Nanogel Characterization

The last section was concerned with describing the experimental parameters (i.e. OMA system and UV source) associated with the synthesis of PNIPAM nanogels. This section serves to present on all the tools for characterization that the PNIPAM nanogels were subject to. First, the nanogels’ morphology and desiccated PSDs will be described via microscopy (namely TEM). Then, the in-situ PSDs, as measured by NTA, will be provided. After that, ATR-FTIR will be used to demonstrate successful polymerization of NIPAM into PNIPAM. Finally, ultraviolet absorbance of the precursor solution and synthesized product will be discussed in an effort to demonstrate the photochemistry involved in the nanogel synthesis. All characterization presented in this section was performed on one sample. The precursor solution had 3 parts NIPAM (monomer) to 1 part MBA (crosslinker) to 1 part Irg 2959 (photoinitiator), by weight, dispersed into filtered DIW. The solution was agitated both mechanically and ultrasonically to ensure homogenous distribution of the solutes. The sample was exposed to UV for approximately 35 minutes. Further details regarding the synthesis are provided in Sect. 2.4 where the time-evolved spectrum of the UV source is provided to monitor the reaction in-situ. The sample that was removed from the chamber was the product upon which all characterization described in this section was performed.

2.3.1. Electron Microscopy

Monodisperse and stimuli-responsive PNIPAM micelles synthesized via emulsion polymerization were characterized in Sect. 2.1.1. That method of synthesis was abandoned for creating stimuli responsive nanogels for biotherapeutic delivery when the micelles were unsuccessful at encapsulating IOMNPs. In this subsection, microscopic characterization of the PNIPAM nanogels synthesized via exposure to UV is presented. Confocal microscopy, SEM, and TEM will be used to describe the morphology of the nanogels and to report on their size. Microscopy is uniquely suited to comment on PNIPAM nanogel morphology since it provides a visual representation of the artifacts. On the other hand, the electron microscopy used here to report on size requires the desiccation of the sample meaning that the reported sizes are likely

smaller than a hydrated sample would be during the intended application. Furthermore, electron microscopy lacked the capability of measuring sizes at varying temperatures. These shortcomings will be resolved later.

The details regarding the mixing of the precursor solution and subsequent exposure of the precursor solution to the UV source were recorded at the beginning of this section. After synthesis, the product was agitated briefly mechanically and ultrasonically just prior to removal of a drop for microscopy.

One drop was placed onto a microscope slide for confocal microscopy, another was dropped onto ITO coated glass for SEM, and a third was pipetted onto a copper coated grid for TEM. Confocal microscopy was conducted by Nawal Khadka in Dr. Jianjun Pan laboratory using the provided wet sample. The sample for SEM analysis was coated with AuPd to improve contrast and the microscope was operated at 6 keV.

As with SEM, the TEM sample was allowed to dry at room temperature just prior to inserting it into the specimen chamber where it was exposed to vacuum further drying out the sample. Then TEM was operated at 60 keV.

A sample micrograph, recorded by Nawal Khadka, is presented in Fig. 2.16 (a). The image shows several of the PNIPAM nanogels, apparently, agglomerating together in the DIW solvent. Note the multiple focal planes in the micrograph causing some smaller artifacts to appear out of focus. It should be noted that confocal microscopy was the first test of the sample after synthesis. Therefore, it wasn’t until later that confirmation of discrete nanogels was obtained. One of the first indications that the sample contained smaller nanogels was provided by SEM. The SEM micrograph in Fig. 2.16 (b) reveals individual nanogels some of which are isolated, but are for the most part in proximity to each other. They appear approximately round and are all relatively close in size to each other. Finally, the micrograph in Fig. 2.16 (c) provides high resolution representations of the nanogels. Again, they are round in appearance with some indication of a bumpy texture on their surfaces. The histogram in Fig. 2.16 (d) reveals the PSD of this sample of PNIPAM nanogels as measured using the TEM software. They have an average size of 203.3 ± 28.4 nm. The histogram was fit with a Gaussian whose peak corresponded to the average size and whose standard deviation from the center corresponded to the error in average size. The nanogels are almost monodisperse in that the error is a little more than one-tenth of the average size.

Like SEM, most TEM micrographs also show the nanogels in proximity to each other. However, in SEM and TEM this can be attributed to sample preparation in that the drop is necessarily desiccated. As

the solvent evaporates, the nanogels are forced together giving the false appearance of agglomerates.

Note, in Fig. 2.16 (b) most nanogels are in proximity to each other, but a few nanogels are, in fact, isolated.

In the case of confocal microscopy, the comparatively lower magnification capability tends to bias the observations toward larger artifacts. Note, in Fig. 2.16 (a) that smaller artifacts are left out of focus while the largest structure is made clear. While these nanogels may not be as monodisperse as the PNIPAM

10 µm (a)

1 μm (b)

(d)

10 100 1000

0 10 20 30 40 50

<D> = 203.3 nm  = 28.4 nm

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Size (nm) (c)

100 nm

Figure 2.16 Displayed here are typical images of the PNIPAM nanogels synthesized via photopolymerization, recorded using (a) confocal microscopy, (b) SEM, (c) TEM, and (d) the PSD of the nanogels as measured using TEM micrographs.

micelles synthesized via emulsion polymerization (see Sect. 2.1.1), their size distribution could easily by cleaned up using techniques such as centrifugation, filtration, and refinement of the final product harvesting after synthesis. The next subsection will demonstrate that these nanogels do not agglomerate when in solution.

2.3.2. Nanogel Tracking Analysis

The last section utilized TEM sizing data of PNIPAM nanogels to report an average size of almost 200 nm. However, those micrographs revealed most nanogels in close proximity to each other with only a few in isolation. In this section, NTA data will be used to establish the fact that the nanogels, synthesized as described at the beginning of this section, are not agglomerating in solution, but exist as discrete entities.

The same data will be used to describe the in-situ PSD of PNIPAM nanogels. These NTA results will be more pertinent to the application of biotherapeutic delivery since the sample was in solution and not desiccated as in TEM. Recall the PNIPAM micelles of Sect. 2.1.1 were revealed, by DLS, to be temperature responsive. Similarly, NTA data will be used in this section to gauge the temperature response of the PNIPAM nanogels.

Detailed specifications regarding the NTA equipment are provided in Malvern’s brochure [166]. The schematic in Fig. 2.17 (a) diagrams how the sample is probed for the size and concentration data that are output by the software. Data sets of this sort allow for the determination of the prevalence, based on concentration, of distinct nanogel size populations. In short, a He-Ne laser propagates through the sample in such a way that the scattered light is captured by a microscope and directed to a digital camera. A video of the nanogels’ scattered light is analyzed for their Brownian motion and the Stokes-Einstein equation can be used to determine their size. The data presented below represents the average of five videos lasting three minutes each. This practice demonstrates repeatability and generates error. The raw data sets range in size from 10 nm to 2000 nm. Samples were allowed to equilibrate for about 15 minutes anytime the temperature was changed in order to ensure a thermally stable sample.

The sample was initially subjected to NTA’s measurements at a temperature of approximately 20.0

°C. The data corresponding to those measurements is plotted in Fig. 2.17 (b) in terms of concentration versus nanogel size. Note that this data has been cropped to exclude data that appeared relatively flat

compared to these peaks. Immediately apparent are five distinct nanogel populations based on size. Each peak was separately fitted with a Gaussian whose peak identified the average size and whose width specified the error after dividing by four. The results of this analysis is collected in Table 2.3. Also in Table 2.3 is an accounting of the prevalence of a particular size population based off their peak concentration measurements. For example, the most prevalent size population measures about 217.7 ± 6.6 nm and it constitutes nearly 25.4 % of the sample. Obviously, this is a narrower PSD than that reported in the previous

50 100 150 200 250 300 350 monodisperse PNIPAM nanogel size populations, (c) whose temperature-dependent concentration versus size study reveal (d) increasing size with elevated temperatures.

section, but it would necessarily grow wider when all five size populations are considered together. The advantage of seeing the size populations resolved in this manner demonstrates that the nanogels are distinct and not agglomerating.

Next, the sample was analyzed by the NTA system again keeping all parameters constant except the temperature. Data sets comparable to that collected at 20.0 °C were also collected at 30.0 °C and 40.0

°C. For the purposes of this temperature study the distinct peaks were ignored and the entire data set was fit with a Gaussian. As before, the peak of the Gaussian identified the average size of all nanogels at that particular temperature and one-fourth of its width gave the error. The Gaussian fits for the sample at each of the three temperatures are plotted in Fig. 2.17 (c). Apparently, the average nanogel increases in size as its temperature is raised. Also, the concentration of a typical size population decreases at increased temperature. In other words, there is a wider distribution of larger sizes at elevated temperature.

Finally, the average sizes and corresponding error were plotted against temperature in Fig. 2.17 (d). As before, there is a clear trend for these nanogels to grow as the temperature is increased.

Unfortunately, the error is rather large in that it overlaps. Recall that this error was the result of considering every size population in the sample. It would be possible separate out the distinct size populations using centrifugation or dialysis. The data was fit with a line to determine that the size of the nanogels is growing with temperature at an approximate rate of 5.8 nm/°C.

In closing, this section served to demonstrate a temperature-dependent, in-situ PSD for the PNIPAM nanogels. Also, the data reveals that the nanogels are not agglomerating as suggested by microscopy data of the previous section. Recall that the PNIPAM micelles synthesized via emulsion Table 2.3 The average size, and corresponding error, for each of the five distinct peaks plotted in Fig. 2.17.

The peaks are consecutively identified with a number 1 – 5 with the leftmost peak being number 1 and the rightmost peak being number 5.