2.1.

MC Preparation

ICG was purchased in powder form under the product name Cardiogreen from Sigma. Stock solutions of $500\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ were prepared by dissolving the powder form into sterile, triple-distilled water, covered with foil, and refrigerated at $3°\mathrm{C}$ to minimize degradation.

ICG-containing MCs were prepared using a simple three-step process. The initial step involved the development of a spherical polymer-salt matrix by lightly mixing $0.5\mathrm{ml}$ poly- (allyamine hydrochloride) (PAH, $2\mathrm{mg}∕\mathrm{ml}$ in deionized ${\mathrm{H}}_2\mathrm{O}$ , $4°\mathrm{C}$ ) with $3\mathrm{ml}$ disodium phosphate ( ${\mathrm{Na}}_2{\mathrm{HPO}}_4$ , $0.01\mathrm{M}$ , $\mathrm{pH}=7.4$ , $4°\mathrm{C}$ ) for $10\mathrm{s}$ in a $15\text{-}\mathrm{ml}$ centrifuge tube. Through ionic crosslinking, a cationic polymer-salt aggregate was formed between the positively charged PAH polymers and the ${\mathrm{HPO}}_4^{-2}$ anions. The ratio of total negative charge of the added salt to the total positive charge of the polymer, denoted as the $R$ ratio, was 6, satisfying the necessary conditions for aggregate formation.^{26, 27}

The second step followed immediately after aggregate formation. The aggregate suspension was mixed with aqueous ICG solution ( $0.5\mathrm{mg}∕\mathrm{ml}$ , $1.5\mathrm{ml}$ ) for 30 s. By electrostatic attraction and hydrophobic interaction, the negatively charged ICG molecules penetrated into the aggregates. By adding a more dilute solution of ICG ( $0.1\mathrm{mg}∕\mathrm{ml}$ , $1.5\mathrm{ml}$ ) in, a batch of MCs containing less ICG was also prepared for the light exposure studies. These MCs with less ICG were compared with a diluted preparation of ICG solution. In the final step, an additional 0.5 ml of dextran polymer ( $40\mathrm{kDa}$ , purchased from Aldrich, $2\mathrm{mg}∕\mathrm{ml}$ in ${\mathrm{H}}_2\mathrm{O}$ ) was added to the mixture and mixed for $30\mathrm{s}$ . The dextran molecules formed a coating around the surface of the aggregate through electrostatic interaction and H-bonding. The capsules were allowed to age for $2\mathrm{h}$ , at which point they were centrifuged at $7000\mathrm{rpm}$ for $30\min$ and resuspended in $6.5\mathrm{ml}$ phosphate-buffered saline (PBS) by sonication. The process was repeated twice to remove excess precursors. The resultant MCs remained suspended in PBS, with pH close to physiological levels in blood $(\mathrm{pH}=7.4)$ , for the duration of the experiments.

For comparison with the MCs, ICG aqueous solution was made by diluting the $500\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ ICG stock solution with sterile PBS. A concentration of $50\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ was chosen to match the ICG concentration contained in the MC suspensions. More dilute ICG solution was made to match the ICG concentration in the less concentrated MC preparations by further diluting down to $10\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ .

2.2.

Absorbance Spectroscopy

Absorbance measurements were taken from 400 to $900\mathrm{nm}$ using a spectrophotometer (Shimadzu UV-2401PC) equipped with a tungsten excitation lamp. For each absorbance reading, $500\mathrm{\mu }\mathrm{l}$ of sample was extracted and placed in an Eppendorf tube. Immediately before taking the absorbance measurement, $50\mathrm{\mu }\mathrm{l}$ of $1\mathrm{M}$ $\mathrm{NaOH}$ was added to each sample to increase the pH. Increasing the pH served two purposes. Earlier experiments revealed that, at a higher pH $(\mathrm{pH}>11)$ , the MCs lost their integrity and ICG was liberated back into solution. Disassembling the capsules eliminated the effect of Mie scattering by the submicrometer-diameter MCs. Additionally, it was found that the nonlinear relationship between ICG concentration and its absorbance spectrum, which results from ICG’s tendency to form molecular aggregates at higher concentrations, was eliminated over a wide concentration range at higher pH (unpublished results). After adding $\mathrm{NaOH}$ , $500\mathrm{\mu }\mathrm{l}$ of the sample was removed and further diluted in $2.5\mathrm{ml}$ of PBS in a disposable cuvette. PBS was used as a reference for all absorbance measurements. Stability of ICG at high pH was also assessed. Preparations of ICG in PBS at high pH $(\mathrm{pH}>11)$ and concentrations ranging from 5 to $50\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ were stored in the dark at room temperature. Absorbance profiles of these samples were recorded at $t=0$ , 20, 40, and $60\min$ . No differences in the absorbance profiles were observed up to $1\mathrm{h}$ after preparation. By taking a standard curve, the relationship between peak absorbance value and ICG concentration was found to be linear over the range of concentrations used in this study, from 1.25 to $25\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ .

2.3.

Calculation of Encapsulation Efficiency

To measure the encapsulation efficiency $\left(\eta \right)$ of ICG within the MCs, a standard absorbance profile was created from the peak absorbance values of several samples of freshly dissolved ICG in PBS at different concentrations. The mass of ICG encapsulated within the MCs was determined by measuring the absorbance value of supernatants and disintegrated MCs and relating the measurements to their corresponding ICG concentrations. It was assumed that the ICG monomers did not degrade during the ICG-MC synthesis. We determined $\eta$ as

2.4.

Size Analysis and Morphology Characterization

Particle size analysis was conducted using brightfield and fluorescence microscopy. Small volumes of MCs $(<100\mathrm{\mu }\mathrm{l})$ suspended in PBS were pipetted onto a glass slide and covered with a coverslip. Scanning electron microscopy was used to characterize the MC morphology.

2.5.

Light Sensitivity

Effect of light exposure on ICG absorbance characteristics was analyzed for MCs containing ICG and freely dissolved ICG at room temperature. Preparations were pipetted into $35\text{-}\mathrm{mm}$ Petri dishes ( $5\mathrm{ml}$ per trial) placed $28\mathrm{cm}$ directly beneath a broadband $250\text{-}\mathrm{W}$ tungsten lamp (Lowel Prolite). Optical power was measured using a Coherent Lasermate 10 power meter with thermal power head. Using an adjustable iris and a collimating lens, the entire dish was illuminated at a uniform intensity of $56\mathrm{mW}∕{\mathrm{cm}}^2$ . With this intensity and exposure duration, the total energy delivered is comparable to light doses used for photothermal therapy.²⁹ At various time points for up to $2\mathrm{h}$ , $500\mathrm{\mu }\mathrm{l}$ was withdrawn from the Petri dish and the absorbance spectrum was recorded. The temperature of the sample was monitored using a thermometer and never exceeded $28°\mathrm{C}$ . Each experiment was conducted in triplicate.

2.6.

Temperature Sensitivity

Preparations of freely dissolved ICG and MCs were aliquotted into microcentrifuge tubes ( $500\mathrm{\mu }\mathrm{l}$ per tube) and remained covered in foil. The samples were either refrigerated at $3°\mathrm{C}$ , maintained at room temperature $22°\mathrm{C}$ , or incubated in a water bath at $40°\mathrm{C}$ . Every $24\mathrm{h}$ , the absorbance spectra for the preparations were recorded and analyzed for up to $96\mathrm{h}$ . The different temperatures were used to investigate the influence of temperature on the optical stability of ICG. Specifically, incubation at $40°\mathrm{C}$ for several days was used to simulate physiological conditions experienced by nanoparticles as they accumulate at a targeted tumor site following a bolus injection. To determine whether ICG escaped from the MCs over the course of our experiments, the capsules were centrifuged and resuspended in PBS. The absorbance profiles were recorded for both the MCs and the supernatant. Three samples were used for each time point.

2.7.

Absorbance Calculations

Alterations in absorbance spectra are a measure of the changes in the concentration of nondegraded ICG molecules as a function of time $\left(t\right)$ and wavelength $\left(\lambda \right)$ . We use equations reported by Holzer to quantitatively express:

ICG is useful for various diagnostic and therapeutic applications in its native, monomeric state. Therefore, we are interested in characterizing the potential spectral changes in the absorption peak of ICG monomers at $780\mathrm{nm}$ that may result from external physical stimuli such as temperature and intense, broadband light exposure, and define the parameter $\varphi \left(t\right)$ as

2.8.

Degradation Kinetics

The change in absorbance over time was used to determine the kinetics of ICG degradation. As the peak absorbance value was linearly proportional to the ICG concentration within the concentration range used in this study, the decrease in absorbance peak over time was assumed to reflect the physicochemical transformations of ICG molecules. The degradation processes were assumed to occur in a single step, and degree of linear fits was used to determine the relationship between the degradation rate and the concentration of ICG. Linear fits for ${\varphi \left(t\right)}^{-1}$ versus time, ${\log }_{10}\mid \varphi \left(t\right)\mid$ versus time, and for $\varphi \left(t\right)$ versus time, were calculated to determine whether the rate of ICG monomer degradation was independent of ICG concentration (zero order), directly proportional to ICG concentration (first order), or proportional to the square of ICG concentration (second order). The reaction order that yielded the best correlation coefficient was considered to be the approximate order.

3.1.

MC Morphology

Figure 1 shows a scanning electron microscopy (SEM) image of the dextran-coated MCs. The MCs are roughly spherical, and ranged from 400 to $800\mathrm{nm}$ in diameter. Brightfield and fluorescence microscopy verified that the MCs did not aggregate appreciably in aqueous solution.

Fig. 1

SEM Image of dextran-coated MCs. Capsules had a spherical conformation and ranged in diameter from 400 to $800\mathrm{nm}$ .

3.2.

Encapsulation Efficiency and ICG Release Kinetics

Freshly prepared MCs were disintegrated with $\mathrm{NaOH}$ , and $\eta$ was evaluated by comparing the peak absorbance values to a standard curve for ICG dissolved in PBS. Our earlier experiments revealed that $\eta$ depends largely on the initial ICG concentration and volume utilized in the MC synthesis process. The ICG concentration and volume used for these studies was selected to optimize $\eta$ , which was determined to range between 48 and 85%. Other experiments showed that when not encapsulated as MCs, PAH, phosphate, and dextran in solution had negligible influence on the absorbance profile of ICG.

The amount of ICG released from the MCs over the course of our studies was determined by absorbance measurements of the supernatants from preparations stored in dark at 3, 22, and $40°\mathrm{C}$ over 2 or 4 days. In the case of MCs stored at 3 and at $22°\mathrm{C}$ , the ICG concentration within the supernatants did not exceed 3% of the initial ICG concentration within the capsules. At $40°\mathrm{C}$ , the supernatant concentration did not exceed 7.5% of the initial capsule concentration. These results suggest that more than 90% of ICG did not escape from the MCs over the course of our experiments. ICG leakage was not considered in the light sensitivity and temperature sensitivity studies. The ICG molecules remain trapped within the aggregate core by electrostatic interaction, and moreover, any change in the absorbance profile at later time points were attributable to the instability of the ICG molecules.

3.3.

Light Sensitivity

Figure 2 shows the normalized change in monomeric peak absorbance value, $\varphi \left(t\right)$ , for both MCs and freely dissolved ICG in the dark when irradiated with broadband light for various exposure durations. At a concentration of $50\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ , freely dissolved ICG was found to degrade significantly in response to broadband light exposure over the course of $2\mathrm{h}$ . MCs containing a comparable amount of ICG were found to degrade by a similar amount.

Fig. 2

Influence of broadband light exposure on, $\varphi \left(t\right)$ , normalized monomeric peak absorbance $\mathrm{abs}(\lambda =780\mathrm{nm})$ of freely dissolved ICG (left) and MCs (right) under different exposure conditions. Freely dissolved ICG and ICG within MCs experienced similar amounts of monomeric degradation at $50\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ . At a more dilute concentration $(10\mathrm{\mu }\mathrm{g}∕\mathrm{ml})$ , ICG monomers within MCs were more stable, while monomers in freely dissolved ICG degraded much more significantly.

As the profiles of $\varphi \left(t\right)$ versus $t$ yielded the greatest correlation coefficients, the rate of degradation for peak absorbance, and therefore the degradation rate for nondegraded monomeric ICG, followed first-order kinetics. The rate constants were determined by the slope of the linear fits and are provided in Table 1 . Interestingly, the degradation rate constants are dramatically different for more dilute preparations (20% of the original concentration) that were irradiated with the same light exposure conditions. When compared to the typical ICG concentrations utilized for this study, the values in Table 1 reveal that the peak absorption value decreases much further for a dilute solution of freely dissolved ICG, with a degradation rate constant nearly 3 times higher than that of the typical solution. This is consistent with previous reports that demonstrate ICG’s increased sensitivity to light exposure at lower concentrations.¹⁸ Conversely, a more dilute suspension of MCs demonstrates greater photostability than the more concentrated preparations of MCs and freely dissolved ICG, evident by a more gradual slope. The results suggest that the protection of ICG from photodegradation provided by encapsulation within MCs is concentration dependent, with more dilute capsule preparations allowing greater stability.

Figure 3 shows the $\zeta (t=120\min ,\lambda )$ profiles for samples that were exposed to broadband light for 120 minutes, the maximum exposure duration. We present results for the wavelength region between 600 and $850\mathrm{nm}$ , as the absorbance outside this region was essentially negligible both before and after light exposure. In all cases, near maximal change in $\zeta$ is found at the monomeric peak, $\lambda =780\mathrm{nm}$ , indicative of the monomers’ sensitivity to light-induced degradation. In the case of freely dissolved ICG at $50\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ , the $\zeta (t=120\min ,\lambda )$ profile exhibits negative values at wavelengths greater than $\lambda =820\mathrm{nm}$ , suggesting that light exposure for $120\min$ resulted in increased molar extinction coefficient $\epsilon$ in this wavelength region. Other investigators have also reported¹⁸ increased absorbance at wavelengths greater than $\lambda =780\mathrm{nm}$ in response to laser light exposure for nearly $1\mathrm{h}$ . As it is assumed that the degraded ICG molecules have minimal absorbance at the monomeric and dimeric peak for nondegraded ICG, the values of $\zeta (\lambda =780)$ and $\zeta (\lambda =680)$ can be regarded as the fraction of ICG monomers and dimers that degraded over the course of the experiment, respectively. Approximately 83% of the monomers of the freely dissolved ICG in the dilute solution $(10\mathrm{\mu }\mathrm{g}∕\mathrm{ml})$ degraded, while roughly 50% of the monomers of the freely dissolved ICG and MCs degraded $50\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ concentrations. In the case of the dilute suspension of MCs, 30% of the monomers degraded when exposed to light for $120\min$ . The freely dissolved ICG samples exhibited dramatic change at the monomeric peak in comparison to the dimeric peak. The flatter profiles of the ICG-MCs suggest that ICG monomers, aggregates, and other oligomer forms of the molecule exhibit comparable sensitivity to light induced degradation.

Fig. 3

Spectral absorbance ratio $\zeta (t,\lambda )$ at $2\mathrm{h}$ of broadband light exposure for freely dissolved ICG (left) and MCs. Freely dissolved ICG degraded more readily at the more dilute concentration $(10\mathrm{\mu }\mathrm{g}∕\mathrm{ml})$ while the converse was observed in MCs.

The $\zeta (t,\lambda )$ values at ( $t=48\min$ , $\lambda =680\mathrm{nm}$ ) for freely dissolved ICG at concentration of $50\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ shows that approximately 20% of the dimers degraded, whereas nearly 70% of dimers degraded in the case of freely dissolved ICG at concentration of $10\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ . The degradation of the monomeric and dimeric molecules resulted in discoloration of freely dissolved ICG at both concentrations. This discoloration was not as pronounced within the MC suspension. For the dilute $(10\mathrm{\mu }\mathrm{g}∕\mathrm{ml})$ MC suspension, only 26% of the dimers degraded, whereas 54% degraded in the MC suspension $50\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ samples.

3.4.

Temperature Sensitivity

To evaluate how encapsulation of ICG within MCs affects ICG sensitivity to temperature, preparations of MCs and freely dissolved ICG at concentration of $50\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ were incubated in the dark at 3, 22, and $40°\mathrm{C}$ for up to $96\mathrm{h}$ . Figure 4 displays $\phi \left(t\right)$ at various times for both systems at the three temperatures. In all cases, the correlation coefficients were $<0.8$ , suggesting that the degradation kinetics did not suitably follow zero-, first-, or second-order trends. Nevertheless, the best-fit profiles for first-order kinetics are included in the figures. Freely dissolved ICG is far more vulnerable to thermal degradation than the ICG within the MCs, as the monomeric peak absorbance decreased by less than 10% for the MCs stored at $40°\mathrm{C}$ after $48\mathrm{h}$ . Freely dissolved ICG also experienced noticeable degradation at 22 and $3°\mathrm{C}$ , whereas virtually no degradation occurred for MCs stored at these temperatures over the course of the experiments.

Fig. 4

Influence of temperature on normalized peak absorbance $\varphi \left(t\right)$ of freely dissolved ICG and MCs.

Figure 5 shows the normalized initial and final absorbance spectra of both freely dissolved ICG and ICG-MCs at $50\mathrm{\mu }\mathrm{g}∕\mathrm{ml}$ incubated at $40°\mathrm{C}$ for $48\mathrm{h}$ . The spectra are normalized by the initial monomeric peak value $\mathrm{abs}(t=0,\lambda =780\mathrm{nm})$ . The spectrum for freely dissolved ICG changed dramatically, exhibiting substantially reduced absorbance and broadening at the peak values as well as the emergence of a new, small peak near $850\mathrm{nm}$ . However, the overall spectrum for the MCs retained the same shape, while the peak values diminished slightly. Similar, but less pronounced, changes in the monomeric and dimeric peaks were observed for the absorbance spectra of freely dissolved ICG incubated at 22 and $3°\mathrm{C}$ , while the spectra for the ICG encapsulated in MCs had little change.

Fig. 5

Absorbance spectra normalized to the monomeric peak $(\lambda =780\mathrm{nm})$ at $t=0$ for both freely dissolved ICG and MCs at $48\mathrm{h}$ of incubation at $40°\mathrm{C}$ . Baseline profiles prior to incubation at $40°\mathrm{C}$ are also presented for comparison.

Figure 6 shows $\zeta (t=48\mathrm{h},\lambda )$ at three different temperatures. All of the spectra display large negative values in the NIR region $(\lambda >800\mathrm{nm})$ . This might be attributable to the increase in absorbance over time that results from the first stages of J-aggregation. Reports have shown that, at room temperatures, J-aggregation of ICG molecules in solution does not set in for several weeks.²⁰ However, because the initial absorbance in this region is so low $(\ll 1)$ , even a slight change is emphasized, i.e., normalizing by ${\mathrm{abs}}_0$ $\left(\lambda \right)\ll 1$ yields a more pronounced value for $\zeta (t,\lambda )$ . With increasing temperature, ICG solution is more prone to degradation, as indicated by the higher values for $\zeta \left(\lambda \right)$ . The maximal change for freely dissolved ICG occurs at the monomeric peak $(\lambda =780\mathrm{nm})$ , indicating that the monomers degraded by 13, 25, and 42% when stored at 3, 22, and $40°\mathrm{C}$ , respectively. ICG dimers also experience noticeable degradation, by as much as 12, 19, and 20% after being stored at 3, 22, and $40°\mathrm{C}$ , respectively. The high values near $\lambda =680\mathrm{nm}$ indicate the degradation of dimers, although the actual local maximum occurs closer to $705\mathrm{nm}$ .

Fig. 6

Spectral absorbance ratios for freely dissolved ICG and MCs, $\zeta (t,\lambda )$ , at $48\mathrm{h}$ of incubation at different temperatures.

Table 1

Calculated degradation rate constants for freely dissolved ICG and MCs in response to broadband light exposure; all constants have units of inverse minutes.

For the MCs, the maximal change in absorbance occurs around the dimeric peak in the samples stored at 3 and $22°\mathrm{C}$ . The negative $\zeta (t=48\mathrm{h},\lambda =680\mathrm{nm})$ values of $-13$ and $-15\%$ suggest that more dimers are present after incubation at 22 and $3°\mathrm{C}$ , respectively. At these temperatures, a slight negative change is also observed at the monomeric peak, however, the change is negligible and most likely a consequence of experimental error. For the sample stored at $40°\mathrm{C}$ , the wavelengths at which there is the most pronounced change in absorbance occurs between 620 and $640\mathrm{nm}$ , with a steady decline in sensitivity at higher wavelengths. At all three temperatures, $\zeta \left(\lambda \right)$ is significantly lower for the MCs than for freely dissolved ICG stored under the same conditions. At all temperatures, less than 7% of the ICG monomers located within the MCs underwent degradation.