why the synthesis and the properties of nanocapsules is a much researched topic. Nanoparticles (a collective name for both nanospheres and nanocapsules) are defined as solid, submicron-sized drug carriers that may or may not be biodegradable [5]. The advantage of nanoparticles over microparticles and liposomes is their relatively higher intercellular uptake because of their submicron size [5,6].

Many methods have been developed for preparing nanoparticles such as nanoprecipitation method, emulsion-diffusion method, double emulsification method, polymer-coating method and layer-by-layer method [5,7]. The latter is used during the research described in this report.

The synthesis of the nanocapsules, with the use of the layer-by-layer method, can be described in three steps. At first the vesicles will be made through extrusion. These vesicles are used as templates for the nanocapsules. Second a RAFT-copolymer is prepared. This RAFT-copolymer is attracted by the vesicle and will surround the interface of the vesicle. At last the encapsulation is done by mixing the vesicles with the copolymer and feeding monomers to it with an initiator for the RAFT-polymerization [3,4,8]. Due to the RAFT-mechanism the polymeric shell can grow layer-by-layer. In figure 1 a schematic view of the synthesis is given.

Figure 1: Schematic view of the nanocapsule synthesis.

In this research the synthesized nanocapsules will be empty. If the capsules have the right properties, they can be used for targeted drug delivery in the future. The capsules must be biocompatible, biodegradable and have a temperature or pH responsive structure to only release their contents under certain circumstances [2,5].

2.1.1

RAFT polymerization

To create a shell for the nano-capsules the reversible addition-fragmentation chain-transfer (RAFT) polymerization method is used. This method is a so-called ‘living’ polymerization. A normal polymerization stops after a termination step, but a ‘living’ polymer can continuously increase its length by adding more monomers.

During this research the used monomers, for creating the RAFT copolymer, are butyl acrylate (BA) and acrylic acid

(AA), see figure 2. The RAFT agent used is dibenzyltrithiocarbonate (DBTTC), as initiator 4,4’-azobis(4-cyanovaleric acid) (V-501) is used.

The RAFT polymerization has 5 stadia, the decomposition of the initiator, the initiation, the propagation, the RAFT pre-equilibrium and the RAFT equilibrium. These stadia are explained in the next few subparagraphs with the above stated chemicals in the reaction equations.

2.1.1.1 Decomposition of the initiator

Like every polymerization, main process starts with the decomposition of the initiator. The initiator, V-501, dissociates in the solvent phase, during which nitrogen gas is formed and leaves the vessel. The dissociation step gives two molecules of 4-cyanovaleric acid, with a tertiary radical on the 4th carbon atom. A schematic view is given in figure 3.

Figure 3: Decomposition of the initiator V-501

2.1.1.2 Initiation

During the initiation the radical initiates the monomers, as the radical moves through the monomer a new tertiary radical is formed and the polymerization can continue, this mechanism is shown in figure 5 [4]. During the next few steps symbols are used instead of the complete molecule structure, an overview of these symbols is given in figure 4.

2.1.1.3 Propagation

The radical initiates another monomer, this step takes place until all the monomers are gone. In the end long polymer chains are synthesized, depending on the added amount of monomer. The propagation is limited in speed by the RAFT agent, the chain grows slower which makes the reaction more controllable than a free radical polymerization. A random copolymer will be formed [4]. The propagation is shown in figure 6.

Figure 6: Schematic view of the propagation step

2.1.1.4 RAFT pre-equilibrium

A pre-equilibrium is formed when the radical of the propagating chain reacts with the RAFT agent’s double bonded sulfur atom. This forms a complex in which the thiocarbonate atom bears a tertiary radical. Then the radical shifts through the molecule to a carbon atom next to an adjacent sulfur atom [4]. In figure 7 the RAFT pre-equilibrium is shown.

Figure 7: The RAFT pre-equilibrium

The radical re-initiates another monomer and starts to form a polymer chain. This propagating polymer chain forms a second equilibrium with the RAFT agent, which is called the main RAFT equilibrium, this is shown in figure 8. The main RAFT equilibrium is explained in the next subparagraph [4].

Figure 8: re-initiation of the monomer

2.1.1.5 Main RAFT equilibrium

When the re-initiated chain reacts with the RAFT agent, the main RAFT equilibrium takes place, see figure 9. The radical reacts with the double bonded sulfur atom and a tertiary radical is formed on the carbon atom of the thiocarbonate. An adjacent sulfur group will eliminate the group bounded to it. This can be the toluene group (1), the polymer chain of the first propagation step (2) or the polymer chain that has just formed (3) [4].

The molecules A, B and C can (re)start propagation, this way the polymerization continues until the monomer supply runs empty, it is even possible to refill the monomer supply to restart propagation. Although the polymers are referred to as ‘living’ polymers, termination is still possible but is less likely to occur. By increasing the relative concentration of RAFT agent to the quantity of free radicals delivered during the polymerization, the selectivity towards the desired product of ‘living’ polymers is increased. This can be done by either increasing the amount of RAFT agent or by decreasing the amount of initiator. Termination will occur when two radical chains meet up, other termination possibilities are chain combination and disproportionation [4].

Figure 9: Schematic view of the main RAFT equilibrium

2.1.2

Vesicle templating

The vesicles are prepared by extruding dimethyldioctadecyl ammonium bromide (DODAB, fig. 10), which bear a cationic charge, due to the ammonium ion within the DODAB molecules. Therefore the vesicles will attract anions of molecules with a negative dipole charge. With this in mind an oligomer, with RAFT-properties, is selected to be used for templating the vesicles. In this research the copolymer acrylic acid-co-butyl acrylate (AAx-co-BAy) is used. Due to the electron rich ester and acid groups the copolymer is attracted to the vesicle and will cover the vesicle. Once the vesicles are covered the monomers and the initiator can be added to the solution so the RAFT-polymerization can start. By doing so a polymer shell will be formed around the vesicles. If the shell isn’t thick enough more monomer can be added, since the RAFT method makes it possible to polymerize by a layer-by-layer approach [2, 4].

2.2

Characterization

2.2.1

DLS

To measure the diameter of the capsules Dynamic Light Scattering (DLS) is used. DLS is also known as Photon Correlation Spectroscopy. For the measurement of the capsules a light beam is used. If the light touches a capsule the light will scatter in every direction. The scatter can be measured and the size of the capsules can be determined [9]. A schematic view is given in figure 11.

2.2.2

GPC

Gel permeation chromatography (GPC) is a type of size exclusion chromatography (SEC), that separates particles on the basis of size. This technique is commonly used to analyze polymers.

GPC separates based on the size or hydrodynamic volume of the particles. Separation occurs via the use of porous beads packed in a column. Small particles can enter the pores easily and therefore spend more time in the column, bigger particles spend little time in the pores and are eluted quickly.

This mechanism is shown in figure 12, the bigger red particles don’t fit in the pores and elute faster than the blue particles who spend a lot of time in the pores and thus elute very slow.

Column have a range of molecular weights that can be separated. To make sure the retention times don’t overlap a good column has to be selected [10].

2.2.3

SEM

A communally used microscope to see particles in the nanometer range is the Scanning Electron Microscopy (SEM). A SEM is a type of electron microscope that produces images of a sample by scanning its surface with a focused beam of electrons. Various signals are produced when the electrons interact with atoms in the sample. These signals can be detected and contain information about the sample’s surface topography and composition. The electron beam scans in a raster scan pattern, the software combines the beam’s positions and the detected signal to produce an image.

Figure 13 shows the schematic parts of a SEM. In this study SEM will be applied to analyze the morphology of the nanocapsules and to measure their size.

Figure 11: DLS principle

Figure 12: GPC principle

2.2.4

(Cryo-)TEM

Transmission electron microscopy (TEM) is a microscopy technique based on the transmission of electrons through a ultra-thin specimen. The beam of electrons interact with the specimen as it passes through. This interaction produces an image, the image is magnified and focused onto an imaging device or it can be detected by a sensor. Respectively a fluorescent screen on a layer of photographic film or a CCD Camera [11].

A significantly higher resolution of the imaging can be obtained by using a TEM instead of a normal light microscope. This enables the examination of fine details, even as small as a single column of atoms. TEM can be used to observe modulations in chemical identity, crystal orientation, electronic structure and sample induced electron phase shift as well as a regular absorption based imaging [11]. A schematic view of a basic TEM is given in picture 14.

Cryo-microscopy is a form of TEM where the samples are studied at cryogenic temperatures (generally liquid nitrogen temperatures). This technique is used to observe the specimens in their native environment, the specimen have not been stained of fixed in any way [12].

3

Materials and methods

3.1

Materials

The following chemicals were used during this research. Dimethyldioctadecyl ammonium bromide (DODAB) 99+% nitrogen flushed was purchased at Acros Organics (USA) , Super-Q water which was purified at the TU/e, sodium hydroxide (NaOH) pellets pure was purchased at Merck KGaA (Germany), all monomers where purchased from Sigma-Aldrich (Germany) methyl methacrylate (MMA) 99% containing ≤ 30 ppm MEHQ inhibitor, tert-Butyl acrylate (t-BA) 98% containing 10-20 ppm MEHQ inhibitor, Ethylene glycol dimethyl acrylate (EGDMA) 98%, 4,4’-azobis 4-cyanovaleric acid (V-501) ≥75% and acrylic acid (AA) anhydrous 99%, contains 180-200 ppm MEHQ inhibitor, isopropylacrylamide (NIPAm) 99%, pure stabilized was purchased at Acros Organics (Belgium), N, N-methylenebisacrylamide (MBA) ≥ 99,5% was purchased at Fluka BioChemika and azobisisobutyronitrile (AIBN) recrystallized by M.A Moradi.

Both the 100 nm and the 200 nm isopore™ Membrane filters are purchased at Millipore (Ireland) Apparatus used during this research were the Microtrac Nanotrac™ Ultra and the Malvern Zetasizer Nano ZS instrument to perform DLS, auto titration of the monomer is done with 665 Dosimat autotitrator from Metrohn swiss made, the used GPC is Waters 2695 Separation Module, SEM pictures are taken with XL30 ESEM-FEG, Fei Co. and TEM pictures are produced on a FEI Tecnai 20, type Sphera.

3.2

Methods

In the next paragraphs the methods for the preparation of the nanocapsules are given. If methods are different from the standard preparations this is also noted and discussed why the deviation is made.

3.2.1

Vesicle preparation

For the vesicle preparation a 10mM DODAB solution was made. Therefore 0,63095 g (1 mmol) DODAB was solved in 100 ml Super Q water. This solution was stirred during a weekend at 200-300 rpm and 70ºC. In table 1 the amounts of DODAB that was solved for the extrusions are given.

For the membrane extrusion the solution was poured into the extruder. The extruder contained a pressure vessel with a nitrogen line and a filter holder containing three stacked polycarbonate filters of appropriate pore size. The whole assembly was temperature controlled at 65ºC ± 10ºC. The extrusion was accomplished by pressurizing the vessel at 5-6 bar using nitrogen. The extrusion was repeated five times filter 2 or first using filter 2 three times and then using filter 1 three times, where filter 2 has 200 nm pores and filter 1 has 100 nm pores. Thereafter the solution was kept stirred at 200-300 rpm at 70ºC during the night, after which the solution was cooled down to room temperature.

Table 1 Used DODAB solutions for vesicle preparations

Name DODAB in gram SuperQ in ml Molarity

S7 0,6304 100 9,99 mM S8 1,5778 250 10,00 mM S9 1,5768 250 10,00 mM S10 0,6529 100 10,35 mM S11 1,5764 250 9,99 mM

3.2.2

Preparation RAFT-oligomer

A premade RAFT-oligomer solution, by R. de Roos, BA6-co-AA9 could be used during the whole extend of this project. To use the solution during the synthesis of nanocapsules the RAFT-oligomer solution was diluted to a 10 mmol mixture. Therefore 1,7150 g RAFT-solution was dissolved in 100 ml SuperQ water, by adding a 1 M NaOH solution the pH was brought to 12. This is needed to dissolve the RAFT-oligomer solution.

3.2.3

Synthesis nanocapsules

To synthesize the nanocapsules a standard reaction scheme is used based on the researches of Ali, S and Roos, R. The reaction setup contained a 50 ml three-necked flask with a magnetic stirrer, a reflux cooler and an Argon inlet. The reaction was heated in an oil bath.

In the next paragraphs the standard reaction scheme is given and in the table an overview of the used monomer solutions is given.

3.2.3.1 Synthesis MMA:t-BA:EGDMA capsules

At first 5 ml of the 10 mmol RAFT-solution was added to the reaction flask. After which 7,5 ml SuperQ water was added. Then 15 ml of DODAB solution was added drop wise, while the solution was continued stirred at 300 rpm. Thereafter 0,007 g of the initiator V-501 was added. Then the solution was flushed with argon for 30 min, after which the solution was heated to 70ºC. At the point of the solution reaching a temperature of 70ºC 0,2 ml of a monomer mixture was added at a speed of 0,01 ml/min using a dosimat autotitrator. The solution was kept stirred and heated for 2 hours after the monomer mixture was completely added. After two hours the solution was slowly cooled down to room temperature. The solution was removed from the flask into a small vessel and a magnetic stirrer was added, so the solution was kept stirred until the analysis where performed. This was done to prevent the capsules from agglomeration. The different monomer mixtures are listed in table 2.

Table 2 Monomer mixtures used during research

Name sample DODAB vesicles solution Monomers in w/w MMA:t-BA:EGDMA V-501 in mg S7C S7 filter 2** 1:2:0,03 7,5 S8C1 S8 filter 2** 1:2:0 7,9 S8C2 1:2:0,03 7,4 S8C3 1:2:0,1 7,6 S8C4 1:2 (gradually added) :0,03 6,9 S8C5 1(gradually added):2:0,03 7,4 S8C6 S8 filter 1* 1:2:0 7,5 S8C7 1:2:0,03 7,2 S9C8 S9 filter 1* 1:2:0,1 7,4 S9C9 1:2 (gradually added) :0,03 7,4 S9C10 1(gradually added):2:0,03 7,2 S10C1 S10 filter 2** 1:2:0 7,4 S10C2 1:2:0,03 7,4 S10C3 1:2:0,1 0

*Vesicles solution that were 3x extruded through 200 nm filters and 3x through 100 nm filters **Vesicles solution that were 5x extruded through 200 nm filters

3.2.3.2 Synthesis NIPAm:MBA:AA capsules

The basics for these synthesis is the same as for the synthesis for the MMA:t-BA:EGDMA capsules. First 5 ml of the 10 mmol RAFT-solution was added to the reaction flask. After which 7,5 ml SuperQ water was added. Then 15 ml of DODAB solution was added drop wise, while the solution was continued stirred at 300 rpm. Thereafter 0,007 g of the initiator V-501 or 0,03 g of AIBN was added. Then the solution was flushed with argon for 30 min, after which the solution was heated to 70ºC. At the point of the solution reaching a temperature of 70ºC 0,2 ml of a monomer mixture was added using a syringe. The solution was kept stirred and heated for 2 hours after the monomer mixture was completely added. Another batch of 0,2 ml monomer mixture was added to some samples to see if the concentration for de encapsulation was right. After the reactions the solution was slowly cooled down to room temperature. The solution was removed from the flask into a small vessel and a magnetic stirrer was added, so the solution was kept stirred until the analysis where performed. This was done to prevent the capsules from agglomeration.

Table 3 Monomer mixtures used during research

Name sample

DODAB solution Monomers in w/w NIPAm:MBA:AA Initiator in g S11N1 S11 filter 2* 4:0,04:0 0,0072 V-501 S11N2 S11 filter 2* 4,0,04:2 0,0074 V-501 S11N3 S11 filter 2* 4:0,04:4 0,007 V-501 S11N4 S11 filter 2* 4:0,04:1 0,0303 AIBN S11N5 S11 filter 2* 4:0:0 0,0073 V-501

4

Results

In this chapter the results stated. The results are divided by the method used to provide them.

4.1

DLS

To measure the average size of the particles Dynamic Light Scattering was used. In table 4 the PDI measured with DLS and the average sizes are given. The second average size shows that the particles agglomerate, most of the time the second average size is an agglomeration of 3 to 10 capsules. In some cases even bigger agglomerations were found, which aren’t stated in this report.

Table 4 DLS PDI and average diameters of vesicles and particles.

Sample PDI (DLS) Avg. diameter 1 Avg. diameter 2 S7 vesicles 0.327 198.4 nm S7C 0.267 172.3 nm S8C1 vesicles 0.683 249.1 nm S8C1 0.446 173.0 nm 419.0 nm S8C2 vesicles 1.221 242.4 nm S8C2 0.599 204.6 nm S8C3 vesicles 0.417 182.1 nm 2006 nm S8C3 1.340 123.4 nm 359.0 nm S8C4 vesicles 0.641 201.3 nm 1357 nm S8C4 0.479 154.5 nm 935.0 nm S8C5 vesicles 0.641 201.3 nm 1357 nm S8C5 0.423 167.0 nm 1756 nm S8C6 vesicles 0.765 223.3 nm 1138 nm S8C6 2.602 170.8 nm 1375 nm S8C7 vesicles 0.242 146.0 nm S8C7 1.889 121.5 nm 579.0 nm S9C8 vesicles 0.610 292.0 nm 1780 nm S9C8 0.999 121.4 nm 416.0 nm S9C9 vesicles 0.331 242.7 nm S9C9 2.727 125.4 nm 414.0 nm S9C10 vesicles 1.073 329.0 nm 1956 nm S9C10 0.538 128.7 nm 364.0 nm

In figure 15 an overview is given of this DLS measurements, only the first average size is given. The vesicles are always bigger than the particles after polymerization. The PDI from the vesicles gained by extruding it with filter 2 and capsules made with these vesicles is around 0,3-1,2, while the scattering of the vesicles gained using filter 1 and capsules made with these vesicles is bigger, between 0,5-2,6, this means that the size distribution is also bigger. The diameter of the vesicles gained using filter 1 that were measures are sometimes very large (300 nm) this is due to the agglomeration.

Figure 15 Average size (left axis) and PDI (right axis) measured with NANOTRAC DLS

Some of the samples were also measured with the DLS Malvern. The found results are given in table 5. The PDI measured with the Nanotrac are 2x or more higher the PDI’s measured with the Malvern device, this is also shown in figure 16. The average diameter is sometimes higher and sometimes lower than the measurement results found with the Nanotrac device. But the average of the capsules made with 200 nm DODAB vesicles have a smaller distribution than the capsules measured with the Nanotrac device.

Table 5 Measured with MALVERN device

Sample PDI (DLS) Z-Average S7vesicles 0,182 181,1 nm S7C 0,11 167,9 nm S8C1 0,266 238,66 nm S8C2 0,101 197,32 nm S8C3 0,256 208,7 nm S8C4 0,22 172,6 nm S8C7 0,296 244,1 nm S9C10 0,19 195,85 nm S10C1 0,129 181 nm S10C2 0,141 177,9 nm S10C3 0,167 182,1 nm 0 1 2 3 4 5 6 7 0 50 100 150 200 250 300 350 S7 ve si cl es S7C S8 C 1 ves ic le s S8 C 1 S8 C 2 ves ic le s S8 C 2 S8 C 3 ves ic le s S8 C 3 S8 C 4 ves ic le s S8 C 4 S8 C 5 ves ic le s S8 C 5 S8 C 6 ves ic le s S8 C 6 S8 C 7 ves ic le s S8 C 7 S9 C 8 ves ic le s S9 C 8 S9 C 9 ves ic le s S9 C 9 S9 C 1 0 ve si cl es S9 C 1 0 P D I (D LS ) D ia met er in n m

Average size of capsules with MMA:t-BA:EGDMA

Avg diameter 200nm Avg diameter 100nm Vesicles

Figure 16 Nanotrac DLS vs. Malvern DLS devices

The goal of this research is to synthesize temperature or pH responsive nanocapsules. Therefore the size of the NIPAm capsules was measured, with the MALVERN device at different temperatures. The data of this measurement is given in table 6 and shown in figures 17 and 18.

Table 6 Measured with MALVERN device

Sample PDI (DLS) Z-Average T in ºC S11N1t 1 0,133 167,7 20 S11N1t 2 0,107 168,8 25 S11N1t 3 0,128 167,6 30 S11N1t 4 0,139 168,2 35 S11N1t 5 0,141 170,4 40 S11N1t 6 0,103 169,8 45 S11N1t 7 0,114 172,3 50 S11N1t 8 0,103 170,3 55 S11N1t 9 0,089 172,9 60 S11N1t 10 0,116 171,3 65 Sample PDI (DLS) Z-Average S11N1t 11 0,092 174,4 70 S11N1t 12 0,104 172,2 75 S11N1t 13 0,122 172,1 80 S11N1t 14 0,11 172,3 85 S11N1t 15 0,089 172,8 90 S11N1t 16 0,085 171,2 95 S11N1t 17 0,064 171,4 100 S11N1t 18 0,08 169,9 105 S11N1t 19 0,053 164,1 110 S11N1t 20 0,096 166,1 115 S11N1t 21 0,061 165 120

The PDI measured are all below 0,14 and have an average of 0,10. As visible in figure 18 a 3th order polynomial trend line is found. The R squared value is 0,99. A R squared value of 1 would be optimal. The trend line shows an size optimum around 37 degrees, after which the size decreases fast. The size of the capsules at the temperature of healthy human tissue (37°C ±0,5) and diseased human tissue (40°C ±1,2) has a very small difference of 0,3 nm.

0 0,6 1,2 1,8 2,4 3 3,6 0 50 100 150 200 250 300 S7 vesicles S7C S8C1 S8C2 S8C3 S8C4 S8C7 S9C10 S10C1 S10C2 S10C3 P D I (D LS ) D ia m et er in n m

Nanotrac DLS vs Malvern DLS

Figure 17 PDI and Z-average measured with MALVERN DLS(average and standard deviation out of 3). Diameter is given on the left axis and PDI is given on the right axis.

Figure 18 Trend line in Z-average (3th order polynomial) when calculating the diameter using the equation use 1, 2, 3 etc. instead of 20, 25, 30 etc. 0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 0,16 0,18 0,2 125 130 135 140 145 150 155 160 165 170 175 20 25 30 35 40 45 50 P D I ( D LS) d ia m et er in n m Temperature in ºC

Temperature Behaviour NIPAm capsules

Diameter PDI y = -0,1361x3 _{+ 0,946x}2 _{- 0,2821x + 167,49} R² = 0,9905 158 160 162 164 166 168 170 172 174 176 20 25 30 35 40 45 50 D ia m et er in n m Temperature in ºC

Temperature behaviour of NIPAm capsules

Diameter Poly. (Diameter)

4.2

GPC

The GPC measurements weren’t usable since the baseline of the chromatograms have a lot of noise. As can be seen in figure 19. The intensity of the peak were also very low, which means that not enough material was dissolved for detection. The chromatograms are taken in a calibration of polystyrene, so the molecular weight found with GPC will be different from the actual molecular weight. Another reason why these measurement aren’t reliable is that the Mn found is in most cases around 1000. But the Mn measured of only the RAFT oligomer is already higher.

Table 7 PDI, Mn, Mw and Mz of synthesized capsules

Sample PDI Mn Mw Mz S7C 1.3 1040 1374 1838 S8C1 1.4 1145 1609 2276 S8C2 1.1 82633 88440 95673 S8C3 1.0 5897 5904 5911 S8C4 1.3 1089 1411 1839 S8C5 1.3 1019 1317 1720 Sample PDI Mn Mw Mz S8C6 1.4 1107 1525 2071 S8C7 1.3 1232 1636 2143 S9C8 1.3 1058 1366 1793 S9C9 1.3 1028 1323 1727 S9C10 1.2 857 1000 1179 S10C1 1.2 939 1135 1387 S10C2 1.3 957 1202 1561 S10C3 1.3 1025 1282 1638

4.3

SEM

In this paragraph the results from SEM are discussed. Some pictures can be found in this paragraph, all other pictures can be found in appendix I.

Capsules made with the S7 DODAB solution are shown in the pictures 20a t/m 20d. In those pictures it looks like the broken parts of the capsules are collapsed to the inside. The size of the particles in the pictures is approximately 2 µm. In picture 20a, 20b and 20d agglomeration can be seen. So probably agglomeration happens faster while drying.

The shape of the particles in the pictures is mostly irregular and sometimes spherical.

(a) scale bar 10 µm (b) scale bar 50 µm

(c) scale bar 1 µm (d) scale bar 5 µm

Figure 20 SEM pictures of sample S7C

In sample S8C2 some capsules could be found, but also some big polymer particles, see picture 21a and 21b. These big polymer parts might explain the high Mw value that was found with GPC. The particles in these SEM pictures are also irregular.

(a) scale bar 5 µm (b) scale bar 20 µm

Figure 21 SEM pictures of S8C2

The capsules made in S10C2 and S10C3 show capsules which aren’t broken. The capsule in picture 22c shows dark spot in the center this is probably caused due to the hollow center of the capsules. The capsules is picture 22c and d are more spherical, but still have some irregularities.

(a) scale bar 10 µm (b) scale bar 2 µm

(c) scale bar 5 µm (d) scale bar 2 µm

4.4

TEM

In this paragraph the results given by TEM are shown. Because this technique take a lot of time, only a few samples are measured.

In figure 23 the TEM pictures of S7C are shown, since this sample gave good results with the other measurement methods. Again spherical capsules are found, sometimes there are some irregularities and agglomeration found. Also sometimes a contrast in the capsules can be seen, those capsules have a darker spot on the inner circle of the capsule. The capsules in these pictures have an average diameter between 100-250 nm.

S7C S7C

The last sample that were viewed with TEM is S11N1, the first NIPAm based capsules. In figure 24a and 24b capsules can be seen, with a size of approximately 100 nm.

(a) (b)

5

Discussion

5.1

Preparation

During the vesicle extrusion it was very hard to extrude the DODAB solution through the 100 nm filters. With most of the extrusions a 50% loss of DODAB solution occurred during the process because the filters got constipated. This method is therefore not very viable. Also during the vesicle extrusion it sometimes took a long time for the vesicles to come through, because of this the solution in the tube to the collection vessel cooled down to approximately 50ºC, which wasn’t desirable. To overcome this problem it was tried to isolate the tube. To prevent agglomeration all samples and DODAB solution were kept stirred. Synthesizing many samples at the same time takes a lot of space in a fume hood and a lot of mechanical stirrers, so isn’t really recommended unless the hardware is readily available.

As the capsules were synthesized a magnetic stirrer was added to the solution. After the polymerization process, it was noticed that part of the RAFT-oligomer had attached itself to the stirrer, so not all of the RAFT agent was attached to the vesicles, so polymerization probably occurred in a different manner in accordance to the attaching of all the RAFT agent to the vesicles. In later experiments the stirrer was only added to the solution right before the DODAB was added, but it would probably be best to use an overhead stirrer.

During the synthesis of the capsules with gradually adding one of the monomers a lot of the solution was pulled out of the reaction to follow the diameter growth during the polymerization. Because of this the ratios of the added monomer aren’t in sync with the amount of vesicles and RAFT-oligomer. Therefore the analysis was sometimes hard and are likely to be incorrect.

5.2

Size

An advantage of the DLS technique is that measurements are fast, use low volume and don’t damage the sample. A disadvantage is that a DLS is not suitable for measuring the size of the particles in high concentrations and it can’t measure dry samples [15,1]. A second pitfall of a DLS is the large influence of dust particles or small amounts of large aggregates in addition to a main component of distinctly smaller size [1]. For the DLS the solution was poured into a vial and couldn’t be stirred, so the mean sizes can be influenced by agglomerations.

The size of the vesicles is bigger than the size after polymerization. This is partly due the shape of the vesicles, which are reported to be ellipsoidal with strong angularities, while the capsules have a spherical shape [16]. Due to the ellipsoidal shape the PDI of the vesicles is also higher. It is assumed that because of the formation of the polymer shell the particles become more dense, with smaller particles as a result.

The average size measured with the Nanotrac device of the vesicles which were prepared with the 100 nm filters was (around 230-340 nm), which is about 2 a 3 times higher than expected. This is probably because the vesicles are unstable and agglomerate very fast. The PDI’s measured for these particles are also very high, which means that there is a wide distribution in particle size. The capsules made with these vesicles are more stable, and are thus reproducible.

During this research the difference between the Nanotrac DLS and the Malvern DLS are examined. The PDI’s found with the Malvern DLS are, with an average of 0.17, in the range with the PDI’s found by Ali [2, 3]. The PDI’s found with the Nanotrac are in most cases 3 times or more higher, with an average of 0.58. If the results with Malvern of the 100 nm vesicle measurements isn’t taken in account it can be said that the diameters of the capsules is more in line with the expectations than the average sizes found with the Nanotrac DLS, since the average capsules size measured with the Nanotrac is sometimes lower and sometimes a lot higher than the expected vesicle size gained by extruding it through 200 nm filters. While the measurements with the Malvern device are more constant, the average found for the capsules made with vesicles that were extruded with 200 nm filters is approximately 190 nm. Therefore it can be said that it is probably best to measure the samples with the Malvern device, since the results are more constant.

When comparing the Malvern and Nanotrac devices in some cases a very large average size is measured. In case of S8C3 the average diameter found with the Nanotrac is 123 nm while the

average diameter. So in cases where a high PDI is measured the average diameter size can’t be compared since the algorithm are different.

5.3

Shape

To see the shape of the particles SEM and TEM pictures were taken. The disadvantage of both method is that they aren’t suitable for measuring the solution sample. Drying the sample can damage the samples, leading to misleading results [15] or the particles shrink because of drying [1]. A SEM can scan a sample very fast in a big area, which is an advantage [15]. The SEM method gives detailed shape and morphological information. A TEM only gives information about the shape.

DODAB vesicles display ellipsoidal structures with strong angularities. The vesicle templates exhibit a wider variety of structures than the capsules. Although this behavior is known for DODAB, it is yet not completely understood [16].

The size of the particles found with the SEM are much bigger than the size found with the DLS. The shape of the capsules as shown in the SEM pictures are irregular. It is assumed that this big size and irregular forms are created during the drying process of the capsules, where the capsules explode and the shell unfolds. As can be seen in figure 20d the capsules have somewhat of an popcorn like structure.

With the TEM a lot of spherical capsules were found. In previous research projects parachute-like structures could be found after the polymerization of DODAB vesicles [2,3,16]. It seems that if a higher amount of EGDMA than 1:2:0.1 with MMA:t-BA:EGDMA is used parachute like shapes occur, while under that ratio spherical shapes are found. It is concluded that the parachute-like structure occurs because of the incompatibility between the synthesized polymer and the bilayer matrix is the driven force for the formation of these peculiar hybrids and that, as a consequence, templating does not occur [16].

5.4

Temperature responsiveness

As shown in figure 17 and 18 the nanocapsules made with NIPAm:MBA:AA show little responsiveness to temperature differences. A 3rd order polynomial made a very good trend line of the diameter size of the capsules. The PDI’s found with the Malvern device have an average of 0.1, which means that the particle sizes are close to the average. It can also be said that the temperature responsiveness is pretty stable since the standard deviation of 3 measurement is very small.

Unfortunately the size change of the capsules in the range of the temperature of human tissue is only 0.3 nm, this means that these capsules are probably not usable for targeted drug delivery.

6

Conclusions

In this report a simple RAFT-based vesicle-templating approach to synthesizing polymeric nanocapsules is presented. These nanocapsules with a size around 190 nm are reproducible and in the range for intravenous administration of human tissue [1]. There were also temperature responsive nanocapsules synthesized with the monomers NIPAm, MBA and AA, but the difference in particle size in the range of human tissue temperature was only 0.3 nm. During the synthesis about 50% of a DODAB solution was lost when extruded through the 100 nm filters.

It can be concluded that the size of polymer capsules is smaller than the vesicles that they’re made of, this happens because the shape of the capsules is less ellipsoidal than the shape of the vesicles. The spherical shape of the capsules also make the particles more dense and therefore smaller.

The shape of the polymer capsules was spherical as expected with these ratios of monomers and the amount of crosslinker [2,3]. During this study no parachute-like structures were observed as found in previous studies [2,3,16].

7

Recommendations

For further research in the synthesis of polymer nanoparticles for targeted drug delivery a few recommendation can be given. To achieve the goal of temperature responsiveness with the NIPAm capsules with good size differences in the temperature range of human tissue, more research about the behavior of polymerization of these capsules is needed. In particular the influence of the amount of crosslinker is interesting. This is because the crosslinker has a big influence on the morphology of the particles.

That the crosslinker has a big influence on the morphology can be seen in the research with MMA,

t-BA, and EGDMA polymer capsules, since with a higher amount of crosslinker parachute-like

structures are found, while spherical structures are found with lower ratios of EGDMA. Therefore it is also interesting to do research the exact behavior of EGDMA. This can be done by adding different amounts of EGDMA, add EGDMA gradually during the polymerization or add EGDMA at different points in time of the polymerization.

To be sure about the size of the particles and which DLS device to use another recommendation is to thoroughly analyze the differences and used algorithms for both devices.

8

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Appendix I – SEM pictures

S7C

S7C

S7C

S7C

S7C

S7C

S8C2

S8C2

S8C2

S8C5

S8C5

S8C7

S9C8

S9C8

S9C8

S9C9

S10C1

S10C2

S10C2

S10C3

Appendix II – TEM pictures

S1C S1C

S1C S1C

S7C S7C

S7C S7C

S11N1 S11N1

S11N1 S11N1