Naphthalocyanine-Based Biodegradable Polymeric Nanoparticles for Image-Guided Combinatorial Phototherapy

Taratula, O., Doddapaneni, B. S., Schumann, C., Li, X., Bracha, S., Milovancev, M., ... & Taratula, O. (2015). Naphthalocyanine-Based Biodegradable Polymeric Nanoparticles for Image-Guided Combinatorial Phototherapy. Chemistry of Materials. doi:10.1021/acs.chemmater.5b03128

10.1021/acs.chemmater.5b03128

American Chemical Society

Accepted Manuscript

http://cdss.library.oregonstate.edu/sa-termsofuse

1

Naphthalocyanine-Based Biodegradable Polymeric Nanoparticles for Image-Guided

Combinatorial Phototherapy

Olena Taratula,§ Bhuvana S. Doddapaneni,

§ Canan Schumann,

§ Xiaoning Li,

§ Shay Bracha,

#

Milan Milovancev, #

Adam W. G. Alani, §,*

Oleh Taratula§,*

§Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University,

Portland, Oregon 97201, United States

#Department of Clinical Sciences, College of Veterinary Medicine, Oregon State University,

Corvallis, OR 97331, United States

* Address correspondence to Oleh.Taratula@oregonstate.edu, Adam.Alani@oregonstate.edu

Abstract (250 words)

Image-guided phototherapy is extensively considered as a promising therapy for cancer

treatment. To enhance translational potential of this modality, we developed a single agent-based

biocompatible nanoplatform that provides both real time near-infrared (NIR) fluorescence

imaging and combinatorial phototherapy with dual photothermal and photodynamic therapeutic

mechanisms. The developed theranostic nanoplatform consists of two building blocks: (1) silicon

naphthalocyanine (SiNc) as NIR fluorescence imaging and phototherapeutic agent and (2) a

copolymer, poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL) as the biodegradable

SiNc carrier. Our simple, highly reproducible and robust approach results in preparation of

spherical, monodisperse SiNc-loaded PEG-PCL polymeric nanoparticles (SiNc-PNP) with a

hydrodynamic size of 37.66 ± 0.26 nm (polydispersity index = 0.06) and surface charge of -2.76

± 1.83 mV. The SiNc-loaded nanoparticles exhibit a strong NIR light absorption with an

2

extinction coefficient of 2.8 x 105 M

-1cm

-1 and efficiently convert the absorbed energy into

fluorescence emission (F = 11.8%), heat (T ~ 25 C) and reactive oxygen species. Moreover,

the SiNc-PNP are characterized by superior photostability under extensive photoirradiation and

structure integrity during storage at room temperature over a period of 30 days. Following

intravenous injection, the SiNc-PNP accumulated selectively in tumors and provided high lesion-

to-normal tissue contrast for sensitive fluorescence detection. Finally, Adriamycin-resistant

tumors treated with a single intravenous dose of SiNc-PNP (1.5 mg/kg) combined with 10 min of

a 785 nm light irradiation (1.3 W/cm2) were completely eradicated from the mice without cancer

recurrence or side effects. The reported characteristics make the developed SiNc-PNP a

promising platform for future clinical application.

KEYWORDS: naphthalocyanine, photothermal therapy, photodynamic therapy, near-infrared

imaging, photostability, theranostic, biodegradability

3

INTRODUCTION

Image-guided phototherapy is extensively considered as a promising, non-invasive therapeutic

modality for intraoperative cancer treatment.1-8

It combines dual photothermal and photodynamic

therapeutic mechanisms, which are different from chemo- and radiotherapies and, therefore, has

significant potential for treatment of resistant cancer cells.7-9

Furthermore, phototherapy results

in minimal side effects because it selectively kills cancer cells by non-toxic photoactive agents

upon activation by a specific wavelength of targeted light.3, 4, 6, 8

Light-activated photoactive

agents can concurrently generate both intratumoral heat and toxic reactive oxygen species

(ROS), leading to cancer cell destruction.3, 4, 6, 8

Moreover, combining phototherapy with real-

time fluorescence imaging enables tracking of accumulation of photoactive agents within tumor

tissue and allows for precise focusing of the laser beam on the tumor targets, to both activate

therapeutic mechanisms and minimize exposure of healthy organs.3, 4

To translate this promising therapeutic modality to the clinic, multifunctional theranostic

nanoplatforms that provide both real-time fluorescence imaging and phototherapy are urgently

needed. Phototherapeutic and imaging agents capable of near-infrared (NIR) light absorption

(~700 -950 nm) and fluorescence have attracted significant attention as the main building blocks

for these nanoplatforms.1, 2, 4, 8, 10, 11

Because biological tissues and body fluids show minimal

light absorption and autofluorescence in the NIR spectral region, NIR phototherapeutic drugs

and fluorophores can provide for an efficient treatment and imaging of deep-seated tumors.12, 13

In recent years, the number of the required nanoplatforms has been developed by encapsulation

of separate NIR imaging and phototherapy agents into a single nanocarrier.2, 7, 10, 11, 14, 15

Despite

their efficiency being demonstrated in preclinical studies, the high complexity and complicated

synthetic procedure could limit further translational potential of these nanoplatforms.3

4

If NIR imaging and phototherapeutic properties could be merged into a single agent,

substantially improved cancer theranostic outcomes and a high translational potential could be

expected. Recently, nanoporphyrin with absorption and emission maximum in a 600 – 700 nm

spectral range have been employed to develop a smart “all-in-one” nanoplatform that

conveniently integrates fluorescence imaging and phototherapeutic functions.3 To further

improve the theranostic efficiency of the “all-in-one” nanoplatforms, there is a high interest in

application of photoactive agents capable to absorb and emit light in the 750 - 900 nm spectral

region. Application of these dyes offers several advantages for image-guided phototherapy

including enhanced tissue penetration of excitation and emission light, lower autofluorescence

from surrounding tissue and body fluids, and decreased skin photosensitivity as a potential side

effect of phototherapy.16-19

The FDA-approved NIR dye, indocyanine green (ICG) with

photophysical properties in a 750 - 850 nm spectral region, has been considered as a main

building block for the construction of theranostic nanoplatforms composed of a single

phototherapeutic agent.4, 20-23

However, ICG exhibits poor photostability and, therefore,

photodegradation of the dye can be misread as an absence of fluorescence signal during the

image-guided phototherapy.24

In addition, photo-degradation of the photoactive agent also

compromises ROS and heat generation abilities, diminishing its therapeutic potential.8, 13, 24, 25

Finally, the quantum yield of ICG is quite low (F = 2.7%), which significantly reduces its

efficiency as the fluorescence imaging agent.26

Recent reports indicated that, owing to its unique intrinsic properties including enhanced

absorption in the spectral range 750 - 800 nm, silicon naphthalocyanine (SiNc) can be efficiently

activated with NIR light to produce fluorescence, ROS and heat for imaging and combinatorial

treatment of tumors.8, 25, 27, 28

Furthermore, it was revealed that dendrimer-encapsulated SiNc

5

molecules with axial substituents exhibit superior photostability compared to ICG.8 Clinical

application of SiNc, however, is limited by low water solubility, aggregation and lack of cancer

specificity.8, 29

Particularly, an intrinsic tendency of SiNc molecules to aggregate results in self-

quenching between the bound dyes, which diminishes their phototherapeutic efficacy and

imaging ability.30

To fully develop its clinical potential and overcome the above-mentioned

obstacles, there is a critical need to develop a highly efficient SiNc-based nanoplatform. Ideally,

this nanoplatform has to be (i) biocompatible and biodegradable, (ii) simple and reproducible in

preparation, and (iii) not complex, in that it contains a small number of components. In addition,

it has to increase the water solubility of the SiNc molecules to clinically relevant concentrations

and decrease their self-aggregation in aqueous solutions to preserve the fluorescence emission

and phototherapeutic properties in the physiological environment. Finally, it should provide

specific delivery of SiNc to the cancer tissue within a short period of time after systemic

administration, while being rapidly eliminated from healthy organs.

Herein, we report the development of a desirable multifunctional nanoplatform for image-guided

phototherapy, which contains only two components: (1) SiNc as NIR imaging and

phototherapeutic agent, and (2) a biodegradable-biocompatible block copolymer, PEG-PCL,31, 32

as the SiNc carrier (Figure 1). The developed procedure is simple, reproducible and results in

fabrication of stable, highly uniform nanoparticles with excellent imaging and phototherapeutic

properties. The obtained characteristics make the fabricated theranostic nanoplatform promising

material for future clinical application.

MATERIALS AND METHODS

Materials. SiNc (2,3-Naphthalocyaninato-bis(trihexylsiloxy)silane) was obtained from Sigma-

Aldrich Co. (Milwaukee, WI). PEG-PCL (methoxy poly(ethylene glycol)-b-poly(ε-

6

caprolactone), MW = 15000 Da, PDI=1.17) was obtained from Advanced Polymer Materials Inc.

(Montreal, Canada). All other reagents were purchased from Fisher Scientific, Inc. (Fairlawn,

NJ) and VWR International, LLC (Radnor, PA).

Preparation of SiNc-based Polymeric Nanoparticles. SiNc nanoparticles were constructed

using modified solvent evaporating method.33

Breifly, PEG-PCL (20 mg) and SiNc (0.6 mg)

were dissolved in 2 mL of THF, The solution was transferred into 20 mL scintillation vial and to

the constantly stirred solution 2 mL of saline was added in a 1-step process. THF was removed

under vacuum using a rotary evaporator and the final volume was adjusted to 2 mL using saline.

The evaporation cycle was divided into three segments, with the first segment lasting 7 min at

420 mbar, the second for 7 min at 320 mbar and the final segment of 6 min at 200 mbar. The

temperature of the water bath was maintained at 45 oC with a rotation of 100 rpm for the round

bottom flask. The final volume was adjusted to 2 mL with saline, the nanoparticles solution was

centrifuged (5 min, 10,000 rpm) and the supernatant was filtered using a 13 mm 0.2 µM nylon

filter.

Characterization. The hydrodynamic diameter, size distribution and zeta potential of

naphthalocyanine loaded polymeric nanoparticle (SiNc-PNP) was determined using dynamic

light scattering (DLS, Malvern ZetaSizer NanoSeries, Malvern, UK) at 25 C as previously

described.7, 8, 34-36

The morphology of the polymeric nanoparticles was observed using cryogenic

transmission electron microscopy cryoTEM. The 3 µl of nanoparticle suspensions (0.3 mg/ml)

were pipetted onto glow-discharged (30 sec, 30 mAmp) copper Quantifoil holey carbon support

grids (Ted Pella 658-300-CU) and vitrified on liquid ethane using a Mark IV Vitrobot (FEI,

Hillsboro, OR). The conditions utilized were 100% humidity, blot force 0 and blotting times

between 2-4 seconds. Low-dose conditions were used to acquire images on a FEI Krios-Titan

7

equipped with a Falcon II direct electron detector (FEI, Hillsboro, OR). Cryo-EM images were

collected with a defocus range of 2-4 µm.

The fluorescence of SiNc-PNP was recorded on UV-1800 Shimadzu spectrophotometer

(Carlsbad, CA) and absorbance spectra were taken on Cary Eclipse R3896 fluorescence Varian

spectrophotometer (Mulgrave Victoria, Australia).

To determine SiNc loading capacity, the SiNc-PNP solution was freeze-dried, and the obtained

pellets were weighted and re-dissolved in THF. SiNc encapsulation efficiency was evaluated as

the percentage of SiNc weight (quantified based on UV–Vis absorption of SiNc at 772 nm)

encapsulated into PNP over the initial weight of SiNc used for loading. The loading capacity (%)

and encapsulation efficiency (%) of the SiNc-PNP were calculated based on the equation

below:37

Loading Capacity (%) = mass of SiNc in SiNC-PNP / total mass of the SiNc-PNP × 100

Encapsulation efficiency (%) = mass of SiNc in SiNc-PNP / initial amount of SiNc × 100.

Fluorescence quantum yield for SiNc-PNP was calculated in aqueous solution (ex = 750 nm)

with indocyanine green (ICG) as reference (0.4)38

as follows:

SiNc = (AbsICG/AbsSiNc) x (AreaSiNc/AreaICG) x ICG x 100

where SiNc is a quantum yield of SiNc-PNP; AbsICG and AbsSiNc are absorbances of ICG and

SiNc-PNP, respectively; AreaICG and AreaSiNc are fluorescence emission areas of ICG and SiNc-

PNP, respectively; where ICG is a quantum yield of SiNc-PNP.

Water was employed as solvent for ICG and SiNc-PNP and, therefore, refractive index was not

used in the equation.

Brightness of SiNc-PNP relative to ICG was calculated by using the previously reported

equation: 39

8

Brightness = (SiNc x SiNc) x (ICG x ICG)

where SiNc and ICG are extinction coefficients of SiNc-PNP and ICG, respectively.

Stability of SiNc-PNP (SiNc encapsulation, size, surface charge) in aqueous media over one

month was assessed at room temperature protected from light.

Singlet Oxygen 1O2 Generation. As previously reported,

1O2 production was measured by

SOSG assay in a 96 well plate.7, 35

. Corresponding wells were treated for 5 min at 785 nm and

1.3 W/cm2 while the dark controls were protected from light. Instantly, the samples were

evaluated on Varian Cary Eclipse R3896 fluorescence spectrophotometer using

excitation/emission of 504/525 nm.

Photothermal Effect. A 785 nm laser diode (CW, ThorLabs, Newton, NJ, 1.3 W/cm2) was used.

For temperature increase evaluation, 300 µL of SiNc-PNP aqueous solutions (0.3 mg/mL) in the

500 L vials was exposed for 20 min to the NIR laser. The thermocouple was used to record

temperature at selected time points. As a control, water was tested under the same irradiation

conditions. Absorption of SiNc-PNP solutions in water at 0.3 mg/mL SiNc concentration was

evaluated prior to and after 30 min laser treatment (1.3 W/cm2, 785 nm) in order to evaluate

photostability.

Dark Cytotoxicity. To determine the dark cellular cytotoxicity of SiNc-PNP a modified cell

viability assay based on Calcein AM (Biotium, Inc., Hayward, CA) was employed. Human

ovarian epithelial carcinoma/Adriamycin-resistant derivative cell line (A2780/AD) were seeded

in two 96-well plates (1 x 104 cells/well) and the next day were incubated with SiNc-PNP (2.0,

5.0, 10.0, 25.0, 50.0, and 100 µg/mL) in RMPI 1640 (10% FBS) in the dark conditions. As a

control, A2780/AD cells only were used. After 36 hours, cell viability was assessed with Calcein

AM solution (10 µM in DPBS buffer) for 1 hour. Fluorescence was recorded on a multiwell

9

Synergy HT plate reader (BioTek Instruments, Winooski, VT) with a 485/528 nm

excitation/emission filters. The cell viability (%) was expressed as a percentage relative to the

control cells.

Cellular Internalization. A2780/AD cancer cells (2 x 105 cells/well) were plated in 6-well

plates followed by 36 hours incubation with SiNc-PNP (30 µg/mL). Cells were washed with

DPBS and imaged with EVOS® FL Cell Imaging System (Life Technologies, Grand Island,

NY).

Quantification of Intracellular SiNc Content. The SiNc content in cancer cells incubated with

SiNc-PNP was estimated via recording UV-Vis absorption. Cells were seeded in T-25 flask to

reach 80% confluency followed by 36 hours incubation with SiNc-PNP (30 g/mL). Next, 5 x

106

cells were lysed with 50 mM NaOH and diluted in THF to dissolve SiNc at 1:9 ratio (100 L

cell lysate/900 L THF). The absorbance was recorded at 772 nm and the amount of SiNc

uptaken by cells was calculated by using the standard curve.

ROS Measurements. DCFH-DA (2´,7´-dichlorodihydrofluorescein diacetate) was employed to

evaluate intracellular ROS levels.7, 35

Cancer cells seeded in 96-well plates (1×104 cells/well)

were incubated with SiNc-PNP (30 µg/mL) for 36 h. After incubation, cell were treated with 10

µM DCFH-DA for 30 min prior to 10 min light treatment (785 nm, 1.3 W/cm2). Separately, the

untreated cells and SiNc-PNP without light treatment were used as a control a dark control,

respectively. Fluorescence was recorded on a plate reader (λabs = 485 nm and λem = 528 nm).

Combinatorial Phototherapy on Cell Pellet. The T-25 cell culture flask with A2780/AD cells

of 80% confluency were incubated for 36 h with SiNc-PNP (50 g/mL) in RPMI 1640 (10 %

FBS). Next, 2 × 106

cells were centrifuged (5 min, 1000 rpm) to give the cell pellet. Samples (in

250 L of culture media) were photoirradiated (785 nm, 10 min, 1.3 W/cm2). Temperature was

10

recorded before and after 10 min light treatment using a fiber optic temperature probe inside of

the pellets. After the light experiment, the cells were seeded in a 96-well plate (1 × 104

cells/well). After 24 h, the cell viability was evaluated by Calcein AM assay along with the

controls: untreated cells, cells incubated with SiNc-NP and untreated cells exposed to the NIR

laser (785 nm, 1.3 W/cm2, 10 min).

In vivo NIR Fluorescence Imaging and Biodistribution. Approval for the animal studies was

given by the Oregon State University IACUC and all procedures were performed according to

IACUC policies. A subcutaneous A2780/AD tumor animal model was developed via injection of

A2780/AD cells (5 × 106) in RPMI 1640 into the flank of 5-week-old athymic nu/nu female mice

(Harlan Laboratories, Inc., USA). The tumor sizes were measured for 25 days and were

calculated as 0.5 × width2 × length. At tumor volume of ~ 40 mm

3, the mice were injected

intravenously with 100 L SiNc-PNP in saline (0.3 mg/mL) at the dose of 1.5 mg/kg. Then, the

whole animal and harvested tumor, heart, liver, spleen, lung, and kidney were imaged using

Pearl Impulse Small Animal Imaging System (LI-COR, Lincoln, NE) with 800 nm channel at

0.5, 2, 24 and 96 h post-injection.

In vivo Phototherapy. With tumor volume of ~ 40 mm3, the mice were distributed to one of four

treatment groups (n=20, 5 mice/group) and injected with saline (control), saline and followed by

exposure to 10 min of NIR light (light control), SiNc-PNP (1.5 mg/mL) (SiNc-PNP dark) or

SiNc-PNP (1.5 mg/kg) followed by NIR (1.3 W/cm2, 785 nm) irradiation (SiNc-PNP light). At

this point, fluorescent images were obtained to determine the SiNc location. At 24 h, the mice in

the specific experimental groups were treated once with phototherapy for 10 min (785 nm, 1.3

W/cm2) under isoflurane anesthesia. Tumor size and body weight were documented for all mice

groups during 25 days after phototherapy. Mice were euthanized when tumor diameter was more

11

than 1200 mm3. Intratumoral temperature was measured in 10-s intervals using a fiber optic

temperature probe as previously described.8

Ex vivo Histology. Tissue samples of control (untreated and NIR light only treated) tumors,

tumor treated SiNc-PNP dark and SiNc-PNP light were dissected from mice 6 h post-

photoirradiation. The formalin-fixed paraffin embedded tissues were sectioned and collected

onto slides as reported earlier.8 Hematoxylin and eosin (H&E, IHC WORLD, LLC Ellicott City,

MD), Hoechst 33342 (NucBlue® Live ReadyProbes® Reagent, Life Technologies, Grand

Island, NY) and caspase-3 (Cell Signaling Technology, Danvers, MA) staining were performed

following manufacturer’s protocol and observed by a Carl Zeiss Axio Imager Z1 fluorescent

microscope (Thornwood, NY).

Data Analysis. Data were analyzed and presented as mean values ± standard deviation (SD)

from three to eight independent measurements. The statistic difference was analyzed using t-test

where P value of 0.05 was considered significant.

RESULTS AND DISCUSSION

Synthesis of SiNc-PNP. To prepare the proposed nanoplatform, we used SiNc molecules

modified with two organic substituents, trihexylsilyloxide (R), through their conjugation to

hydroxyl groups at the silicon center of naphthalocyanine (Figure 1). The application of

substituted SiNc provides several important advantages for our nanoplatform. First, we

previously demonstrated that the appropriate hydrophobic substituents can facilitate

encapsulation of phthalocyanine and naphthalocyanine dyes into the hydrophobic interior of the

nanoparticles.7, 8, 35

Second, these substituents provide steric hindrance of these dyes and reduce

intermolecular aggregation inside of the nanoparticles, minimizing the self-quenching effect and

12

thus improving their fluorescence imaging and ROS generating properties.7, 8, 35

Third, several

reports have shown that SiNc with the substituents in axial positions exhibits superior

photostability in comparison to non-modified molecules. 8, 30

To overcome in vitro and in vivo challenges associated with low intrinsic solubility of the SiNc

(<1 ng/mL, evaluated by Advanced Chemistry Development ACD/Universal LogSw GALAS

Module, Percepta 14.0.0 (Build 1996)), amphiphilic copolymer, PEG-PCL, which consist of a

hydrophilic poly(ethylene glycol) (PEG) and a hydrophobic poly(-caprolactone) (PCL) blocks

(Figure 1), has been employed as a carrier. The PEG is nontoxic material and cleared by the US

Food and Drug Administration (FDA) for internal use in the human body.40

The PCL is

biocompatible and biodegradable polyester approved by the FDA for biomedical applications in

humans.31

The PEG-PCL copolymer was extensively tested for various biological applications

and all studies have reported low cytotoxicity and complete clearance from the body.32, 41, 42

As a

result, this co-polymer is characterized by controlled biodegradability, excellent biocompatibility

and self-assembly in aqueous solution to form polymeric nanoparticles (PNP) with a

hydrophobic core for loading non-water soluble drugs.32

In addition, this copolymer, while

forming nanoparticles, decreased drug uptake by the liver and kidney, and also prolonged drug

retention in the blood.43

A simple, highly reproducible and robust approach for transformation of hydrophobic SiNc into

water-soluble nanoplatforms has been developed through loading of SiNc into the hydrophobic

core of PEG-PCL-based polymeric nanoparticles (Figure 1). The encapsulation procedure is

based on solubilizing SiNc and PEG-PCL in THF followed by addition of saline to produce an

emulsion. After evaporation of the organic solvent under vacuum, stable SiNc-PNP are obtained

with drug loading capacity and encapsulation efficiency of 3% and 98%, respectively. According

13

to the quantitative UV–Vis absorption measurements the formulated nanoparticles were able to

solubilize 0.30 ± 0.01 mg/mL (3 x 105

ng/mL) of SiNc and retain the compound at the initial

concentrations for tested 30 days period. Thus, incorporation of SiNc into the polymeric

nanoparticles (PNP) increased its intrinsic aqueous solubility (<1 ng/mL) by 3 x 105 fold, thereby

achieving therapeutically relevant dosing concentrations for in vivo assessment. Our findings

were consistent with published results, where the incorporation of a hydrophobic compound into

amphiphilic diblock copolymers like PEG-PCL increases their aqueous solubility and stability.32,

44

According to the DLS analysis, the hydrodynamic size of the SiNc-PNP was 37.66 ± 0.26 nm

with highly monodisperse size distribution as indicated by polydispersity index (PDI) of 0.06 ±

0.02 (Figure 2A). PDI values of less than 0.1 specify unimodal distribution of nanoparticles.

Cryogenic transmission electron microscopy (cryo-TEM) images revealed a spherical

morphology for SiNc-PNP with an average size of 19.3 ± 1.5 nm (Figure 2B) and further

confirmed preparation of highly monodisperse nanoparticles by using the developed approach

(Figure S1). The observed discrepancy between SiNc-PNP sizes measured by TEM and DLS is

in good agreement with the previous studies and it is related to the fact that DLS yields a larger

average size by measuring the hydro-dynamic diameter of the particle including the solvation

layers, while cryoTEM detects their dehydration morphology.4, 34

Nevertheless, both techniques

demonstrated that the developed nanoplatforms have a required size to minimize their rapid

clearance by the kidneys (<10 nm), identification by macrophages (<100 nm), and to increase

blood circulation time and accumulation in cancer tumors via passive targeting (<200 nm).45-47

Moreover, the recent studies indicated that nanoparticles with a size ≤50 nm have an advantage

in terms of the deep penetration and retention in the tumor tissue.48

14

In addition to the size, surface modification and charge of nanoparticles are also important

factors affecting their clearance and biodistribution after systemic administration.47, 49, 50

The

employed amphiphilic copolymer, PEG-PCL, introduced a biocompatible, non-charged PEG

layer to the nanoparticles surface, which provides for almost neutral zeta potential of the

prepared SiNc-PNP (-2.76 ± 1.83 mV). The number of reports indicated that PEG layer and

neutral/slightly negative surface charge significantly reduce non-specific interaction with plasma

proteins and, therefore, minimize opsonization and rapid uptake of nanocarriers by the

reticuloendothelial system.47, 49, 50

Of note, the developed nanoparticles are characterized by a high stability during storage. Thus,

no noticeable change in nanoparticle size distribution, zeta potential or drug loading was detected

over the one month period at the room temperature, implying that SiNc-PNP formulation is

stable in aqueous environment (Figure 2A and Table 1). The detected long shelf life of the

nanoplatform further indicates its high translation potential. The stability of SiNc-PNP in the

solution system similar to physiological environment such as phosphate buffer solution (PBS)

was also evaluated. No significant changes in nanoparticles hydrodynamic diameter (37.01 ±

0.18 nm vs 36.58 ± 0.20 nm) and UV-Vis absorption spectra (Figure S2) were detected over 24 h

period at the room temperature.

Theranostic Properties of SiNc-PNP. The effective photoactive theranostic agents should

exhibit a strong NIR absorption to assure efficient conversion of the absorbed light into

fluorescence emission, ROS and heat. SiNc loaded in the polymeric nanoparticles revealed a

strong NIR absorption in water with extinction coefficient of 2.8 x 105 M

-1cm

-1 which is 2.8-fold

higher as compared to the FDA approved clinical imaging agent, ICG (1.0 x 105 M

-1cm

-1).

39

Upon excitation with NIR light, the SiNc-PNP demonstrated intense fluorescence emission in the

15

760-810 nm wavelength range (Figure 2C). It is known that most NIR dyes including SiNc are

characterized by the small Stokes shift.27

In the current studies to avoid crosstalk between the

excitation light and resulting fluorescence signal, the SiNc-loaded nanoparticles were excited at

750 nm and the fluorescence was recorded in 760 – 850 nm spectral range. Fluorescence

quantum yield (F) of 11.8% was calculated for SiNc-PNP in water using ICG as reference

solution.38

The fluorescence quantum yield of free SiNc was also evaluated in THF and F of

30.0 % was calculated. Aggregation of SiNc molecules inside of the polymeric nanoparticles

could induce quenching effect. To minimize this effect, we employed SiNc molecules modified

with trihexylsilyloxide. These substituents provide steric hindrance of these dyes and reduce

intermolecular aggregation inside of the PEG-PCL nanoparticles, minimizing the self-quenching

effect. Therefore, the relatively high quantum yield of 11.8% for SiNc-PNP in water was

achieved. Of note, the nanoplatform-loaded SiNc has a higher quantum yield in aqueous

environment (11.8%) than NIR dyes such as ICG (2.7%) and IRDye800 (~4-10%), currently

employed in clinical trials.26, 51, 52

Furthermore, SiNc-PNP are approximately 10 times brighter

than ICG as determined according to the previously reported equation.39

Jin et al. recently developed highly fluorescent SiNc-doped polymer dots (Pdots) and the

quantum yield of the doped NIR dye was reported to be 11%.27

The high fluorescence intensity

of SiNc was achieved by excitation of Pdots with 457 nm light and following energy transfer

from Pdot matrix to NIR dye. However, the fluorescence of the SiNc-loaded Pdots excited with

NIR light at 763 nm was substantially dimmer (F = 7%) than the Pdots excited at 450 nm. It is

important to indicate that SiNc-loaded in our polymeric nanoparticles exhibit high fluorescence

emission (F = 11.8%) following excitation with NIR light at 750 nm, which is strongly required

for the deeper tissue penetration. In addition, SiNc-PNP in water revealed an intense NIR

16

fluorescence signal detected by Pearl Imaging System (Figure 2C, inset) using 800 nm imaging

channel, which eludes tissue autofluorescence. Thus, the PEG-PCL-based nanocarrier can

efficiently solubilize SiNc molecules in water, prevent them from aggregation, and, therefore,

preserves the fluorescent properties of the dye.

To test properties of SiNc-PNP as an agent for photodynamic therapy (PDT), production of

singlet oxygen (1O2) was evaluated by the SOSG assay.

53 After treatment with a laser diode (785

nm, 5 min 1.3 W/cm2,), SiNc-PNP demonstrated a 20-fold increase in

1O2 generation when

compared to the water control (Figure 2D). Thus, SiNc within the developed PEG-PCL

polymeric nanoparticles is capable of an efficient 1O2 generation and therefore demonstrates

great potential as a PDT agent.

To assess heat generation properties of SiNc loaded in the polymeric nanoparticle, the SiNc-PNP

solution was irradiated with the 785 nm NIR light (1.3 W/cm2) and temperature variations were

recorded during 20 min. Figure 2E indicates that the nanoparticles demonstrate rapid heating

under photoirradiation, reaching 40 °C in 2.5 min and 50

°C in 5.5 min. The attained temperature

could be sustained for the required duration when the laser diode is on. We also demonstrated

that the desired temperature increase can be achieved by varying laser power density (Figure S4),

providing the possibility for controlling intratumoral temperature during treatment. In the same

experimental settings, water did not trigger a significant temperature increase (∆T = 1.1 °C),

suggesting that the combination of SiNc-PN and NIR light was responsible for the heat

generation (Figure 2E). Our results are in a good agreement with the previous reports. For

example, Sheng et al reported that temperature of PBS only increased by 1.9 °C upon exposure to

a 808 nm laser irradiation with a power density of 1.0 W/cm2.4 The similar result was reported

by Li et al after irradiation of PBS only with 808 nm laser diode (8.0 W/cm2).

24

17

Recently, Yang et al reported a highly efficient PTT agent, the polypyrrole organic

nanoparticles.54

It was demonstrated that the temperature of aqueous solution containing

polypyrrole organic nanoparticles (0.25 mg/mL) increased from ~ 15 °C to ~ 35

°C (~∆T = 20

°C)

under 808 nm laser irradiation at a power density of 0.5 W/cm2. The Figure S4 indicates that the

developed SiNc-PNP demonstrate slightly lower photothermal conversion properties, which

could be related to a fact that SiNc-PNP can simultaneously generate fluorescence, heat and

ROS. The temperature of aqueous solution of SiNc-PNP (0.30 mg/mL) increased from ~ 25 °C to

~ 40°C (~∆T = 15

°C) under 785 nm laser irradiation at the same power density (Figure S4).

Photostability. The NIR organic dyes, including ICG, undergo irreversible photobleaching upon

photoirradiation, which may significantly affect the efficiency of fluorescence imaging and

phototherapy.8, 13, 25, 55

Therefore, we evaluated the potential photobleaching of SiNc loaded into

the PEG-PCL nanoparticles under extensive photoirradiation. The 30 min exposure of the SiNc-

PNP solution to NIR light (785 nm, 1.3 W/cm2) did not result in a visible reduction of SiNc

fluorescence or absorption intensity (Figure 2F). In addition, SiNc-PNP were capable of

elevating and upholding the temperature of a water solution at 54-55 oC upon irradiation for the

extended period of time (Figure 2E). In contrast, previous reports revealed that exposure of an

ICG solution to the laser light of a similar power density resulted in a significant reduction in

absorbance, fluorescence and photothermal properties, indicating photodegradation of ICG.8, 25

It

was also reported that encapsulation of the NIR dyes into the micelles only reduces their

photobleaching under photoirradiation as compared to free dyes.1 Finally, Sheng et al. indicated

that free ICG decomposes under storage conditions and loading of this dye into nanoparticles

only delays its degradation process.4

18

SiNc encapsulated into the developed PNP demonstrated a high optically stable behavior under

both extensive photoirradiation and storage conditions (Figure 2F and Figure S3), and thus this

nanoplatform has a high potential for theranostic applications. The superior photostability of

SiNc-PNP could be related to the following two reasons. First of all, the employed SiNc with

axial substituents is characterized by minimal photobleaching compared to unsubstituted

naphthalocyanines.8, 30

It was reported that absorption of free substituted SiNc was only

decreased by 10% upon exposure to a 785 nm laser diode (1.3 W/cm2) for 30 min.

8 Additionally,

the loading of SiNc into the hydrophobic core of the polymeric nanoparticles further protects the

dye molecules from photodegradation and decomposition.4, 8

In vitro Theranostic Efficiency of SiNc-PNP. The Adriamycin-resistant A2780/AD human

ovarian carcinoma cells were employed to evaluate theranostic properties of SiNc-PNP in vitro.

Negligible cytotoxicity, a critical parameter for safe and effective phototherapy and bioimaging,

was observed for SiNc-PNP (2 – 100 g/mL) under dark conditions (Figure 3A). Since

fluorescence imaging, PDT and photothermal therapy (PTT) efficiency strongly rely on the

amount of phototherapeutic drug delivered into cancer cells,56

we quantitatively measured the

cellular uptake of SiNc-PNP with absorption spectroscopy. According to these measurements,

the quantity of SiNc delivered into cells by the polymeric nanoparticles was found to be 9.1 pg

per cell. Cellular internalization of SiNc-PNP was also verified with fluorescence microscopy by

taking advantage of SiNc intrinsic fluorescence (Figure 3B) and thus, confirming its imaging

ability. As shown in Figure 3B, the cancer cells emit bright fluorescence after incubation with

the SiNc-PNP.

To assess the effect of combinatorial phototherapy mediated by the developed SiNc-PNP in

vitro, A2780/AD cancer cells (5 ×106 cells) were incubated with the non-toxic concentration of

19

SiNc-PNP (50 g/mL, Figure 3A) and exposed to NIR irradiation (785 nm, 1.3 W/cm2) for 10

min. Cell viability measurements revealed more than 95% cancer cell death when compared to

the appropriate controls (Figure 3C). In contrast, laser irradiation alone or SiNc-PNP under dark

conditions were not capable of substantial killing of cancer cells, indicating safety and selectivity

of the combinatorial phototherapy. The obtained results are supported by the earlier publications

which pointed out that PTT has a synergistic effect with PDT, and only combinatorial

phototherapy can substantially induce cancer cell death as compared with PDT or PTT alone.2-4,

8, 14

To validate that both therapeutic mechanisms contributed to the destruction of SiNc-PNP-

transfected cancer cells under exposure to a NIR laser, we evaluated alterations in intracellular

ROS and temperature during the combinatorial therapy. Following photoirradiation the

temperature inside the cell pellets increased by 17 oC reaching a value of 50

oC. In contrast,

temperature increase in non-treated cell pellets exposed to NIR light was minimal (<1 0C). In

addition to the photothermal therapy, it was further confirmed that the PDT mechanism was also

activated during the treatment. As indicated in Figure 3D, intracellular ROS level increased 26-

fold following irradiation of SiNc-PNP-treated cancer cells for 10 min with the 785 nm laser

light.

In Vivo Evaluation of Fluorescence Imaging and Phototherapy. Successful in vitro

assessment of SiNc-PNP as a NIR theranostic agent provided us with expectations for effective

in vivo bioimaging and cancer phototherapy applications. To assess the imaging feasibility and

biodistribution behavior of the developed SiNc-PNP, the intrinsic fluorescence of nanoparticle-

encapsulated SiNc after intravenous injection into nude mice bearing subcutaneous A2780/AD

tumors was monitored in vivo and ex vivo in various tissues at different time points. Figure 4

20

demonstrates that only weak autofluorescence was detected in the liver before SiNc-PNP

administration. After 30 min post injection, the entire body of the mouse emitted bright NIR

fluorescence indicating the distribution of SiNc-NP (Figure 4A). In addition, ex vivo images

revealed strong fluorescence intensity in major organs such as the liver, heart, kidneys, and lungs

(Figure 4B). After 2 h, strong fluorescence was still observed in the entire mouse body; however

fluorescence intensity significantly decreased in the liver, heart, kidneys and lungs,

demonstrating rapid clearance of SiNc-PNP from these normal organs (Figure 4). After 24 h, the

SiNc-loaded nanoparticles accumulated predominantly in tumors and provided high lesion-to-

normal tissue contrast for sensitive fluorescence detection (Figure 4). The developed SiNc-PNP

have the optimal characteristics including small hydrodynamic size, neutral charge and

PEGylated surface, which provide an efficient passive tumor targeting and penetration.57

Li et al.

reported significant accumulation of PEGylated theranostic nanoparticles with a diameter of 32

nm in ovarian cancer tumors 24 h after intravenous administration.3 The cancer cell targeting

ligands could potentially improve internalization of the nanoparticles by cancer cells. However,

it will also increase complexity, complicate synthetic procedure, raise manufacturing cost and

therefore limit translational potential of these nanoplatforms.

Despite a fact that SiNc is not biodegradable molecule, SiNc was cleared from healthy organs

within 96 h after intravenous injection. This is an important characteristic for the developed

nanoplatform, because the US FDA requires all injected contrast agents to be completely cleared

from the body in a reasonable time period.45, 58

The tumor targeting capability and rapid

clearance of SiNc-PNP highlights the possibility of developing this unique nanoplatform for

phototherapy treatment.

21

To investigate the in vivo anticancer efficacy of the combinatorial phototherapy, mice bearing

subcutaneous xenograft of Adriamycin-resistant ovarian cancer cells were intravenously injected

with SiNc-PNP at the dose of 1.5 mg/kg. Under NIR fluorescence imaging guidance, which

confirmed SiNc accumulation in the tumor at 24-h post-injection, the laser beam (785 nm, 1.3

W/cm2) was precisely focused on the tumor and maintained for 10 min. The growth rate of

tumors was monitored for up to 25 days every 2-3 days and the day of injection was considered

as day 0 (Figure 5A). The untreated tumors and tumors treated with NIR light only and SiNc-

PNP without light (dark) grew rapidly, indicating that the A2780/AD tumor growth was not

affected by NIR laser alone or non-activated SiNc-PNP (Figure 5A). When the SiNc-PNP-

treated tumors were exposed to the continuous irradiation with NIR laser beam for 10 min, the

tumor tissue was completely eradicated on day 3-5 post-injection, and there was no tumor

recurrence detected during 25 days.

Acute toxicity due to treatment with SiNc-PNP was determined in female athymic mice. Post-

injection, mice were monitored for 25 days for changes in behavior, weight loss > 15 % and

death. During the course of the study none of the mice treated exhibited weight lost, changes in

behavior or died. Weight changes between the treatment and control groups are presented in

Figure 5B. As seen in the figure, none of the mice in the treatment groups were significantly

different as compared to the control mice, indicating that SiNc-PNP treatment shows no adverse

effects.

As demonstrated in Figure 5C, after exposure to NIR laser irradiation (1.30 W/cm2), the

intratumor temperature increased up to ~ 47 C, which is sufficient to ablate malignant cells. In

contrast, SiNc-PNP-free tumors did not demonstrate significant change in intratumoral

temperature under the same laser irradiation (∆T = 1.3 °C). Finally, not even slight skin burn was

22

noticed after irradiating the tumors for 15 min, suggesting that 785 nm laser light at a power

density 1.3 W/cm2 is safe for phototherapy. It is important to mention that NIR lasers with a

similar power density are widely used to conduct combinatorial phototherapy on living body. For

example, Li et al. reported that a 690 nm laser diode with a power density of 1.25 W/cm2

was

employed to perform combinatorial phototherapy in vivo.3 In another study, Li et al. employed a

808 nm laser at a power density of 1.4 W/cm2 to generate phototherapy in mice and the

temperature changes in saline-treated tumors was only ~ 2 °C.

24

Based on our in vitro studies (Figure 3D) and the observed strong anticancer effect in vivo

(Figure 5A), we suggest that PDT contributed to the therapeutic effect of PTT and the

photoirradiation of SiNc-treated tumors resulted in generation of intracellular ROS. However,

the in vivo real time measurements of intratumoral ROS during the combinatorial phototherapy is

challenging to experimentally support this point and future studies are needed to further elucidate

this potential mechanism of tumor ablation.

The observed therapeutic outcome of the combinatorial phototherapy is in good agreement with

a previous report, which indicated that simultaneous PDT/PTT therapy mediated by ICG-loaded

nanoparticles resulted in complete inhibition of tumor growth with no detected cancer

recurrence.4

To investigate morphological changes and apoptotic processes in tumor tissues during the

phototherapeutic treatment and for corresponding controls, histological studies were carried out

in tumors excised from the mice. Changes in morphology in tumor tissue were evaluated using

Hematoxylin and eosin (H&E) staining for tumor sections. H&E staining showed that the

phototherapy treated sample had more condensed nuclei (dark purple) suggesting an increase in

cellular apoptosis as well as marked increase in tissue damage when compared to control

23

samples (Figure 6A). The Hoechst stained images in the control and light only tissue samples as

well as in dark sample all demonstrated a minimal amount of cells positive for nuclear

condensation and membrane blebbing all of which are hallmarks of cells undergoing apoptosis.59

The phototherapy treated sample conversely showed a pronounced increase in the number of

cells positive for apoptosis as indicated by the highly fluorescent cellular nuclei (Figure 6B).

Tumor tissue staining with a caspase-3 antibody further verified that only combinatorial

phototherapy result in the substantial apoptosis (red fluorescence) (Figure 6C).

Cumulatively, the above experiments demonstrated that PEG-PCL polymeric nanoparticles can

safely and selectively deliver SiNc to tumors after systemic administration while preserving its

fluorescence imaging and phototherapeutic properties. Due to high photostability, SiNc-PNP

offers accurate detection of the tumor tissue during prolong imaging time, which also allows for

precise treatment with NIR light with minimal side effects toward healthy organs.

CONCLUSIONS

Herein we have reported the development and use of biodegradable-biocompatible SiNc-loaded

polymeric nanoparticles for image-guided combinatorial phototherapy, which consist of only two

building blocks. The developed preparation procedure is simple, reproducible, and provides

highly uniform nanoparticles with the required structural features for enhanced passive tumor

targeting after systemic administration. SiNc-PNP demonstrated excellent photostability and

efficient generation of NIR fluorescence, ROS and hyperthermia under NIR light illumination

needed for tumor imaging and cancer eradication via combined PDT and PTT mechanisms. After

efficient tumor accumulation of SiNc-NP following intravenous injection, ovarian cancer tumors

were delineated with NIR fluorescence and completely eradicated after irradiation with a single

24

dose of 785 nm NIR light. Thus, our strategy provides cancer imaging with a single-agent

theranostic nanoplatform and subsequent phototherapeutic treatment with great potential for

clinical translation. We also expect that the developed simple approach can be employed for a

variety of NIR phototherapeutic and imaging agents with limited translational potential due to

their hydrophobic nature.

Acknowledgement. The authors are grateful for funding support provided by the OSU College of

Pharmacy, OSU Venture Development Fund, OSU General Research Fund and the Medical

Research Foundation of Oregon Health and Science University. Electron microscopy was

performed at the Multiscale Microscopy Core (MMC) with technical support from the Oregon

Health & Science University (OHSU)-FEI Living Lab and the OHSU Center for Spatial Systems

Biomedicine (OCSSB).

Supporting Information. Cryo-TEM-based size distribution histograms of SiNc-PNP,

absorption spectra of SiNc-PNP aqueous solution before and after storing at room temperature,

and temperature profiles of SiNc-PNP aqueous solution exposed to the 785 nm NIR laser diode

with various power densities. This material is available free of charge via the Internet at

http://pubs.acs.org.

25

Table of Contents

26

Figures and Tables

=

+

PEG-PCL

SiNc

SiNc-PNP

PhototherapyImaging

leaky blood

vessel

Figure 1. Schematic illustration of a single-agent based theranostic nanoplatform (SiNc-PNP)

designed for image-guided combinatorial phototherapy. The nanoplatform is prepared by

encapsulation of a theranostic agent, SiNc, into the hydrophobic interior of PEG-PCL polymeric

nanoparticles. After intratumoral accumulation of SiNc-PNP, presumably facilitated by passive

targeting, the tumor tissue can be detected via NIR fluorescence imaging. When irradiated with

the NIR laser, SiNc-PNP transform the absorbed light energy into toxic ROS and heat to

eradicate tumor tissue.

27

Figure 2. (A) Size distribution of SiNc-PNP measured by Dynamic Light Scattering before and

after storing for 30 days at room temperature. The inset shows the actual photo of vial with 0.3

mg/mL SiNc-PNP in aqueous solution. (B) Representative cryo-TEM image of SiNc-PNP. (C)

Fluorescence spectrum (ex = 750 nm) of SiNc-PNP water solution (SiNc = 0.05 mg/mL). The

inset represents fluorescence image of SiNc-PNP solution recorded with Pearl Imaging System at

800 nm. (D) Singlet oxygen generation by SiNc-PNP (0.02 mg/mL) without (dark) and in the

presence of NIR irradiation (light) (785 nm, 1.3 W/cm2, 5 min). **P < 0.005 when compared

with water as control. (E) Temperature profiles of water (black) and SiNc-PNP (0.3 mg/mL)

aqueous solution (red) exposed to the NIR light (785 nm, 1.3 W/cm2). (F) Absorption spectra

and fluorescence images (Insets) of SiNc-PNP aqueous solution before and after irradiation with

NIR light (30 min, 1.3 W/cm2).

10 20 30 40 50 60 70 80

0

5

10

15

20

25 Freshly prepared

30 days

Vo

lum

e,

%

Size, nm

600 700 800 900

0.0

0.5

1.0

Before

After

Wavelength, nm

Ab

so

rba

nc

e, a

.u.

750 800 850 9000

200

400

600

800

1000

Flu

ore

sc

en

ce

, a

.u.

Wavelength, nm

C

Dark Light0

200

400

600

Water

SiNc-PNP

SO

SG

Flu

ore

sc

en

ce

, a

.u.

* *

0 5 10 15 2020

30

40

50

60

70

80

90 Water

SiNc-PNP

T

em

pe

ratu

re,

C

Time, min

FED

A

50 nm

B

28

0

30

60

90

120

150

* *

Control

NIR light

SiNc-PNP dark

SiNc-PNP light

Ce

ll v

iab

ilit

y, %

0

20

40

60

80

100

120

SiNc concentration, g/mL

Ce

ll v

iab

ilit

y,%

B

Dark Light0

500

1000

1500

2000* *

Cells control

SiNc-PNP

R

OS

in

cre

as

e, a

.u. DC

A

2.0 5.0 10.0 25.0 50.0 100.0

E cellular

internalization

ROS

increase

T ~ 50 C

cell pellet

formation

SiNc-PNP

Figure 3. (A) Viability of A2780/AD ovarian carcinoma cells treated with SiNc-PNP under dark

conditions. (B) Representative fluorescence microscopy image demonstrating intracellular

accumulation of SiNc-PNP. (C) Combinatorial (PDT+PTT) therapeutic effect against A2780/AD

ovarian carcinoma cells. The cells were treated with 50 μg/mL of SiNc-PNP and exposed to a

laser light for 10 min (785 nm, 1.3 W/cm2). **P < 0.005 when compared to the controls. (D)

ROS generation in SiNc-PNP-treated A2780/AD cells following NIR light irradiation (785 nm,

1.3 W/cm2). **P < 0.005 when compared the controls. (E) In vitro evaluation of the

combinatorial photherapy mediated by the SiNc-PNP.

29

TumorLungsHeartSpleenKidneysLiver

Before

0.5 h

2 h

24 h

1.0

0.0

96 h

A B

Figure 4. The biodistribution of SiNc-PNP in nude mice. In vivo NIR fluorescence imaging of

the mice bearing A2780/AD tumor (A) and ex vivo imaging of SiNc-PNP in liver, kidneys,

spleen, heart, lungs, and tumor (B) after i.v. injected with SiNc-PNP (1.5 mg/kg) before and at

0.5, 2, 24 and 96 hours post-injection, respectively.

30

0 250 500 750 100030

35

40

45

50

55 NIR light

SiNc-PNP light

Intr

atu

mo

ral

tem

pe

ratu

re,

oC

Time, s

0 5 10 15 20 25

0

300

600

900

1200

1500

Control

NIR light

SiNc-PNP dark

SiNc-PNP light

Tu

mo

r v

olu

me

, m

m3

Time, days

0 5 10 15 20 2510

15

20

25

Control

NIR light

SiNc-PNP dark

SiNc-PNP light

Time, days

Bo

dy

we

igh

t, g

C

Laser “on”

Laser “off”

A

B

Figure 5. (A) Tumor growth profiles in mice with A2780/AD xenografts after the following

treatments (a) Control (black curve): no treatment, (b) NIR light (red curve): tumors irradiated

with a laser light (785 nm, 1.3 W/cm2) for 10 min, (c) SiNc-PNP dark (blue curve): mice

intravenously injected with SiNc-PNP (1.5 mg/kg) without light exposure and (d) SiNc-PNP

light (green curve): mice intravenously injected with SiNc-PNP (1.5 mg/kg) and tumors were

irradiated for 10 min with a laser light (785 nm, 1.3 W/cm2). (B) Changes in body weights of

treated mice compared to control groups. Means ± SD are shown. Arrows represent one injection

on day 0. (C). Intratumoral temperature profile during the phototherapy mediated by SiNc-PNP

31

(1.5 mg/kg) in combination with NIR light (red curve). Tumors irradiated with the NIR laser

light only were employed as the control (black curve).

A Control NIR light SiNc-PNP dark SiNc-PNP light

H&

EH

oech

st

Casp

ase-3

B

C

Figure 6. Representative images of H&E (A), Hoechst (B), and caspase-3 (C) stained tumor

sections harvested from the mice after the following treatments: control (no treatment), NIR light

(tumor irradiated with a laser (785 nm, 1.3 W/cm2)), SiNc-PNP dark (mice treated with SiNc-

PNP without light exposure) and SiNc-PNP light (phototherapy).

Table 1. Stability of the SiNc-PNP

Time Size, nm PDI Zeta potential, mV SiNc loading, mg/mL

Day, 0 37.66 ± 0.26 0.06 ± 0.02 -2.76 ± 1.83 0.30 ± 0.01

Day, 30 37.16 ± 0.83 0.08 ± 0.01 -2.84 ± 1.68 0.29 ± 0.02

32

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