self healing starch capsules

Self-Healing Waxy Maize Starch (WMS) loaded PLGA Microcapsules Research Project MingouZhang.MEngMaterialsScience&EngineeringCornellUniversity

AbstractThe purpose of this research project is to apply the concept of using microcapsules to impact self-healing characteristics to starch resin films. The microcapsules were fabricated through double emulsion process using degradable polymer poly (lactic-co-glycolic) acid (PLGA) and food grade Waxy Maze Starch (WMS). Microcapsules with core shell structures were observed in solution state via confocal imaging technique. Microcapsule size analysis was done after varying in PVA, PLGA content and homogenization speed. Microcapsule size showed both increasing and decreasing trend between 1.72-4.76w/w% of PVA. Decreasing PLGA content from 0.3g to 0.2g increased the capsules size to around 30-40 𝜇m, while no capsule formation was observed with 0.1g PLGA. The increase in homogenization speed from 10000 rpm to 30000 rpm first increased and then decreased the microcapsule sizes recording the largest capsule size of 2-9 𝜇m at 20000 rpm homogenization. Less than 1 𝜇m capsules were observed in capsule solution at 30000 rpm. Further confocal image analysis showed potential sub microcapsules embedded within larger capsule agglomerations, which created uneven surfaces. The calculated starch load was around 78v/v% for a single capsule agglomeration. Starch load is expected to be varied across different size capsule agglomerations. Crack healing test for the capsule loaded WMS film failed due to weak bonding between the fracture surfaces. It is possible that higher microcapsule loading may result in self-healing.

Introduction: Recent research on starch based composites has shown excellent mechanical properties which makes them potential replacement for synthetic polymer materials such Polyethylene (PE), Polypropylene (PP), Polymethylmethacrylate (PMMA).[1,11,14] Most conventional synthetic polymers used at present, are derived from petroleum, which lack sufficient end-of-life treatment.[1] Due to their relative low cost and abundant supply, starch composites may be easy to obtain and process. Pure starch consists of amylose and amylopectin, with an average molecular weight of 107-109g/mol. [1] Both amylose and amylopectin chains are made up from individual D-glucopyranose with three hydroxyl groups, which make them reactive to crosslinking agent. Typical starch gelatinization temperature occurs around 80oC, but the actual temperature depends on the ratio of amylose and amylopectin. High amylose content tends to lower the gelatinization temperature.[1] Crosslinking starch with bi- or poly-functional reagents such as polycarboxylic acid is critical to transform the starch from thermoplastic to thermosets polymers.[1] Ester linkage is formed between starch chains and carboxylic acid as a result of condensation reaction. Figure (1) shows typical crosslinking reaction of starch molecule with polycarboxylic acid.

Figure (1) Crosslinking of cellulose with poly carboxylic acid.[1]

Micro-encapsulation of bio healing agents has been done in various biomedical and pharmaceutical researches, as healing agent can be delivered through the breakage of capsules vehicles.[2][3][4][5] In fragrance and textile industry, fragrance content was encapsulated and released when applied on human skins or garments.[15] In food & Agriculture industry, microencapsulation was used to improve shelf life and ingredient protection, as active agent can be encapsulated and controlled released to mask undesirable taste, color and odor.[15] There are different techniques to fabricate microcapsules used in different industries and applications.[14] Some example processes can be in situ polymerization, single/double emulsion, suspension polymerization,

interfacial polymerizations molecular encapsulation using cyclodextrins, coacerations, etc.[14] The double emulsion process will be described in detail since it was the technique adopted in this study.

With an interest of exploring same encapsulation concept, capsules loaded with starch solution or crosslinking agent has been designed and envisioned. Poly (lactide-co-glycolide) (PLGA) micro particles have been investigated as carriers for drug controlled release due to its biocompatibility and biodegradability.[2][3][4][5] However, the most common materials encapsulated are proteins, peptides and antigens designed for living organisms [2][3[4][5] the encapsulation of starch within complex composites environment is not well understood. The inspiration of this project was to apply the same principle of drug delivery carriers such as micro-encapsulation of healing agents and place them into cross-linked starch composites. Instead of releasing biological healing agent, in the present study, encapsulated starch content was released to fill the gaps of micro-cracks within the composites or resin. When polymers are stressed, micro-cracks form and lead to significant reduction in mechanical properties of composites. Such phenomenon is commonly evident in composite structures such carbon fiber/epoxy composites. Micro-cracks can be observed when composites materials are subject to tensile loading, fatigue loading or thermal loading. [6]

In this experiment, an attempt to create spherical polymer microcapsules was done by using PLGA, which encapsulate waxy maze starch (WMS) as healing agent. The method used was water-in-oil-in-water double emulsion.[7] An emulsion is commonly done by dispersing minute droplets of a certain liquid whether or not it is soluble or miscible.[7] This method is composed of a continuous liquid phase of solution in which oil globules are dispersed.[7] Smaller aqueous droplets are suspended within the oil phase, which makes up a different phase of its own.[7] The Generalized double emulsion process is shown in Figure (2). The gelatinized waxy maize starch (WMS) solution was dispersed within the in a PLGA matrix, which undergoes two emulsion processes. The first step involves dispersing WMS solution in PLGA-dichloromethane solution through homogenization. Homogenization is the process of converting two immiscible liquids into emulsions through the use of homogenizer.[8] This process generates the first degree of emulsion (water – in – oil emulsion). Once the first degree of emulsion is achieved, the combined system was then be mixed with PVA (Polyvinyl Alcohol) which acts as the surfactant in the oil-water interface and optimally stabilizes the emulsion.[8] By completing the two steps of double emulsion process, the encapsulation of starch solution droplets in microsphere PLGA can be obtained after evaporation of dichloromethane. The remaining components of the solution should be PVA solution and starch loaded PLGA microcapsules. After evaporation, the solution was hardened and freeze-dried to remove the water. For the purpose of the self-healing experiment, the prepared microcapsules were added into the WMS films and crack-healing test was performed. Additional crosslinking reaction occurs within the micro cracks after the breakage of the microcapsules. The prepared WMS films were expected to be loaded with excess amount of crosslinking agent. According to the previous studies by Ghosh Dastidar and Netravali, the initial concentration of BTCA is linearly related to the percentage of cross-linking of cellulose.[11] However their FTIR results seemed to be plateaued at around 25 %w/w BTCA.[11] Thus, a higher percentage of BTCA content is suggested for additional crosslinking during crack healing.

Figure (2) Schematic Illustration of Typical W/O/W double emulsion process.[13]

Both optical and confocal images were used to characterize the obtained capsules and their internal starch loading content. Confocal florescent imaging was primarily adopted in this experiment to determine the internal structure of the starch-loaded microcapsules. The core principle of confocal imagery involves detecting the florescent chemicals present within the specimen of interest, where the entire specimen is flooded with the light source from the microscope. [9] The florescent dye within the specimen is excited by the light source at a specific range of wavelength and the resulting emitted light from the florescence are collected by a photo detector [9] Compared to other types of optical microscopy, confocal florescent microscopy applies point scanning process, which only one point in the sample is illuminated at a time. [9] Confocal microscopy also allows users to have complex 3D structural profiling through a process called Z-stacking. Z-stacking is done by recording images at different focal planes of the samples.[10] The distance between each plane is usually much smaller than the thickness of the sample allowing the internal structure of the sample to be visualized.[10] Experimental Procedure: Materials. Food grade Waxy Maize starch powder was obtained from Now® Sport (90% amylopectin). 1,2,3,4 - Butanetetracarboxylic acid (99% purity), Sodium hypophosphite monohydrate (99% purity), Polyvinyl Alcohol (99% purity), Dichloromethane (> 99.5% purity), were purchased from Sigma Aldrich (St. Louis, MO). 50:50 poly (DL-Lactide -co- glycolide) (10 grams) ester terminated was purchase from LACTEL® absorbable polymers, (Birmingham, AL). Cross-linked Waxy Maize Starch Film Fabrication. The cross-linked WMS film was fabricated according to the method developed by Ghosh Dastidar & Netravali [11] with slight modifications. The process was as follows: 3.4g WMS was added to a beaker containing 120 ml DI water with magnetic stirring at 300 rpm. The starch solution was heated to 90oC for 30 min ensuring maximum gelatinization. 40 w/w% of BTCA was

added to the gelatinized starch solution along with SPH (50 w/w% of BTCA) as catalyst. The solution was further heated with magnetic stirring at 90oC for 1 h to ensure maximum crosslinking. The starch solution was poured on to 5in * 5in Teflon coated glass plate for cooling and drying for 2-3 days. The pealed-off starch films were hot pressed under 120oC and 1000 lb. for 20 min, (Carver® Hydraulic Press Test System, 230 VAC 60Hz). The final films obtained were washed with DI water and dried at room temperature. WMS loaded PLGA Micro-Capsules fabrication & characterization. The WMS starch loaded microcapsules were fabricated based on w/o/w double emulsion method. WMS solution was initially prepared by dissolving 10g of WMS in 100ml of DI water with continuous magnetic stirring and heating at 90oC for 1h. The gelatinized WMS solution was stored in multiple 20ml glass vials. 1-2 drops of Rhodamine B were pipetted into each glass vial for staining of the starch solution. 3ml of stained starch solution was pipetted into a beaker along with 20 ml of PLGA/DCM solution. The first degree of homogenization speed was set at 10000 rpm. (VWR-10032-338, 250 Homogenizer) The combined solution of WMS and PLGA/DCM were homogenized for 1 min. After the mixing was complete, 37.5 ml of PVA/water solution was added into the beaker. The combine mixture (WMS, PLGA/DCM, and PVA) was homogenized for additional 1min, (Second degree of homogenization).

In order to determine the average size and starch loading of the microcapsules, effect of three variables (PVA, PLGA, and Homogenization speed) was characterized. All PVA variations were dissolved in 37.5 ml of DI water and PLGA in 20ml of Dichloromethane. Table (1a-1c) summarizes all the parameters and variation of tests. The characterization of size and internal structure of the starch-loaded microcapsules was done by using optical and confocal microscopy (Zeiss 710 Confocal). Table (1a) Experimental parameters for determining effect of PVA concentration PVA in water w/w%

0 1.72 2.44 4.76 6.97

PLGA (g) 0.4 0.4 0.4 0.4 0.4 WMS solution (ml)

3 3 3 3 3

Homogenization Speed (RPM)

10000 For both processes





Table (1b) Experimental parameters for determining effect of PLGA concentration PVA in water w/w%

4.76 4.76 4.76

PLGA (g) 0.1 0.2 0.3 WMS solution (ml) 3 3 3 Homogenization speed (rpm)




Table (1c) Experimental parameters for determining effect of homogenization speed PVA in water w/w%

4.76 4.76 4.76

PLGA (g) 0.4 0.4 0.4 WMS solution (ml) 3 3 3 Homogenization speed (rpm)

10000 Only for the second homogenization process



WMS films with microcapsules fabrications & Fracture/healing testing. The fabrication of WMS microcapsules films was performed similar to the fabrication of the pure WMS films. For this 3.4g of WMS, 40-w/w% BTCA, (50 w/w% of BTCA SHP) were mixed with 120ml of water and heated at 90oC for 2h. PLGA/WMS microcapsules (Prepared by 3ml pure WMS solution, 0.476 w/w% PVA, 0.3g PLGA) were harvested by centrifuging and then freeze–dried. A total of 0.6g of PLGA capsules were harvested and added to the cross-linked WMS solution. The ratio of the capsule within the starch film was set to be 15 w/w%. Magnetic stirring at 300 rpm was applied to speed up (40 min) the dissolving process while no heat was supplied. The cooled solution was cast 5in * 5in Teflon® coated glass plate for cooling and drying for 2-3 days before pilled off and cured at 120oC/3000 psi pressure. The fracture testing sample was fabricated according to the design from J.R.Kim and Netravali.[12] Laser cutting was used to cut the film (Both capsule loaded and pure WMS) with specific shape (3.2cm*2.4cm) shown in Figure (9a) & Figure (9b). In Figure (9c), the samples were allowed to crack completely through. These samples were placed in room temperature with two objects gently placed on both of the sample with no force applied to compress the sample. These two objects were used to restrict the motion of the cracked sample as shown in Figure (9d). The cracked sample was placed at room temperature for 24 h and the same testing was performed to determine the self-healing. In both testing cases 5 samples were tested and the load vs displacement curves were plotted.

Results & Discussion Physical Appearance of Obtained PLGA Microcapsule Solutions Figure (3a) shows the homogenized microcapsule solution of 6.97 w/w% and 0% PVA concentration right after 24h magnetic stirring. The 0 % solution showed sign of de-mixing, which might attribute to lack of PVA stabilization. Hard clear beads could be seen on the wall of the glass beaker. The solution was clear and pink with black precipitate at the bottom. Comparing the 0 % solution with 6.97 w/w% PVA solution, the latter showed cloudy milk like solution indicating emulsification. Figure (3b) shows the obtained PLGA microcapsules solution with varying concentrations of PVA before centrifuging. As shown in Figure (3b) the four solutions obtained were stored in 10ml plastic tubes. They were pink or grey cloudy solutions with varying degree of precipitations, which suggest some de-mixing, occur after they were placed stationary for few hours. The cause of this de-mixing behavior was unclear and subjected to further investigation. Visually, these precipitations are black or grey viscose dense liquid appearing at the bottom of 6.97 w/w%, 4.76w/w% and 1.72 w/w% tubes. Figure (3c) shows the 4.76 w/w% and 6.97 w/w% solution after centrifuging. Similar separation was obtained for 1.72 w/w% and 2.44w/w% solutions. As can be seen in Figure (3c), there are three components, the upper clear solution with slight pink color, and the bottom top residue with grey and black color. Other PLGA capsule containing solutions with variation of parameters shared similar physical appearance.

Figure (3a) Top left, homogenized PLGA capsules solutions after 24 hours magnetic stirring and evaporation of organic solvent. (6.47 w/w% solution on the left, 0 w/w% solution on the right) Figure (3b) Top right, homogenized PLGA capsules solution stored in 10ml plastic tubes. (From right to left: 1.72 w/w%, 2.44 w/w%, 4.76 w/w%, and 6.97 w/w%) The labels are incorrect because during experiment, 1.875g of PVA was dissolved in 37.5 ml

of water. For convenience reason the incorrect ratio was calculated to be 1.875/37.5=5%. The same argument applies for all the labeling.

Effects of Varying PVA Concentration on the Size of Capsules Confocal and optical microscopy images of 1.72-6.97 w/w% solution (imaging samples were made before de-mixing and precipitates were observed) are presented in Figures (4a-4h). As shown in all the images, red areas were the Rhodamine b florescence dye detected by the confocal microscope indicating the location of the dye, which is associated with the presence of the WMS. Although the exact binding between the starch molecule and Rhodamine b was unclear, the dye molecules were expected to be linked through either hydrogen bonding or weak ether linkage (since both molecules have hydroxyl groups present) with the starch molecules without separating into the oil phase. In this experiment the amount of florescence dye detected was assumed to proportional to the amount WMS starch present.

Figure (4a) & (4b) show starch agglomeration with some capsules embedded within the agglomerates (As indicated by the arrow). Large area of agglomerates indicated lacking of stabilizing effect from the 1.72 w/w% PVA resulting in coagulation of WMS starch capsules. No shell structure was observed. Figure (4c) & (4d) showed capsules with wide distribution of sizes. Although densely packed, the capsules were not coagulated or agglomerated. Core-shell structure could be seen in some of the microcapsules with larger diameters, as labeled. The number of microcapsules observed within one specific area was highly dependent on the sample that was extracted from the parent solution (observed after comparing different images), for example the densely occupied sample image of Figure (4c) & (4d) might have lower number of microcapsules if different sections of liquid was extracted from the 2.44 w/w% solution. Due to capsule weight differences, heavy capsules could be located at the bottom of the tube while having lower number of the capsules observed within the image frame. Light

Figure(3c)left,centrifuged4.76w/w%(lefttube)and6.97w/w%(righttube)PLGAcapsulessolutions

capsules would be dispersed on the top and having more of them displayed. Also sub capsules might be imbedded within the larger capsules agglomeration, which were not easy to identify and separate the actual single capsule from the larger aggregations. Thus the actual distribution of particles was not analyzed due to this complexity and inconsistency of sample measuring. However a rough estimate of capsule size of corresponding solution could be identified by visual inspection of the most commonly appeared size range of capsules present the solution. The corresponding capsule size results are shown in Table (2).

(4a) (4b)

(4c) (4d)

Capsulesclusters

Shellstructure

Starchtrappedwithinawell-definedsphericalstructure

(4e) (4f)

(4e) (4f)

(4g) (4h)

Figure (4a-4h) Confocal (left) and corresponding optical (right) images of (3a-3b) 1.72 w/w % PVA, (4c-4d) 2.44 w/w% PVA, (4e-4f) 4.76 w/w% PVA, (4g-4h) 6.97 w/w% PVA.

Effects of Varying Homogenization Speed on the Size of Capsules The effect of homogenization speed could be seen in Figure (4e), Figure (5a) & Figure (5b). The increase of homogenization speed first increased and then decreased the overall size. The most commonly appeared capsule size range in 20,000-rpm solution increased to around 2-9 𝜇m as opposed to 1-3 𝜇m observed in 10,000-rpm solution. When the homogenization speed was increased to 30,000 rpm as shown in Figure (5b), Capsule sizes reduced to around 1 𝜇m. Some of the capsules were too small to be visualized, which could be attributed to two reasons: 1) The homogenization process damaged the particles, thus no capsules formed 2) The inconsistency of sample from different sections of liquid might suggest larger particles exist elsewhere in the solution. However, the later observations with varying PLGA content seemed to suggest second assumption to be true. The Rhodamine-b dyed starch solutions seemed to be dispersed uniformly in 30,000 rpm homogenized solution forming particle-like structure as seen in previous images. The detail size information regarding variation in homogenization speed is presented in Table (2). When comparing the obtained capsule size with previous experiment done by H Jeffery, S.S Davis and D.T. O’ Hagen [4] (1 w/v % PVA stabilization with 150 mg of PLGA in 2.5ml of DCM, >10,000 rpm), their reported particle size obtained was around 12 𝜇m, which was about four to ten times bigger to the particle size range prepared in the present study, with 4.76 w/w% PVA at 10,000 rpm. Although Jeffery group’s highest homogenization speed was not clearly specified [4]

Figure (5a) & (5b) the confocal images of 4.76 w/w% PVA stabilized at 2000- rpm (left) 30000 rpm (right) homogenization speed

Effects of Varying PLGA Content on the Size of Capsules Figure (6b) and Figure (6c) show the effect of varying PLGA content on the microcapsules obtained. With the decrease of the PLGA added to the solution, the capsule size seemed to be increasing with the most commonly found capsule range between 30 𝜇m and 40 𝜇m (Compared to the 4.76 w/w% sample observed in previous images). The shell wall of the capsule became thinner, which suggest less PLGA surrounding the starch/dye solution. No significant capsule size difference was observed between Figure (6b) and Figure (6c). When the PLGA concentration was at 0.1g in 20ml, no capsules were observed as shown in Figure (6a). Unlike Figure (5b) where the capsules were scattered uniformly across the image, Figure (6a) had almost no detection of fluorescence suggesting that the starch solution and dye might had been already leaked before they were contained within the water phase. This observation also suggests that in Figure (5b) the red florescence were the actual capsules with even smaller diameters as opposed to being damaged by the mechanical action of homogenization. The detailed size information regarding the PLGA variation is presented in Table (2).

Figure(6a)Topleft,0.1gPLGAcapsulessolutionsamplewith4.76w/w%PVAand10000-rpmhomogenization.Figure(6b)Topright,0.2gPLGAcapsulessolutionsamplewith4.76w/w%PVAand10000-rpmhomogenization.Figure(6c)bottomleft,0.3gPLGAcapsulessolutionsamplewith4.76w/w%PVAand10000-rpmhomogenization.

Microcapsule Size Analysis Summary Microcapsule size distribution was not analyzed due to the inconsistency and

complexity of the samples obtained from different sections of the solution. The capsule size determinations were highly subjective and inaccurate due to the constraints described in the text. For PVA concentration below 1.72 w/w% no particle could be stabilized and, hence, observed. Particle size decreased in 4.76 w/w% PVA solution, where smaller capsules sizes of 1-3 𝜇m were found. Capsule size increased to around 8-17 𝜇m at 6.97w/w% PVA. Higher homogenization speed first increased and then decreased the overall sizes of the microcapsules with the 10,000 rpm producing microcapsule size of about 1-3𝜇m ranges. 20,000 rpm homogenization speed produced capsule sizes between 2-9 𝜇m. At 30,000 rpm microcapsules were hard to quantify under optical microscope. Decreasing PLGA content increased the microcapsules size to around 30-40 𝜇m due the formation of thinner shell structure compared to capsules formed with higher PLGA. No significant size difference was detected between 0.2g and 0.3g PLGA capsule solutions. At PLGA loading content of 0.1 g, no spherical capsules could be formed. Table (2) PVA, Homogenization Speed, PLGA variations on capsule size. *

PVA w/w% (Based

on 10000rpm, 0.4g PLGA)

Most common capsules

size range) (𝝁m)

Homogenization Speed (RPM) (Based on 4.76

w/w% PVA, 0.4g PLGA)

Most common capsules size range (𝝁m)

PLGA (g) (Based on

4.76 w/w% PVA, 10000

rpm)

Most common capsules

size range (𝝁m)

0 N/A 10000 ~1-3 0.1 N/A 1.72 N/A 20000 ~ 2-9 0.2 ~30-40 2.44 ~4-10 30000 <1 0.3 ~ 30-40 4.76 ~1-3 0.4 ~ 1-3 6.97 ~ 8-17

*The data recorded for the size of particles in the table above were approximate values based on visual inspection of the capsule shown in the images only. The size calculations were highly inaccurate due to the constraints described in the text. WMS Capsule Surface/Inner Structure & Starch Loading Determination Due to the resolution limit of the microscope, individual capsule loading was hard to identify. Figure (7) shows the Z-stack images of a capsule agglomeration of size around 50 𝜇m from 4.76 w/w% PVA, 0.3g PLGA, 10,000 RPM solution. The shell wall could be seen in the figure as the black rim changes its thickness through different images. (No florescence detection) Although there were total of 50 images only 12 were selected to represent the sequence of evolution. As indicated in the picture, there were smaller sub capsules embedded within the larger capsule, which shared similar structure & morphology to what J.R.Kim & Netravali reported in BSM loaded microcapsules.[12]

These sub capsules could potentially have PLGA shells that were not sliced to expose internal starch content. Thus these capsules were shown as the black or grey small particles in some of the pictures (4-9). The starch loading was determined by using image (11) as indicated. The shell thickness was determined to be around 2 𝜇m. If the internal structure was assumed to be solid and perfectly spherical, the WMS volume can be calculated to be:

𝑉!"# =43𝜋𝑟

! =43𝜋(25𝜇m− 2𝜇m)! = 50965 𝜇m!

The total volume of the capsule is:

𝑉!"#$%&' =43𝜋(25𝜇m)

! = 65449 𝜇m! Thus, the volume percentage of the internal starch is:

𝑉!"#𝑉!"#$%&'

=50965 𝜇m!

65449 𝜇m! = 0.7786 𝑜𝑟 77.9𝑣𝑣%

(1) (2) (3) (4)

(5) (6) (7) (8)

(9) (10) (11) (12)

Individualsubcapsulesorvoidsembeddedwithinthelargeragglomerations

Figure (7) (1-12), Z-Stack confocal images showing the individual horizontal slices of a single capsule agglomeration from 4.76 w/w% PVA, 0.3g PLGA, 10000RPM solutions.

The images were taken layer by layer with inter-layer distance of 1 𝜇m.

Since there was broad distribution of capsule sizes within the solution as well as variations in shell wall thickness, the exact volume fraction corresponding to each size category was not established. The internal surface structure of a capsule cluster (70 𝜇m) from the same sample solution is presented in Figure (8). As shown in the image, the surface structure of the capsule is uneven and highly porous. This could be attributed to the random agglomeration of the sub capsules within the structure. The grey spots shown in some images of Figure (7) could be the voids that were located within the capsules due to the agglomeration. This highly porosity and unevenness suggest that the actual volume fraction of the starch content within the capsule could be lower. Thus, the calculation above only describes the starch load of one specific capsule agglomeration.

Figure (8) Internal WMS surface structure of a 70 𝜇m capsule agglomeration from 4.76

w/w% PVA, 0.3g PLGA, 10000RPM solutions.

Determination of Self-healing Efficiency of the PLGA/WMS Loaded Films.

The cross-linked PLGA capsule loaded film and pure cross-linked WMS film are presented in Figure (9a) and Figure (9b). Before curing, the film thickness obtained for both films were around 0.5mm with slight variation across different sections of the surface. In Figure (9a), capsules agglomerates (Small scattered particle like substances) could be seen throughout the film suggesting precipitation during the drying process. The heavier suspension in the cross-linked solution deposited at the bottom of the Teflon® coated glass slide. The rest of the film surface exhibiting uniform pink color suggesting even finer capsules suspended in the solution before they were trapped in the dry cross-linked starch. In Figure (9b), no traces of pink color can be seen. After curing, thickness of both films reduced to around 0.4mm, with major change in internal appearance of the capsule loaded film. The majority of agglomerates disappeared after hot pressing as shown in Figure (10a). The pink color of the film intensified as compared to the pink color in the pre-cure film. The disappearance of the agglomerates mentioned earlier could be attributed to two causes: 1) The capsules agglomerates decomposed into smaller capsules or capsule agglomerates after hot curing and 2) The capsules were not able to withstand the curing temperature at 120oC or pressure and get damaged as the result. Without verifying the presence of capsules within the cross-linked starch matrix, the test proceeded based the assumption of the former.

Figure (9a) Left, Cross-linked WMS film loaded with 0.6g of PLGA/WMS capsules before heat curing process. Figure (9b) Right, Cross-linked Pure WMS film before heat curing process.

Figure (10a) Top Left, PLGA capsule loaded & cured WMS film fracture testing sample. Figure (10b) Top Right, Reference cured fracture testing sample with no PLGA capsule. Figure (10c) Bottom Left, fractured testing sample setup. Figure (10d), Bottom Right, room temperature (23C, 65% humidity) healing placement. The Load versus Crack displacement diagram for capsule loaded films is presented in Figure (11a). The load versus Crack displacement curves exhibited increasing trend with fluctuations, suggesting rubbery behavior with expected high strain and extension before breaking. The highest initial cracking load was recorded at 0.05 Kgf. The constant fluctuation of the curve might be attributed to the water molecule trapped within the starch films and incomplete crosslinking. Amyloses, BTCA, SHP within the starch films all have hydroxyl groups, which are extremely hydrophilic. [1] These hydroxyl groups can easily pick up the moisture from the air, Which can act as plasticizers.[1] In this test the conditioning room is maintained at 65% relative humidity level, which could reduce the effectiveness of the test. After 24 hr of healing, no crack

healing was observed in 2 of the samples. For the rest 3 samples, the bonding was too weak and the crack reopened during mounting process before the test. Thus, the healing load curves were not established for the capsule loaded film. Figure (11b) shows load versus crack displacements for pure WMS films before and after healing. Similar trend of fluctuations were observed for both curves, suggesting possible water plasticizing effect. The initial crack opening load was similar to that of the capsule loaded film at around 0.05 kgf. However, the crack opening process before healing was considered to be brittle due to the low extension and large value initial slope, which correlate to higher elastic modulus as indicated by the trend line. The crack opening curve after 24 hours has shown alternate loading force fluctuating around -0.04 kgf and 0.2 kgf. This behavior could be attributed to the incomplete bonding between the crack surfaces, in which some sections along the crack showed higher bonding than other sections. Since no capsules were added to the film, the nature of this bonding force was suspected to be hydrogen bonding due to the presence of hydroxyl groups on starch molecules as well as hydroxyl groups in BTCA and SHP. In summary, the crack-healing test of capsule loaded films was incomplete. Numerous flaws can be found before and during the testing processes. First, the determination of microstructure before and after curing was not performed due to time constraint of the research project. Second, the testing film samples were made too thin, which affects the accuracy of loading force as well the process of mounting cracked samples. Ideally the sample should be made at least 1mm thick as mentioned in similar tests done by J.R.Kim & Netravali [12]. A third cause may be that the testing room humidity level had a tremendous impact on the results. Hydrophobic treatment is suggested to apply on the surface of the samples before testing.

Figure (11a) PLGA capsules loaded WMS film cracking test result.

-0.03

-0.02

-0.01

0

0.01

0.02

0.03

0.04

0.05

0.06

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

Load(Kgf)

Extension(mm)

PLGAcapsuleloadedWMSJilmcrackloadvsdisplacement

Figure (11b) Pure WMS film cracking test result (Blue: First Initial Crack. Red: Crack test after 24 hours) Conclusions In this research project, double emulsion method was employed to the fabrication of WMS loaded PLGA microcapsules. The confocal imagery results have shown the existence of the capsules in the liquid suspension state. The size of the forming microcapsules or capsule agglomerations was influenced by various parameters such as PVA, PLGA contents as well as homogenization speed. The capsule size analysis was incomplete and highly inaccurate due to the constraints present in the actual experiments. Sub-microcapsules were observed and assumed to be embedded within larger agglomerations. For starch microcapsule with agglomerations, surface structure was observed to be uneven and highly porous with the approximate calculation of starch loading to be around 78%. Starch load might be associated with the size of the capsule agglomeration. The microcapsule loaded film self-healing (fracture) test showed no healing. Potential flaws with laboratory setup and fracture testing methods still need

-0.08

-0.06

-0.04

-0.02

0

0.02

0.04

0.06

0 2 4 6 8 10 12 14 16 18 20

Load(Kgf)

Extension(mm)

PureWMSJilmOriginalvsHealedCrackloadvsDisplacement

WMSFilmHealed

WMSFilm

further investigation. Overall, this research project was considered to be incomplete, with only partial information being obtained through the laboratory results due to time constraint. Future endeavors might involve exploring new variables (eg. Starch loading) that might influence the capsule formation and further application SEM images to analyze the dry capsule samples. Acknowledgements I want to pay special thanks to Professor Anil Netravali and Dr. Joo Ran Kim for their kind support and directing of my research project. I would also thank the Cornell Center for Materials Research, Department of Fiber Science & Apparel Design for kindly allowing me the use of their facilities. Reference

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self healing starch capsules

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