**7. NIR flourescent proteinoid-PLLA particles**

#### **7.1. Synthesis of the NIR fluorescent Prot8 particles**

The optimal Prot8 particles were used to encapsulate ICG, a well-known NIR dye already in use in medical diagnostics. The NIR fluorescent particles were prepared by self-assembly of the crude Prot8, in the presence of ICG. Briefly, 100 mg of the dried fabricated Prot8 were resuspended in 10 mL of 10-5N NaCl solution. The mixture was then heated to 80°C while stirring for 15 min. To this solution, 1 mg (1% of the proteinoid polymer) of ICG was added. The mixture was then removed from the hot plate and was allowed to return to room tem‐ perature. During the cooling process hollow particles were formed and precipitated from solution. The obtained NIR fluorescent particles dispersed in water were then dialyzed versus 4 L of 10-5 NaCl aqueous solution overnight at room temperature.

#### **7.2. Determination of the encapsulated ICG concentration in the NIR fluorescent Prot8 particles**

A calibration curve of free ICG was obtained by measuring the integrals of absorbance peaks of standard solutions (0.5–10 μg/mL) in PBS, at wavelengths 630–900 nm. The concentration of the encapsulated ICG was determined by measuring the integral of the absorbance spectrum at 630-900 nm of a 1 mg/mL dispersion of the NIR fluorescent particles in PBS. An estimation of encapsulated ICG per mg of particles was determined according to the calibration curve.

#### **7.3. Characterization of the NIR fluorescent Prot8 particles**

Hydrodynamic and dry particle size and size distribution were determined by DLS and SEM, as mentioned above. For the SEM study, the diameter of more than 200 particles with image analysis software (AnalySIS Auto, Soft Imaging System GmbH, Germany). The self-assembly procedure produced spherical proteinoid particles of 145 ± 20 nm hydrodynamic diameter and 70 ± 15 nm dry diameter, as shown in Figure 7. The hydrodynamic diameter of these particles dispersed in water is illustrated by the typical light scattering measurement shown in Figure 7A. The dry diameter of the proteinoid particles is illustrated by the typical SEM photomicro‐ graph shown in Figure 7B.

In addition, absorbance spectra were obtained using a Cary 100 UV-Visible spectrophotometer (Agilent Technologies Inc.). Excitation and emission spectra were recorded using a Cary Eclipse spectrofluorometer (Agilent Technologies Inc.). As indicated in Figure 8, no shift of absorbance of the ICG after encapsulation is observed compared to that of the free ICG.

particles at both concentrations produced the highest LDH levels (up to 13% toxicity), when compared to untreated (blank) cells, indicating minor toxicity of these proteinoids to this cell line. Prot8 had the lowest cytotoxic effect on the cells treated with both concentrations, almost zero toxicity. This proteinoid is therefore the most suitable for treating cells, considering its

The optimal Prot8 particles were used to encapsulate ICG, a well-known NIR dye already in use in medical diagnostics. The NIR fluorescent particles were prepared by self-assembly of the crude Prot8, in the presence of ICG. Briefly, 100 mg of the dried fabricated Prot8 were resuspended in 10 mL of 10-5N NaCl solution. The mixture was then heated to 80°C while stirring for 15 min. To this solution, 1 mg (1% of the proteinoid polymer) of ICG was added. The mixture was then removed from the hot plate and was allowed to return to room tem‐ perature. During the cooling process hollow particles were formed and precipitated from solution. The obtained NIR fluorescent particles dispersed in water were then dialyzed versus

**7.2. Determination of the encapsulated ICG concentration in the NIR fluorescent Prot8**

A calibration curve of free ICG was obtained by measuring the integrals of absorbance peaks of standard solutions (0.5–10 μg/mL) in PBS, at wavelengths 630–900 nm. The concentration of the encapsulated ICG was determined by measuring the integral of the absorbance spectrum at 630-900 nm of a 1 mg/mL dispersion of the NIR fluorescent particles in PBS. An estimation of encapsulated ICG per mg of particles was determined according to the calibration curve.

Hydrodynamic and dry particle size and size distribution were determined by DLS and SEM, as mentioned above. For the SEM study, the diameter of more than 200 particles with image analysis software (AnalySIS Auto, Soft Imaging System GmbH, Germany). The self-assembly procedure produced spherical proteinoid particles of 145 ± 20 nm hydrodynamic diameter and 70 ± 15 nm dry diameter, as shown in Figure 7. The hydrodynamic diameter of these particles dispersed in water is illustrated by the typical light scattering measurement shown in Figure 7A. The dry diameter of the proteinoid particles is illustrated by the typical SEM photomicro‐

In addition, absorbance spectra were obtained using a Cary 100 UV-Visible spectrophotometer (Agilent Technologies Inc.). Excitation and emission spectra were recorded using a Cary Eclipse spectrofluorometer (Agilent Technologies Inc.). As indicated in Figure 8, no shift of absorbance of the ICG after encapsulation is observed compared to that of the free ICG.

**7. NIR flourescent proteinoid-PLLA particles**

4 L of 10-5 NaCl aqueous solution overnight at room temperature.

**7.3. Characterization of the NIR fluorescent Prot8 particles**

**7.1. Synthesis of the NIR fluorescent Prot8 particles**

low toxicity.

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**particles**

graph shown in Figure 7B.

**Figure 7.** Hydrodynamic size histogram (A) and SEM image (B) of the P(EF-PLLA) NIR fluorescent nanoparticles.

However, due to the dye encapsulation process, the maximal absorbance peak of the free ICG changed from 779 nm to 718 nm, probably since the ICG molecules get close to each other inside the nanoparticle interior and aggregation of the dye may occur causing this change in absorption peaks [56,57]. Furthermore, a 12 nm blue-shift of the emission spectrum of the NIR fluorescent particles compared to the free dye in solution is also observed.

The estimation of encapsulated ICG showed that the complete quantity of ICG used in the encapsulation procedure was encapsulated within the Prot8 nanoparticle interior. Following particle formation, leakage of the encapsulated ICG into PBS not-containing and containing 4% albumin at room temperature was not observed, indicating that the dye is strongly associated within the Prot8 particles, probably due to physical interactions between the dye and the polymer hydrophobic portions assembled in the core of the particles.

As suggested before, the proteinoid forms particles of different sizes according to the nature of its surrounding [40]. When discussing Prot8, the hydrophobic portions, in this case mainly the PLLA segments and the aromatic rings of the phenylalanine portion are assembled within the particle matrix, while the polar hydrophilic groups (mainly carboxylates) are exposed to the aqueous environment. This way, the self-assembly yields particles that encapsulate within them the ICG associated with the hydrophobic core via hydrophobic interaction, as illustrated in Figure 9.

#### **7.4. Optimization of the ICG concentration entrapped within the Prot8 particles**

In order to optimize the particles fluorescence intensity, different concentration (0.5, 1, 2 and 5% w/w relative to Prot8) of ICG were added to the Prot8 hot solution, prior to the formation of the particles through the self-assembly process, as described above. The NIR fluorescent

**Figure 8.** Absorbance and emission spectra of free ICG (dotted lines) and ICG-encapsulated Prot8 particles (solid lines) dispersed in water.

**Figure 9. Schematic representation of the self-assembled NIR fluorescent particles.** Hydrophobic moieties are repre‐ sented by scribbled lines, ICG is represented by the interior green dots.

Prot8 particles dispersed in PBS were diluted to 1 mg/mL and their fluorescence intensities were measured at 809 nm. The encapsulated ICG concentration that gave the maximum fluorescence intensity of the resultant NIR fluorescent particles was 1% w/w relative to Prot8. At higher dye concentrations, quenching of the fluorescence was observed, as the dye molecules encapsulated within the Prot8 nanoparticle are close to each other, resulting in nonemissive energy transfer between them.

#### **7.5. Photostability of the NIR fluorescent Prot8 particles**

In order to examine the photostability of the encapsulated ICG as opposed to free ICG, photobleaching experiments were performed for both the encapsulated and the free dye. For this purpose, an aqueous solution of ICG (0.05 M) in PBS was prepared, and the

fluorescence intensity with λex set at 780 nm and λem set at 800 nm was measured. A dispersion of NIR fluorescent Prot8 particles in PBS was prepared, and diluted to give similar fluorescence intensity to the dye at the same wavelengths. The excitation and emission slits were opened to 20 nm and 5 nm, respectively. Each of the samples was illuminated continuously with a xenon lamp, and the fluorescence intensity was meas‐ ured over a period of 20 min by a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies Inc.). Intensity values were normalized for comparison. Figure 10 illustrates that during illumination, the fluorescence intensity of the ICG-containing Prot8 particles remains intact while that of the free ICG decreased significantly. The photobleaching of ICG is significantly reduced by the encapsulation within the proteinoid-PLLA particles. The encapsulation probably protects the free dye from light-inducing factors such as oxygen, oxidizing or reducing agents, temperature, exposure time and illumination levels, which may reduce the fluorescence intensity irreversibly [58,59].

**Figure 10. Photostability of the ICG-containing Prot8 particles (A) and free ICG (B) as function of time.** Samples of ICG-containing Prot8 particles and free ICG were illuminated with a Xenon flash lamp for 20 min, as described above.

#### **7.6. In vitro cytotoxicity of the Prot8 particles**

Prot8 particles dispersed in PBS were diluted to 1 mg/mL and their fluorescence intensities were measured at 809 nm. The encapsulated ICG concentration that gave the maximum fluorescence intensity of the resultant NIR fluorescent particles was 1% w/w relative to Prot8. At higher dye concentrations, quenching of the fluorescence was observed, as the dye molecules encapsulated within the Prot8 nanoparticle are close to each other, resulting in non-

**Figure 9. Schematic representation of the self-assembled NIR fluorescent particles.** Hydrophobic moieties are repre‐

**Figure 8.** Absorbance and emission spectra of free ICG (dotted lines) and ICG-encapsulated Prot8 particles (solid lines)

In order to examine the photostability of the encapsulated ICG as opposed to free ICG, photobleaching experiments were performed for both the encapsulated and the free dye. For this purpose, an aqueous solution of ICG (0.05 M) in PBS was prepared, and the

emissive energy transfer between them.

dispersed in water.

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**7.5. Photostability of the NIR fluorescent Prot8 particles**

sented by scribbled lines, ICG is represented by the interior green dots.

In order to revoke cell toxicity of the NIR Prot8 particles, in vitro cytotoxicity of the particles was tested by using human colorectal adenocarcinoma LS174t, SW480 and HT29 cell lines. The cell lines are adherent to the used culture dishes. LS174t cells were grown in Minimum Essential Medium (MEM) eagle supplemented with heat-inactivated fetal bovine serum (FBS, 10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). SW480 cells were maintained in Dulbecco's MEM supplemented with heat-inactivated fetal bovine serum (FBS, 10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). HT29 cells were maintained in McCoy's 5A medium supplemented with FBS (10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). Cells were screened to ensure they remained mycoplasma-free using Mycoplasma Detection Kit [54]. Cell cytotoxicity was assessed by measuring the release of cytoplasmic lactate dehydrogenase (LDH) as described above.

Figure 11 exhibits the cytotoxicity levels of the Prot8 particles at two different concentrations (1.25 and 2.5 mg/mL). It can be seen that at both concentrations, the Prot8 particles have no significant cytotoxic effect on all three cell lines, compared to untreated (blank) cells, meaning that the particles may be used for biomedical applications as suggested, including drug delivery.

**Figure 11.** Cytotoxic effect of the NIR fluorescent Prot8 particles on human colorectal adenocarcinoma LS174t, SW480 and HT29 cell lines measured by the LDH assay. Cells (3×105 ) were incubated for 24 and 48 h with the Prot8 particles (1.25 and 2.5 mg/mL in PBS). Cells were incubated with 1% Triton-x-100 as positive control (100% toxicity). In addition, cells were incubated with Triton-x-100 1% and the Prot8 particles (2.5 mg/mL) to revoke any interaction of the particles with the LDH kit components. Untreated cells (negative control) were similarly incubated. Each bar represents mean ± standard deviations of 4 separate samples (originally published in [23]).

#### **7.7. In vivo biodistribution in a mouse model**

In order to examine the biodistribution in a living body, the NIR fluorescent Prot8 particles (2 mg/mL, 0.01 mg/kg body weight per mouse) were injected i.v. into mice through the tail vein and checked at several time intervals over 24 h. Male BALB/C mice (Harlan Laboratories, Israel) were utilized in this study under a protocol approved by the Institutional Animal Care and Use Committee at Bar-Ilan University. The biodistribution of the NIR fluorescent Prot8 particles was studied in normal 8-weeks-old mice, weighing 20-25 g at the time of experiment. Prior to the experiment, mice were anesthetized by intraperitoneal injection of Ketamine (40-80 mg/kg body weight) and Xylazine (5-10 mg/kg body weight), and the mice's skin was shaved with an electric animal clipper.

Essential Medium (MEM) eagle supplemented with heat-inactivated fetal bovine serum (FBS, 10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). SW480 cells were maintained in Dulbecco's MEM supplemented with heat-inactivated fetal bovine serum (FBS, 10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). HT29 cells were maintained in McCoy's 5A medium supplemented with FBS (10%), penicillin (100 IU/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). Cells were screened to ensure they remained mycoplasma-free using Mycoplasma Detection Kit [54]. Cell cytotoxicity was assessed by measuring the release of cytoplasmic lactate dehydrogenase (LDH) as described

Figure 11 exhibits the cytotoxicity levels of the Prot8 particles at two different concentrations (1.25 and 2.5 mg/mL). It can be seen that at both concentrations, the Prot8 particles have no significant cytotoxic effect on all three cell lines, compared to untreated (blank) cells, meaning that the particles may be used for biomedical applications as suggested, including drug

**Figure 11.** Cytotoxic effect of the NIR fluorescent Prot8 particles on human colorectal adenocarcinoma LS174t, SW480

(1.25 and 2.5 mg/mL in PBS). Cells were incubated with 1% Triton-x-100 as positive control (100% toxicity). In addition, cells were incubated with Triton-x-100 1% and the Prot8 particles (2.5 mg/mL) to revoke any interaction of the particles with the LDH kit components. Untreated cells (negative control) were similarly incubated. Each bar represents mean ±

In order to examine the biodistribution in a living body, the NIR fluorescent Prot8 particles (2 mg/mL, 0.01 mg/kg body weight per mouse) were injected i.v. into mice through the tail vein

) were incubated for 24 and 48 h with the Prot8 particles

and HT29 cell lines measured by the LDH assay. Cells (3×105

**7.7. In vivo biodistribution in a mouse model**

standard deviations of 4 separate samples (originally published in [23]).

above.

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delivery.

100 μL of either nanoparticle dispersion or free ICG solution (0.01 mg/kg body weight, dissolved in PBS) were administered to the mice through tail vein injection at a concentration of 2 mg/mL. During image acquisition, mice remained anesthetized by the intraperitoneal injection of Ketamine/Xylazine. Image cubes were obtained from the mice at several time points up to 24 h after injection. Each treatment group includes 3 mice for each time point (5 min, 20 min, 1 h and 24 h); 2 uninjected mice served as negative control. The experiment was repeated twice, testing a total of 52 mice. At the end of the experiment, the mice were euthan‐ ized by cervical dislocation, and organs were taken for imaging (liver, spleen, kidney, duode‐ num, colon, brain, heart, tibia bone and blood).

Whole body fluorescence images were acquired using a Maestro II in vivo fluorescence imaging system (Cambridge Research &Instrumentation, Inc., Woburn, MA). The system is equipped with a fiber-delivered 300W xenon excitation lamp, and images can be acquired from λ=500-950 nm by a 1.3 megapixel CCD camera (Sony ICX285 CCD chip). Each pixel within the image cube therefore has an associated fluorescence spectrum. The software for the Maestro system (Maestro 2.10.0) contains several algorithms to process the spectral data cubes to remove undesired auto-fluorescence signal and generate overlaid images for multiple fluorophores. A deep red excitation/emission filter set was used for our experiments (λex: 700-770 nm, λem>780 nm). The liquid crystal tunable filter (LCTF) was programmed to acquire image cubes from λ=780 nm-860 nm with an increment of 10 nm per image. The camera was set to 150 ms (whole body image), 15 ms (liver), 500ms (spleen), 7000ms (kidney), 10 ms (duodenum), 500 ms (colon), 1000ms (brain), 1000ms (tibia bones), 200ms (heart) and 1000ms (blood) exposure times. Fluorescence intensity measurements were performed using ImageJ NIH (National Institutes of Health) software.

Figure 12 shows whole body images of mice injected with the particles over time: at 5 min, 20 min, 1 h and 24 h from injection. 5 min post injection, there is an initial burst of fluorescence which subsided quickly, while the majority of the fluorescent particles concentrated in the liver, at 20 min. 24 h post injection, the fluorescence is almost non-existent, signifying the nanoparticle clearance from the body over 24 h. Biodistribution was tested for free ICG as well, and no significant differences in distribution and kinetics were found between particles containing ICG and free ICG up to 24 h post injection. These findings were in complete agreement with previous reports of ICG and ICG-containing particles pharmacokinetics and biodistribution, as the free dye in solution, derivatives of the free dye and ICG-containing particles are all evacuated from the body after 1 h and completely vanished 24 h after i.v. injection [60,61].

**Figure 12.** Typical whole body fluorescence images of the NIR fluorescent Prot8 particles at 5 min, 20 min, 1 h and 24 h after i.v injection. 12 mice (each experiment group contained 3 mice) were anesthetized and treated with NIR fluores‐ cent Prot8 particles (2 mg/mL, 0.01 mg/kg body weight per mouse). Blood was drawn and organs were harvested at each time point. 2 uninjected mice served as negative control. 12 mice were injected correspondingly with free ICG solution, giving similar results (not shown). The experiment was repeated twice with similar results (originally pub‐ lished in [23]).

**Figure 13.** Fluorescence intensities of different organs taken at 5 min, 20 min, 1 h and 24 h post i.v. injection into mice tail veins. 12 mice (each experiment group contained 3 mice) were anesthetized and treated with NIR fluorescent Prot8 particles (2 mg/mL, 0.01 mg/kg body weight per mouse). Blood was drawn and organs were harvested at each time point. 2 uninjected mice served as negative control. The experiment was repeated twice with similar results (originally published in [23]).

*Ex vivo* fluorescence images of specific organs and blood were also obtained. Organs from mice were harvested and blood was drawn 5 min, 20 min, 1 h and 24 h post injection of the particles into the tail vein. Figure 13 shows the calculated fluorescence intensities of the lungs, bones, brain, colon, duodenum, heart, liver, kidney, spleen and blood screening. Evidently, this analysis shows that the particles penetrated and were found in all checked organs. It is shown clearly that by 20 min most of the inserted quantity of the fluorescent particles is cleared from the blood. The particles concentrate mostly at the liver and are probably evacuated from the body. Interestingly, it is also apparent that the particles pass the blood-brain barrier (BBB), since they are found in the brain at 20 min post injection. This may open up a scope of drug targeting to the brain for drug molecules which are usually blocked. Overall, it was demon‐ strated that following a single i.v. injection of the particles, fluorescence intensity at all organs decreased over time, and only traces of fluorescence could be seen after 24 h.

#### **7.8. Conjugation of the tumor-targeting ligands to the particles**

PNA was covalently conjugated to the NIR fluorescent Prot8 particles by the cabodiimide activation method [62]. Briefly, EDC (1 mg) and Sulfo-NHS (1 mg) were each dissolved in 0.1 M MES (pH 6.0, 1 mL) containing 0.5 M NaCl. The EDC solution (1 mg/mL, 10 μL) was added to an aqueous solution of PNA (0.25 mg, 62.5 μL), followed by the addition of the sulfo-NHS solution (1 mg/mL, 25 μL). The mixture was then shaken for 15 min, followed by the addition of the NIR fluorescent Prot8 particles (2.5 mg in 1 mL PBS). The mixture was then shaken for 90 min. The obtained PNA-conjugated fluorescent particles were then washed from excess reagents by dilution and filtration through a 30-kDa filtration tube (VS2021 VIVA SPIN) at 1000 rpm (Centrifuge CN-2200 MRC) for 2 min, repeated three times. FITC–PNA, anti-CEA and anti-rabbit IgG were conjugated to the NIR fluorescent particles through a similar procedure. The concentration of bound PNA was determined with FITC–PNA by a calibration curve of FITC–PNA fluorescence using a multiplate reader (TECAN SpectraFluor Plus, Neotec Scientific Instruments). The concentrations of bound anti-CEA and anti-rabbit IgG were determined using a mouse IgG ELISA kit (Biotest, Israel). The calculated quantities of bound PNA and anti-CEA were 3.2 and 1.9 μg per mg particles, respectively.

### **7.9. Optical detection of human colon tumors in a chicken embryo model**

#### *7.9.1. The chicken embryo CAM model*

**Figure 12.** Typical whole body fluorescence images of the NIR fluorescent Prot8 particles at 5 min, 20 min, 1 h and 24 h after i.v injection. 12 mice (each experiment group contained 3 mice) were anesthetized and treated with NIR fluores‐ cent Prot8 particles (2 mg/mL, 0.01 mg/kg body weight per mouse). Blood was drawn and organs were harvested at each time point. 2 uninjected mice served as negative control. 12 mice were injected correspondingly with free ICG solution, giving similar results (not shown). The experiment was repeated twice with similar results (originally pub‐

**Figure 13.** Fluorescence intensities of different organs taken at 5 min, 20 min, 1 h and 24 h post i.v. injection into mice tail veins. 12 mice (each experiment group contained 3 mice) were anesthetized and treated with NIR fluorescent Prot8 particles (2 mg/mL, 0.01 mg/kg body weight per mouse). Blood was drawn and organs were harvested at each time point. 2 uninjected mice served as negative control. The experiment was repeated twice with similar results (originally

*Ex vivo* fluorescence images of specific organs and blood were also obtained. Organs from mice were harvested and blood was drawn 5 min, 20 min, 1 h and 24 h post injection of the particles into the tail vein. Figure 13 shows the calculated fluorescence intensities of the lungs, bones, brain, colon, duodenum, heart, liver, kidney, spleen and blood screening. Evidently, this

lished in [23]).

66 Advances in Bioengineering

published in [23]).

A chicken embryo CAM model was used to test the specific tumor detection by both the nonconjugated and the bioactive (PNA, anti-CEA or anti-rabbit IgG) conjugated NIR fluorescent Prot8 particles. Among most commonly used animal models, the chicken egg model allows the imaging of several tumors in a short time period and is less expensive [62]. Tumor cells were grafted on CAM according to the literature [62,64]. Briefly, fertile chicken eggs obtained from a commercial supplier were incubated at 37°C at 60–70% humidity in a forced-draft incubator. On day 3 of incubation, an artificial air sac was formed, allowing the CAM to drop. After 8 days of incubation, a window was opened in the shell and the CAM was exposed. Tumor cells were collected by trypsinization, washed with culture medium and pelleted by gentle centrifugation. Following removal of the medium, 5x106 cells were resuspended in 30 μL ice-cold Matrigel and inoculated on the CAM at the site of the blood vessels. Eggs were then sealed and returned to incubation. On day 6 post-grafting, day 14 of incubation, the tumor diameter ranged from 3 to 5 mm with visible neoangiogenesis.

Figure 14 shows typical SW480 cell line derived tumors bordered by plastic rings on a chicken embryo CAM inside the egg.

**Figure 14. Light photograph of SW480 cell line derived tumors bordered by a plastic ring on chicken embryo CAM.** Suspensions of 5×106 SW480 cells suspended in Matrigel formed compact structures (asterisk) 8 days after transplanta‐ tion with attraction of host blood vessels (arrow).

#### *7.9.2. CAM tumor detection*

Chicken embryos with 6-days-old human adenocarcinoma tumors (LS174t and SW480 cancer cell lines) implanted on the CAM were treated with the non-conjugated, PNA-conjugated and anti-CEA-conjugated NIR fluorescent Prot8 particles (40 μL, 2 mg/mL). Additionally, nonpathological CAM treated with non-conjugated particles and untreated tumors served as control groups. After 40 minutes, the nanoparticle dispersions were removed and the tumors were washed with PBS. Then, the tumors and the non-pathological CAM were removed from the eggs, washed again with PBS and spread on a mat black background for observation using a Maestro II™ in vivo imaging system (Cambridge Research & Instrumentation, Inc., Woburn, MA). A NIR excitation/emission filter set was used for the experiments (λex: 710–760 nm, λem > 750 nm). The Liquid Crystal Tunable Filter (LCTF) was programmed to acquire image cubes from λ = 790 nm to 860 nm with an increment of 10 nm per image. Fluorescence intensity measurements were calculated as average intensity over the tumor surface area, using ImageJ software.

#### *7.9.3. In vivo optical detection of human colon tumors in a CAM model*

LS174t and SW480 colorectal cell lines were used to demonstrate the possible use of the NIR fluorescent Prot8 particles in tumor detection. As mentioned before, LS174t cells express certain receptors (β-D-galactosyl-(1-3)-N-acetyl-D-galactosamine and CEA) at a much higher extent than SW480 cells [62,65,66]. This way, the chosen bioactive ligands PNA and anti-CEA, once conjugated to the Prot8 particles, can lead the particles specifically to the LS174t cancer cells. As shown in Figure 15, the LS174t tumors treated with bioactive-conjugated particles (B and C) gained higher fluorescence than SW480 tumors, compared to those treated with nonconjugated particles (A). This is accurate both for Prot8 particles conjugates with PNA (B) and anti-CEA (C), probably as a result of effective ligand-receptor interactions. In addition, the SW480 tumors treated with bioactive-conjugated Prot8 particles gained less fluorescence compared to those treated with the non-conjugated particles. The relative fluorescence intensities of the treated tumors by the conjugated and non-conjugated Prot8 particles are summarized in Figure 16. LS174t cells compared to SW480 cells gave fluorescence intensity ratios of 4:1 and 8:1 for PNA-conjugated particles and anti-CEA-conjugated particles, respec‐ tively. The non-conjugated Prot8 particles also labeled the tumors, however, the difference in the intensities between the types of tumors were not statistically significant. In this case, the overall fluorescence in both types of cells was higher than when treated with PNA-conjugated particles. This fact shows that even the bare non-conjugated particles penetrate the human cancer cell lines with a good extent. The possible reason is that the Prot8 particles can penetrate and label the cancerous cells specifically by either receptor-ligand interaction or utilization of these particles as nutrients for tumor growth, as they resemble biological proteins. The fluorescence intensity ratios between the types of cells show the significance of the nanoparticle surface. As seen in Figure 15D, no autofluorescence was observed in untreated tumors, signifying that all fluorescent signals are related to the fluorescent Prot8 particles labelling. Figure 15E shows that no non-specific labelling of non-pathological CAM tissue was observed, indicating the specificity of the Prot8 particles towards the tumor tissue.

Figure 14 shows typical SW480 cell line derived tumors bordered by plastic rings on a chicken

**Figure 14. Light photograph of SW480 cell line derived tumors bordered by a plastic ring on chicken embryo CAM.** Suspensions of 5×106 SW480 cells suspended in Matrigel formed compact structures (asterisk) 8 days after transplanta‐

Chicken embryos with 6-days-old human adenocarcinoma tumors (LS174t and SW480 cancer cell lines) implanted on the CAM were treated with the non-conjugated, PNA-conjugated and anti-CEA-conjugated NIR fluorescent Prot8 particles (40 μL, 2 mg/mL). Additionally, nonpathological CAM treated with non-conjugated particles and untreated tumors served as control groups. After 40 minutes, the nanoparticle dispersions were removed and the tumors were washed with PBS. Then, the tumors and the non-pathological CAM were removed from the eggs, washed again with PBS and spread on a mat black background for observation using a Maestro II™ in vivo imaging system (Cambridge Research & Instrumentation, Inc., Woburn, MA). A NIR excitation/emission filter set was used for the experiments (λex: 710–760 nm, λem > 750 nm). The Liquid Crystal Tunable Filter (LCTF) was programmed to acquire image cubes from λ = 790 nm to 860 nm with an increment of 10 nm per image. Fluorescence intensity measurements were calculated as average intensity over the tumor surface area, using ImageJ

LS174t and SW480 colorectal cell lines were used to demonstrate the possible use of the NIR fluorescent Prot8 particles in tumor detection. As mentioned before, LS174t cells express certain receptors (β-D-galactosyl-(1-3)-N-acetyl-D-galactosamine and CEA) at a much higher extent than SW480 cells [62,65,66]. This way, the chosen bioactive ligands PNA and anti-CEA, once conjugated to the Prot8 particles, can lead the particles specifically to the LS174t cancer cells. As shown in Figure 15, the LS174t tumors treated with bioactive-conjugated particles (B and C) gained higher fluorescence than SW480 tumors, compared to those treated with non-

*7.9.3. In vivo optical detection of human colon tumors in a CAM model*

embryo CAM inside the egg.

68 Advances in Bioengineering

tion with attraction of host blood vessels (arrow).

*7.9.2. CAM tumor detection*

software.

**Figure 15.** Fluorescent (upper) and greyscale (lower) images from a typical experiment of tumor cell lines LS174t and SW480 implants on chicken embryo CAM treated with non-conjugated (A), PNA-conjugated (B) and anti-CEA-conju‐ gated (C) NIR fluorescent Prot8 particles. Images of untreated tumor cell lines are shown in (D). Images of non-patho‐ logical CAM treated with non-conjugated, PNA-conjugated and anti-CEA-conjugated particles are shown in (E). The experiment was repeated 5 times with similar results.

**Figure 16.** Relative fluorescence intensities of LS274t and SW480 tumors labeled with non-conjugated, PNA-conjugated and anti-CEA-conjugated particles. Data is presented as the mean value ± SE. Values not sharing a common letter (a, b, c or d) differ significantly from each other (p<0.05). The calculations are an average of 3 experiments.

In another set of *in vivo* experiments on LS174t and SW480 tumors implanted on the CAM model, the specific biomarker anti-CEA was tested against anti-rabbit IgG, serving as a nonspecific agent, as well as a control group of non-conjugated particles. As clearly illustrated in Figure 17, LS174t tumors treated with anti-CEA-conjugated particles (B) gained greater fluorescence compared to those treated with non-conjugated particles (A) or anti-rabbit IgGconjugated particles (C). This can be explained by the effective ligand-receptor interaction. Furthermore, the SW480 tumors treated with the anti-CEA-conjugated particles gained less fluorescence (about 3.5 times) compared to the LS174t tumors treated the same way. The fluorescent signal of LS174t tumors labeled by anti-CEA-conjugated particles was 4 times higher than that of the the tumors labeled by the anti-rabbit IgG-conjugated particles. Antirabbit IgG "blocks" the particle from interacting with the tumor receptors by the conjugation to the surface active moieties, thus serving as a negative control in colon tumor labelling.

**Figure 17.** Fluorescent and grayscale images from a typical experiment of LS174t and SW480 human tumor cell lines implanted on chicken embryo CAM treated with the non-conjugated (A), anti-CEA-conjugated (B) and anti-rabbit IgGconjugated (C) NIR fluorescent Prot8 particles. Images of untreated tumors are shown in (D). The experiment was re‐ peated 3 times with similar results.

#### **7.10. Optical detection of human colon tumors in a mouse model**

**Figure 16.** Relative fluorescence intensities of LS274t and SW480 tumors labeled with non-conjugated, PNA-conjugated and anti-CEA-conjugated particles. Data is presented as the mean value ± SE. Values not sharing a common letter (a, b,

In another set of *in vivo* experiments on LS174t and SW480 tumors implanted on the CAM model, the specific biomarker anti-CEA was tested against anti-rabbit IgG, serving as a nonspecific agent, as well as a control group of non-conjugated particles. As clearly illustrated in Figure 17, LS174t tumors treated with anti-CEA-conjugated particles (B) gained greater fluorescence compared to those treated with non-conjugated particles (A) or anti-rabbit IgGconjugated particles (C). This can be explained by the effective ligand-receptor interaction. Furthermore, the SW480 tumors treated with the anti-CEA-conjugated particles gained less fluorescence (about 3.5 times) compared to the LS174t tumors treated the same way. The fluorescent signal of LS174t tumors labeled by anti-CEA-conjugated particles was 4 times higher than that of the the tumors labeled by the anti-rabbit IgG-conjugated particles. Antirabbit IgG "blocks" the particle from interacting with the tumor receptors by the conjugation to the surface active moieties, thus serving as a negative control in colon tumor labelling.

**Figure 17.** Fluorescent and grayscale images from a typical experiment of LS174t and SW480 human tumor cell lines implanted on chicken embryo CAM treated with the non-conjugated (A), anti-CEA-conjugated (B) and anti-rabbit IgGconjugated (C) NIR fluorescent Prot8 particles. Images of untreated tumors are shown in (D). The experiment was re‐

peated 3 times with similar results.

70 Advances in Bioengineering

c or d) differ significantly from each other (p<0.05). The calculations are an average of 3 experiments.

Experiments were performed according to the protocols of the Israeli National Council for Animal Experiments by Harlan Biotech, Israel. Cancerous cells (30 μL containing 2×106 LS174t cells) were injected into the mouse intestinal wall. 2 weeks later the nude mice were anaes‐ thetized and treated with the bio-conjugated NIR fluorescent Prot8 particles (0.1%, 200 μL), through the anus, using the guidance of a mini-colonoscope. 20 min later each colon was washed with PBS (5 × 1 mL) and mice were allowed to recover for 4 h. The mice were sacrificed and the colons were removed. Each colon was spread on a solid surface and imaging was performed using the Odysey Infra-red Imaging System (Li-Cor Biosciences, Lincoln, NE, USA) with excitation wavelength of 780 nm and emission wavelength of 800 nm.

Figure 18 shows typical (8 out of 10 mice) fluorescent and grayscale images of the mice colons after treatment with anti- CEA (A) and anti-rabbit IgG (B) conjugated particles. As illustrated in Figure 18A, the anti-CEA-conjugated particles detected the tumors specifically and selec‐ tively with good signal to background ratio (SBR), the background refers to the surrounding non-pathological tissue. Moreover, as illustrated in Figure 18B, the "inactive" anti-rabbit IgGconjugated particles did not produce a significant signal of the tumors.

**Figure 18.** Fluorescent and grayscale images of typical LS174t colon tumors treated with anti-CEA (A) and anti-rabbit IgG (B)-conjugated NIR fluorescent Prot8 particles. 20 mice (10 in each experiment group) were anesthetized and treat‐ ed with 0.1% particle dispersion in PBS, as described in section 3.2.5.14. 2 untreated mice served as a control group.
