PNIPAM encapsulated QD nano-spheres were synthesized in three steps [26]. This three-step process consists of (1) PNIPAM particle synthesis; (2) CdTe nanocrystal preparation; (3) encapsulation of nanocrystals in PNIPAM nano spheres. Specifically, PNIPAM particles were synthesized by mixing NIPAM monomer, N(3-aminopropyl) methylacrylamide chloride monomer, methylene-bis-acrylamide and N,N-cysteine-bis-acrylamide in deionized, distilled water. The solution was sparged with nitrogen and heated to 70 °C for 40 min. Potassium persulfate was then added to the solution to initiate the reaction. The reaction was carried out at 70 °C for 4 h. CdTe nanocrystals were synthesized from sodium hydrogen telluride (NaHTe) and Cd(ClO₄)₂·6H₂O with proper pH adjustment. PNIPAM encapsulated QDs were synthesized by breaking the S-S bonds and forming reactive thiol groups and cross-linking those with free thiols on the QDs. PNIPAM particles were mixed with 1,4-dithiothreitol to break S-S bonds and to form reactive thiol groups. The mixed solutions (PNIPAM particles and the QDs, 24 h) were centrifuged at 3,000–10,000 rpm for 1 h at 25 °C. By decanting the supernatant, followed by repeating the cycle of re-dispersion with water and centrifugation (six to eight times), the unloaded and surface attached QDs were removed and PNIPAM spheres loaded with CdTe QDs were obtained, denoted as CdTe-PNIPAM. It is observed that after four washings, almost all surface attached QDs get detached. A schematic is provided in Figure 1.

An FEI Nova Nanolab 200 Scanning Electron Microscope (SEM) was used to observe the morphology of the hydrogel encapsulated quantum dot nanospheres. In order to prevent collapsing of the polymer network, cryo-immobilization was achieved through plunge-cooling the sample in liquid nitrogen. Accelerating voltage for SEM imaging was kept between 20–30 kV, which enables secondary electron signal generation from the sample. SEM imaging was performed at a magnification of ∼120,000×. To observe the encapsulated QD nanocrystals, high resolution transmission electron microscopy (HRTEM) was carried out using an FEI Tecnai F20 HRTEM.

E. coli (K 12) was grown aerobically at 37 °C in an incubator/shaker at 100 rpm overnight to stationary phase. Luria-Bertani (LB) medium (10 gm/L of Tryptone, 5 gm/L yeast extract and 10 gm/L sodium chloride) was used for the bacterial culture. A small but equal amount of saturated culture (1:100 by volume) was seeded into each freshly prepared PNIPAM-QD-LB Broth solution. For spectrophotometric reference, unseeded PNIPAM-LB Broth solutions were used. The positive control was the same growth media without PNIPAM-QD nanospheres. These cultures were incubated at 37 °C, shaking at 100 rpm and the optical density (OD) was measured using A_600nm at selected times to determine growth rates. 600 nm is selected, because to measure bacterial growth by light scattering it is best to pick a wavelength where absorption is at a minimum and for most bacterial cultures wavelengths around 600 nm are a good choice [24]. This procedure was followed for all concentrations of QD/PNIPAM gels evaluated.

E. coli K 12 cells were made competent using treatment with a calcium chloride solution. The transformation process introduced a commercial 5.4 kb plasmid encoding green fluorescent protein (GFP) regulated by an arabinose promoter and conferring ampicillin resistance (pGLO, BioRad, Inc., Hercules, CA, USA). Transformed cells were re-suspended in the nutrient broth and spread on plates containing LB agar, ampicillin, and arabinose, and then incubated at 37 °C overnight. Another control plate was streaked using LB and ampicillin, without arabinose. Both plates contained 100 μL PNIPAM-QD nanosphere solutions. LB-Amp-Ara plates without nanospheres were used as a positive control.

The QD-PNIPAM nanospheres exhibited good colloidal stability in aqueous solution with excellent luminescent efficiency and no obvious precipitation after several days (Figure 2(A)). Under proper storage conditions (2–6 °C, in a dark vial) the nanospheres can be stored for up to two years. In order to extract the nanosphere morphology and size distribution, scanning electron microscopy (SEM) was performed. The sample preparation involved plunge cooling inside liquid nitrogen, thus preventing them from collapsing after freeze-drying. Spherical to oval shaped particles were observed (Figure 2(B)); analyzing several images and constructing the nanosphere size distribution histogram (not shown here), the mean diameter was found to be 382 ± 45 nm, which was re-confirmed by the Dynamic Light Scattering (DLS) measurement.

It should be noted that, during sample preparation for SEM imaging, some spheres become slightly oval shaped because of surface roughness of the carbon tape (on the mounting stub, seen in the background). It is important for the designed system that the polymer encapsulates the nano-sized QDs, which not only facilitate the solubility and bioavailability, but also provides a linker for bioconjugation of peptides, antibodies, oligonucleotides, or small molecule drugs. The SEM micrographs did not reveal any QD nanocrystals on the surface of the hydrogel nanoparticles. The absence of QDs on the surface provided indirect proof of the polymer encapsulation around the semiconductor nanocrystals and not surface attachment, which is extremely important for biomedical applications. The lower electron beam energy (5–30 kV) of SEM imaging was insufficient to penetrate the outer polymer shell, so we directly assessed particle encapsulation by HRTEM. At an accelerating voltage of 200 kV, electrons were able to pierce through the outer polymer shell. Using Performing High Resolution Transmission Electron Microscopy (HRTEM) analysis (not shown here), the loading of CdTe QDs was found as ∼0.4 × 10⁴ inside a typical 400 nm diameter PNIPAM sphere for the medium density packing. For high and low density packed spheres, these numbers stand at ∼0.7 × 10⁴ and 0.2 × 10⁴ per sphere, respectively. HRTEM micrographs (Figure 2(C)) further reveal that the QDs did not form large scale agglomerations and were quite uniformly distributed throughout the spheres. The average QD diameter was found to be 2.5 ± 0.2 nm, excitation wavelength was 400 nm, and the emission maximum was 530 nm. Simultaneous imaging of the polymer shell and the encapsulated QDs were not possible here. The problem in imaging core-shell nanostructures has been reported by other research groups as well [27,28]. It should be noted that, in our previous work, the QDs encapsulated in the PNIPAM nanospheres showed a 200% enhancement in the PL efficiency as the nanogels shrank at a temperature above the LCST of PNIPAM [26]. This PL enhancement is presumably due to the change of the configuration of the nanogels that confined the movement of QDs. The temperature induced refractive index change also led to a novel gain mechanism in these systems. It is suspected that the dots come closer once the temperature goes above the LCST, moving into the range of FRET.

To address concerns regarding nanosphere stability in the growth media, DLS measurements were taken of the nanospheres in distilled water and in LB growth media. With this procedure, the difference between the behavior of the nanospheres in solutions with and without salt was compared. We did not see any difference in diameter. Furthermore, as the nanoparticles have a tendency to agglomerate in LB media, we have performed a time dependent size measurement experiment. PNIPAM-coated QDs remained stable in LB media, thus retaining their size without forming large agglomerates. DLS measurements were taken over the time frame correlating with the growth measurements to determine how the particles behaved during that process. Figure 3 reveals DLS measurements of QD-PNIPAM nanospheres in suspension for a duration of five hours in LB media. During this run, the nanospheres remained stable and showed little change in hydrodynamic radius. Stability of PEG coated gold particles in LB media has previously been observed as well [24].

Growth curves (Figure 4(A–C)) were generated for E. coli growing in 20 mL of LB media containing QD-PNIPAM nanospheres (400 nm diameter) with high, medium, and low packing densities. For each loading scenario, toxic (red), borderline (blue), and safe (green) limits have been evaluated.

The overall results indicate that even densely packed QD-PNIPAM spheres exhibit a safe limit (around 0.1 mg/mL) for E. coli growth. Uncoated CdTe solution having 25% fluorescent intensity to that of the low packing density nanospheres (borderline level, i.e., 0.5 mg/mL) is used as negative control and growth rate is compared (black). It is observed that the encapsulated CdTe-PNIPAM nanospheres reduce cytotoxicity effects significantly. This is a significant improvement over the existing findings with QD cytotoxicity studies [20]. Although Helbig and coworkers have observed E. coli growth at a relatively higher Cd concentration [29], it must be noted that CdTe QDs (encapsulated or bare) are a completely different system. The reason for high dosages of Cd (75 μM) permitting E. coli growth may be related to lower intracellular uptake of the Cd²⁺ ions in the absence of surface modification and subsequently, less ROS generation. Moreover, QD size and surface charge has played a huge role in toxicity and uptake, as observed by Lovric and coworkers [30]. In our study, the growth of E. coli exposed to significantly higher concentration was inhibited but at moderate or lower concentration, the growth was normal. This is particularly important for high density packing, as the dots are close to each other and volumetric shrinkage of the PNIPAM shell brings them even closer at temperatures above the LCST, thus the condition becomes ideal for FRET. For medium and low packed QD-PNIPAM spheres, higher concentration levels (0.17 mg/mL for medium packed and 0.5 mg/mL for low packed) proved to have negligible cytotoxic effects. Bacterial growth depends on available nutrition and environment. At higher QD concentrations the mechanism for growth inhibition may be that quantum dots have some effects on replication machinery, slowing DNA synthesis and leading to increased time before binary fission.

A preliminary gene expression study was conducted by transforming E. coli cells with plasmid pGLO, which expresses GFP. Green fluorescent colonies were observed after overnight incubation of spread LB-amp-ara plates with a high density QD-PNIPAM nanospheres (0.1 mg/mL, borderline level) (Figure 5(A)). Encapsulated CdTe QDs did not suppress arabinose promoter regulated pGLO gene product GFP expression. A similar number of fluorescent colonies were present in the positive control (without nanospheres, not shown here); however, cell growth was prohibited drastically with bare QDs, even from dosages having as low as one-fourth of the fluorescent intensity as that of the previously administered PNIPAM encapsulated QDs. As a result, no colony formation took place and no signal was detected (not shown). Similar results (about 50% inhibition ratios after 135 min exposure) were observed during toxicological assessment of TiO₂ nanoparticles with recombinant E. coli [25]. This indicates that the PNIPAM-QD nanospheres are not cytotoxic to the procaryotic cells at certain concentrations and do not inhibit the GFP gene expression. Furthermore, healthy cells were observed (Figure 5(B)) after overnight incubation with high density QD-PNIPAM nano-spheres at this dose level; again cell growth was not observed with bare QD exposure (previously mentioned dose level).