Indocyanine green dye based bimodal contrast agent tested by photoacoustic/fluorescence tomography setup.

Multimodal imaging systems are in high demand for preclinical research, experimental medicine, and clinical practice. Combinations of photoacoustic technology with other modalities including fluorescence, ultrasound, MRI, OCT have been already applied in feasibility studies. Nevertheless, only the combination of photoacoustics with ultrasound in a single setup is commercially available now. A combination of photoacoustics and fluorescence is another compelling approach because those two modalities naturally complement each other. Here, we presented a bimodal contrast agent based on the indocyanine green dye (ICG) as a single signalling compound embedded in the biocompatible and biodegradable polymer shell. We demonstrate its remarkable characteristics by imaging using a commercial photoacoustic/fluorescence tomography system (TriTom, PhotoSound Technologies). It was shown that photoacoustic signal of the particles depends on the amount of dye loaded into the shell, while fluorescence signal depends on the total amount of dye per particle. For the first time to our knowledge, a commercial bimodal photoacoustic/fluorescence setup was used for characterization of ICG doped polymer particles. Additionally, we conducted cell toxicity studies for these particles as well as studied biodistribution over time in vivo and ex vivo using fluorescent imaging. The obtained results suggest a potential for the application of biocompatible and biodegradable bimodal contrast agents as well as the integrated photoacoustic/fluorescence imaging system for preclinical and clinical studies.


Introduction
A powerful emerging field of photoacoustic (also called optoacoustic) imaging combines the high optical contrast provided by optical absorption and high spatial resolution due to the use of ultrasound detection techniques [1,2]. This method demonstrated impressive applications for intraoperative imaging of pathological tissue [3]; visualization of sentinel lymph nodes in oncology [4][5][6]; and angiography of blood vessels [7]. Photoacoustic imaging of endogenous chromophores, such as melanin and haemoglobin, has been also used to visualize vascularization [1,[8][9][10]. In many applications, verification of the signal origin among artefacts and endogenous noise sources can be enhanced by integrating the multimodal system with bimodal contrast agents. An advantage of photoacoustic imaging compared to multiphoton microscopy or optical coherence tomography (OCT) is much deeper tissue penetration -up to 30-50 mm vs 0.4-1 mm and ≤ 2 mm, respectively [11]. There are multimodal imaging systems, which utilize a combination of photoacoustics with ultrasound [12,13], OCT [14,15] or multiphoton microscopy [16]. A combined photoacoustic and fluorescence tomography excels in spatial resolution (up to tens of microns), deep tissue penetration (several centimetres), and high sensitivity at low concentrations of contrast agents down to nM [17]. There are many photoacoustic contrast agents such as multilayer nanocomposites [18], spacers [19], magnetic bacteria nanoparticles [20]; some of those are bimodal and also provide fluorescence response such as liposomes [21] and micelles [22], inorganic nanoparticles [23][24][25], perfluorocarbon droplets [26], organic nanoparticles [27] and fluorescent dyes [28,29]. The synthesis of bimodal fluorescent and photoacoustic contrast agents based on the polyelectrolyte capsules with a fluorescent dye as a signalling compound was previously demonstrated [30]. The bimodality of such contrast agents is usually achieved by combining several signalling compounds for each modality, but the number and type of the components (e.g. inorganic nanoparticles) increase the time required for clinical trials. There are some bimodal contrast agents, which use only one signalling compound: micelles made of nonionic surfactant and encapsulated with fluorescent dye [22] or perfluorocarbon droplets with hydrophobic fluorescent dyes [26], where useful volume is occupied by signalling compound and little to no place is left for encapsulation of a drug or active substances. Another examples are organic nanoparticles made of antibiotics with fluorescent dye [27] and inorganic nanoparticles [31,32], which are highly specialized for chemo-photothermal therapy, T-cells and liposomes labeled with fluorescent dye [33,34]. Fluorescent dyes by themselves can also be used as molecular probes [28,29], but they are quickly excreted from the body making it difficult to maintain imaging contrast for long time. Having only a fluorescent dye as a contrast agent limits their multifunctionality. An autofluorescence imaging technique is also being developed, but its spectral range is beyond the transparency window of biological tissues [35].
Recently, we utilized the self-quenching effect to achieve bimodality using only indocyanine green (ICG) fluorescent dye as a signalling compound and found that the photoacoustic signal of those particles depends on the amount of dye in the shell [30]. However, the explanation of fluorescence signal dependency on the dye concentration and localization has not been explored. Furthermore, bimodal fluorescent and photoacoustic imaging in the study mentioned above was performed using independent separate fluorescent and photoacoustic setups and different sample holders like plastic cuvettes and polyvinyl chloride plastisol (PVCP) blood vessel phantoms. Separate imaging setups for photoacoustic and fluorescence modalities were conventionally used [6,29] and some progress in simultaneous imaging has been made [36,37]. Therefore, the main goal of this study was to explore contrast agent particles with a higher amount of dye in the shell using the unique commercially available integrated photoacoustic and fluorescence imaging instrument (TriTom, PhotoSound Technologies, Houston, TX).

Contrast agent preparation
Spherical porous vaterite particles with an average diameter of 5 ± 1 µm were used as a core for preparation of the contrast agent. That size of particles was chosen for its convenience to work with as a model of contrast agent as the size of particles for clinical application e.g. cancer treatment should be in submicron range and the size decrease will be the next step for development of a contrast agent [38]. Vaterite synthesis protocol [39] was modified as described further. Calcium chloride dihydrate and anhydrous sodium carbonate (Sigma-Aldrich) were used to prepare the solutions. All of the solutions were made using deionized (DI) water (specific resistivity, higher than 18.2 M cm) from a Milli-Q Integral 3 water purification system (Millipore). The synthesis started with adding of 2 ml of 1M Na 2 CO 3 solution to 10 ml of H 2 O and after that 2 ml of 1M CaCl 2 solution were injected under vigorous agitation. Then, 30 seconds later, agitation was halted and the dispersion of the particles was separated by centrifugation and washed two times with DI water.
Vaterite cores were loaded, as showed in Fig. 1(a), by freezing-induced loading (FIL) [40] with ICG and bovine serum albumin (BSA) conjugate. The loading cycle includes mixing of template particles with the loading substance and freezing it under rotation ( Fig. 1(a), step 1) and thawing after complete freezing ( Fig. 1(a), step 2). The amount of dye loaded into the core was controlled by the number of freezing-thawing cycles of FIL method. We prepared five types of cores applying a different number of freezing cycles: 0, 3, 6, 9, 11. The complex is further referred as ICG&BSA and the concentrations of BSA and ICG in the complex are 0.8 mg/ml and 0.05 mg/ml respectively. ICG&BSA was prepared via solution with concentrations of 14.3 mg/ml of BSA and 1 mg/ml of ICG. Bovine serum albumin dissolved in 0.01 M phosphate-buffered saline (PBS) (Sigma-Aldrich) and indocyanine green (MP Biomedicals), dissolved in DI were used to prepare complex. The dialysis was performed to remove the unbound dye using cellulose dialysis tubing with 20 µm pore and 28 mm tube diameters (Orange Scientific, Belgium) Step 1 is the mixing the template particle suspension with the loading ICG&BSA solution; step 2complete freezing of the mixture. Sample preparation scheme: (b) Layer-by-Layer assembly. 1 -adsorption of positively charged polyelectrolyte on the surface of the vaterite core particle; 2 -adsorption of negatively charged polyelectrolytes; 3 -adsorption of ICG fluorescent dye; 4 -dissolving the core particle to obtain a hollow shell; (c) the sample without core load, (d) the sample with loaded core and (e) the sample shell structure The contrast agent preparation consists of the four main steps. Those are core preparation, core loading ( Fig. 1(a)), core-shell formation and dissolution of the core ( Fig. 1(b), 1-4 steps). As a result, five types of samples were prepared: control sample without core load ( Fig. 1(c)) and samples with different dye amount loaded into the core, controlled by the number of freezing cycles ( Fig. 1(d)).
After the core loading, the polyelectrolyte shell was formed using layer-by-layer (LbL) approach. Dextran sulphate sodium salt (DEX; MW = 100 kDa) (Fluka), poly-L-arginine hydrochloride (PARG; Mw = 15-70 kDa) and sodium chloride (Sigma-Aldrich) were used to prepare PARG and DEX in 0.15 M NaCl solutions. The concentration of polyelectrolytes in the solutions was 1 mg/ml. To form a polyelectrolyte layer, CaCO 3 cores were suspended in a test tube with PARG solution and left in the rotator for 15 minutes. After that, the suspension was centrifuged and washed three times with DI water. Every layer of the forming shell was deposited in a similar way in order to obtain the following composition of the shell: (PARG/DEX)(PARG/ICG)(PARG/DEX) 2 ( Fig. 1(e)). The deposition of the ICG layer was done using an aqueous dye solution with the concentration of 1 mg/ml.
To finish the preparation of the contrast agent after forming the shell, the vaterite core was dissolved. We used ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich) and sodium hydroxide (NPO «Ecros») to prepare 0.2M EDTA solution with pH = 7. This solution was added to the water suspension of core-shell particles and after that the core was dissolved, leaving polyelectrolyte microcapsules. After the core dissolution, microcapsules were washed with DI water five times to remove the excess of EDTA and dissolution products.

Tomography setup description and measurements
The photoacoustic and fluorescence imaging system (TriTom) layout: the imaging chamber ( Fig. 2(b)) consists of a glass cylinder (1); centred rotary scanning stage (2); optical/photoacoustic orthogonal excitation (3); optical/photoacoustic epi-excitation (4); curve-linear 96-element photoacoustic (PA) transducer array (5); and a fluorescence-enabled sCMOS camera (6). The sample holder ( Fig. 2(a)) was mounted onto the shaft embedded in the rotary stage and centred with respect to the photoacoustic detector enabling a spherical tomographic configuration. Orthogonal to the plane of the curve-linear transducer array there are two fibre bundle outputs positioned outside the imaging chamber 45°with respect to the curve-linear transducer plane there are two other fibre bundle outputs, designed to induce epi-excitation of photoacoustic imaging. All fibre bundle outputs are vertically aligned with the centremost element of the transducer array (48th element out of 96 elements). The fibre bundles are pulsed with a laser at 10 Hz using a wavelength range of 670-1064 nm. The fibre bundle input is connected to the laser (optical parametric oscillator or OPO) output and the arrangement of individual fibres in each bundle is randomized with respect to the fibre bundle output. In the case of a superficial imaging scan, a separate 532-nm excitation wavelength input is used providing output only to the epi-excitation fibre bundle pair. The epi-excitation fibre bundle outputs have a slightly increased width (∼1.5 mm) to accommodate the randomized fibres of both OPO input and 532-nm input.
The samples were loaded into the polytetrafluoroethylene (PTFE) tubes of inner diameter d = 0.81 mm and wall thickness of 0.05 mm and arranged in the sample holder as shown in Fig. 2(a). DI water was used as a negative imaging control and CuSO 4 100 mM solution was used as a reference providing photoacoustic-only response [41].
Fluorescent images were captured using a sCMOS camera with the emission filter set for ICG imaging. Only the excitation wavelength of 780 nm was used for dual-modality fluorescence/photoacoustic imaging. Fluorescence signals of the samples were determined by averaging image intensity values of each tube's cross-section over multiple the z-sections, the data obtained was then used in statistical computation (mean and max values, 8-bit scale). The gain of photoacoustic detection electronics was set at 65 dB for all PA scans. The PA probe has a 6 MHz ± 10% central frequency, bandwidth > 55% at -6 dB in Transmit/Receive. To obtain PA spectra of the sample, image intensity values of each tube's cross-section at every wavelength were averaged over multiple the z-sections. The water extinction correction was applied according to [42]. OPO output laser energy was set to E = 60 ± 5 mJ per pulse. The fluence at a target for the experimental setup was calculated as 2.5 ± 0.2 mJ/cm 2 from a single fiber output, estimating illumination area with full width at half maximum of the beam profile at 680 nm, the highest water transmission wavelength used. The maximal energy of the peak beam profile was 5.0 ± 0.4 mJ/cm 2 for max energy. The maximum possible fluence is the sum all four fiber outputs, which is equal to 20.0 ± 1.6 mJ/cm 2 .

Extinction measurements
Extinction spectra of microcapsules were measured by Thermo Scientific GENESYS 30 Visible Spectrophotometer (USA) using a polystyrene cuvette (Mfr. No. 759075D, Thomas Scientific, USA). The extinction spectra of ICG and ICG&BSA were measured by Tecan Infinite M Nano + (Tecan Trading AG, Switzerland) using a plastic 96-well plate.

ICG loading evaluation
ICG stock solution was diluted to ICG concentrations of 23, 11, 6, 3, 1.4, 0.7, 0.3, 0.2, and 0.1 µM. ICG&BSA stock solution was diluted to ICG concentrations of 65, 32, 16, 8, 4, 2, 1 and 0.5 µM. The extinction spectra of those solutions were used to calculate calibration baselines [30]. The supernatant liquid was collected every time after ICG&BSA was loaded into the core and ICG deposited into shell and extinction was measured. The extinction data were used to estimate ICG concentration in the supernatant. The mass of loaded ICG was calculated using the initial concentration of the dye and concentration in the supernatant. The total load is a sum of loaded masses in each load cycle. Particle concentration was measured via haemocytometer. ICG amount per particle was calculated by dividing the total amount of dye loaded in the sample by the particle concentration.

Cell toxicity evaluation and in vivo measurements
Cell toxicity evaluation of free ICG solution and capsules without core load (sample 0) was conducted using 4T1 cell line by MTS-assay. Cells were seeded in a 96-well culture plate in RPMI medium (Thermo Scientific) at concentration of ∼10 4 cells per well, cultured at 37 •C in humidified air with 5% CO 2 for 24 h. Bio-Rad TC20 Automated Cell Counter was used to measure cell concentration in the used cell suspension. After seeding the well-plate, an appropriate amount of sample 0 suspension and free ICG solution was added to each well in order to get the concentration of 5, 10 and 20 capsules per cell and 8, 16, and 33 µg/mL for free ICG solution. The well-plate was subsequently incubated for 48 h. Non-treated cells were used as a control. Then the CellTiter 96 AQueous One Solution (G3582 Promega) was added to cells and incubated for 4 h according to the manufacturer's protocol. The absorbance was measured at 490 nm by using a Tecan Infinite M Nano + (Tecan Trading AG, Switzerland).
In vivo studies were performed using FvB male mice (6-8 weeks old, 4 mice in group). All experiments were performed in accordance with relevant guidelines and regulations, animal ethics clearance was approved by the Animal Ethics Committee of Saratov State Medical University. For this in vivo experiment, the capsules suspension concentration of (24 ± 5) · 10 6 /ml was chosen based on previous work [38], where the concentration of capsules no more than 10 8 /ml, and on the previously used concentrations in the TriTom setup measurements. The mice were anesthetized using 1.5-2% isoflurane in the induction chamber, then 100 µL of the sample 0 suspension in PBS was administered via tail vein injection. In vivo biodistribution study was conducted on a group of mice and two untreated mice taken as a negative control. The mice were imaged at the 0 h, 0.1 h, 1 h, 3h, 5 h, 8 h, 24 h and 48 h timepoints with the IVIS SpectrumCT in vivo imaging system (PerkinElmer, USA) by using the excitation/emission ratio at 745/810 nm. Photons were quantified with the LivingImage software (PerkinElmer, USA). At each time point, blood was collected using heparinized capillaries via retro orbital puncture to assess the time of capsule circulation in the blood. The fluorescence intensity of blood at each time point was determined by measuring the fluorescence signal at 780/830 nm using the Infinite M Nano + . Non-treated mice (PBS injection) were used as control for all fluorescent measurements.
Ex vivo biodistribution study. Mice were euthanized at 48 h after intravenous administration of capsules followed by harvesting of internal organs including heart, liver, lung, kidney, spleen. The organs were imaged with the IVIS SpectrumCT by using the excitation/emission ratio at 745/810 nm. Photons were quantified with the LivingImage software.

Contrast agent spectra
The resulted extinction (combined contribution from absorption and scattering) and PA spectra measurements of the samples are shown in Fig. 3(a) and (b) respectively. The extinction spectra were used instead of the absorption spectra because the average diameter of indocyanine green dye/polymer based particles is 5 ± 1 µm. Thus, the light scattering caused by these particles is required to be considered during extinction spectra measurements. The shape and relative magnitude of extinction and PA signals for each sample are qualitatively comparable. Figure 3(c) is the comparison of extinction spectra of the samples with PA spectra normalized to the extinction maxima, providing better visualization of the correlation between PA and extinction spectra.
The PA spectrum of CuSO 4 closely matches the extinction spectrum. All the PA spectra of the samples closely match their extinction spectra up to 820 nm, at which point the PA values drop with respect to the extinction data. The discrepancy of extinction and PA spectra of the samples beyond 820 nm excitation could be explained by types of the containers used for measuring: plastic cuvettes for extinction and PTFE tubes for PA imaging setup. The wavelength range of both extinction and PA spectra of the samples lies within biological tissue transparency window [43]. Figure 4(a-c) shows cross-sections of fluorescent and photoacoustic images of the samples. All contrast sample tubes are visible in both modalities, except CuSO 4 which is not fluorescent and a control DI water. In the PA image ( Fig. 4(b)) we can see that the samples 1-5 differ in shape from sample 6 which is CuSO 4 solution. The fluorescent image (Fig. 4(a)) reveals similar incomplete circular shapes of sample images cross-sections. This effect can be explained by the visually observed adsorption of the particles on the tube walls. Apparently, this is caused by the hydrophobic effect and an indirect confirmation for this is that the more dye is present in the core, the stronger the observed adsorption. Because of this, the particles are densely packed at the surface of the tube producing ring-type images. Sample 2 shows little fluorescence but has the most intensive PA signal among contrast agent samples. Samples 3-5 show the opposite effect: low PA signal and high intensity fluorescence. Figure 4(c) shows the overlap of the PA and fluorescence cross-section images.  Figure 4(d) is the photograph of the sample holder with samples loaded into PTFE tubes. The obtained data were used to reconstruct the 3D image of the samples (Fig. 4(e), Visualization 1). The 3D image shows in greater detail that the sample particles precipitated on the tube walls.

Extinction, photoacoustic signal, fluorescent intensity and mass values correlation
The obtained particle concentrations, which appeared to be (24 ± 5) · 10 6 /ml for samples 0, 6, 9 and (23 ± 5) · 10 6 /ml for samples 3 and 11, along with calculated core loads were used to estimate the amount of dye loaded per particle (Fig. 5(a)). The figure shows that the amount of ICG loaded in the shell depends on the amount of ICG loaded into the core: the more dye present in the core, the less is adsorbed onto the shell. The dye is affected by the concentration gradient and electrostatic force, which attracts or repels the molecules, depending on the core's content. ICG in solution is charged negatively and is attracted to a positively charged polyelectrolyte layer of the shell. The sample without dye in the core has a concentration gradient directed from the core which causes diffusion toward the core, thereby increasing the amount of loaded ICG. Both forces increase the adsorption of ICG in the shell, which results in a larger amount of ICG in the shell. In samples with a loaded core the high concentration of ICG inside the core creates the overall negative charge, which repels ICG from the shell and creates a concentration gradient directed into the core, with subsequent diffusion outwards from the core. This phenomenon was observed before [18] and could be explained by the coulombs forces and concentration difference affecting the adsorption rate [30].
Comparing the amount of loaded ICG mass into the sample's shell (Fig. 5(a)) and the PA signals of the samples (Fig. 5(b)) suggests that PA signal is mainly produced by the shell and has dependency on the dye loaded amount into the shell. Additionally, the amount of loaded ICG&BSA complex influences the shell thickness with increasing concentration of complex, probability of adsorption of that complex to the inner positively charged layer of the shell is increased and thus decreases the PA signal from the shell [30]. Comparison of the fluorescence signals of the samples (Fig. 5(c)) and the total ICG mass loaded into the samples (Fig. 5(a)) allows us to conclude that fluorescence signal depends on the total amount of dye in the samples, except for sample 3. Figure 5(c) shows mean and maximum fluorescence intensity as it has uneven distribution in the tube cross-section due to the adsorption of the samples to the tube walls.
The outlier fluorescence result for sample 3 can be explained by the highest dye amount in the shell. At this dye concentration, reabsorption of the core-emitted fluorescence by the overlapping excitation and emission spectra was achieved, which resulted in the decrease of overall sample fluorescence signal. The schematics of this phenomenon is shown in Fig. 5(d) for the sample 0 without loaded core and sample 3 (Fig. 5(e)) and 11 ( Fig. 5(f)). Fluorescence emission is denoted as I, photoacoustic response -PA, shell thickness -d, thickness of the ICG layer -d FL . For samples 0, 3 and 11 indexes in the figure denoted as 1, 2 and 3 respectively. As it was shown before [30], the amount of dye loaded into the core has an impact on the shell thickness and therefore d 3 > d 2 > d 1 , considering the effect of decreasing the amount of dye in the shell with an increasing amount of dye in the core, d FL1 > d FL2 > d FL3 .

Cytotoxicity, in vivo and ex vivo studies
We estimated the concentration-dependent toxicity of particles loaded with ICG loaded in the shell (sample 0) compared to free ICG solution and non-treated cells as controls. A series of concentration dilutions of samples were added to the 4T1 cell culture, incubated for 48 h (5% CO 2 , 37°C), cell viability was measured using the MTS test. As a result, absorbance measurements after MTS assay for sample 0 and free ICG solution were normalized to non-threated cells values and plotted in Fig. 6. According to the article by De Koker et. al. that even 30 capsules per cell led to a decrease in viability down to 80% [44]. Therefore, we took concentrations of 5, 10 and 20 capsules per cell, which in our case showed no significant cytotoxic effect, which is also corresponds to this article.
In vivo experiments were performed using the sample 0 without core loads and results are shown below in Fig. 7. Capsules with ICG (sample 0) were intravenously administered to mice to identify organs that trap and accumulate capsules as well as to assess their further clearance from the bloodstream. Capsule biodistribution was monitored over time in vivo and ex vivo using live fluorescent imaging ( Fig. 7(a)). A strong fluorescent signal from the injected capsules appeared in organs within the first 5 min. Dynamic analysis of the whole-body fluorescence in living mice showed that capsules moved at approximately the same speed in all treated animals with a fluorescent signal peak at 3 h. The majority of capsules accumulated in the liver and lungs 1-3h after injection displayed by the strongest fluorescence in the projection of this organ ( Fig. 7(d)). After 5 h, the fluorescence intensity decreases. The capsules were nearly cleared from the bloodstream 1 h after injection, indicated by the reduced fluorescent intensity of blood at this time point (Fig. 7(b)). Mice were sacrificed respectively 48h after injection for ex vivo studies aiming for more precise identification of the capsule-trapping organs. Imaging of isolated organs revealed that liver accumulated the highest amounts of capsules at both time points, whereas the lowest quantity of capsules retained in the heart (Fig. 7(c, d)). Similar biodistribution was observed before for particles with shell formed by the same polyelectrolytes [45].

Conclusion
In this study the bimodal contrast agent based on organic dye/polymer particles was synthesized and characterized using a commercial photoacoustic-fluorescent tomography instrument (TriTom). All sample tubes with contrast agent are seen in both photoacoustic and fluorescent modes, but high intensities are concentrated near the tube walls. Only the reference CuSO 4 sample is shown as a solid (not hollow) cross-section. It is hypothesized that the organic dye/polymer particles were adsorbed to the tube walls. As a result, particles were densely packed changing the spatial distribution and intensity of the imaging signals. Cytotoxicity studies of the particles and free ICG solution have shown no significant cytotoxic effect at different concentrations on the 4T1 cells line. In vivo and ex vivo studies shown that sample quickly cleared from the blood stream and the most capsules were accumulated in the liver.
Also, it is important to note that the samples showed a known tendency of decreasing the amount of the shell loaded dye with an increasing amount of dye loaded into the core [18,30]. The contrast agent demonstrates significant signal levels of both modalities in the biological tissue transparency window. The samples 0 and 3 have high PA signal due to higher amount of dye in the shell in comparison with samples 6-11, which additionally contain a significant amount of ICG&BSA that makes the shell thicker. This allows us to conclude that the shell contributes the most to the PA signal generation. It is shown, that PA signal is proportional to the amount of ICG in the shell of the particles. This correlation was observed before [18,30]. The fluorescent signal is proportional to the total amount of dye in the particles, except for sample 3, that has the highest amount of dye. In the case of that sample, high dye concentration in the shell produces self-quenching effect [29,46] and the fluorescence emitted by the core is reabsorbed in the shell [47][48][49]. The obtained results are extremely important for the field of multifunctional particle contrast agent combining bimodal imaging characteristics optimal for photoacoustic and fluorescent (PA&FL) modalities caused by ICG containing polymer shell and drug delivery function caused by encapsulating drug into the hollow volume formed by polymer shell as well as monitoring of such type particles using PA&FL commercially available setup TriTom. It opens new avenues for preclinical studies, clinical diagnostics and therapy using nanostructured particle constructs based on the FDA approved ICG dye.