Redox-responsive porphyrin-based polysilsesquioxane nanoparticles for photodynamic therapy of cancer cells

International Journal of

Molecular Sciences

Article

Redox-Responsive Porphyrin-Based

Polysilsesquioxane Nanoparticles for Photodynamic

Therapy of Cancer Cells

Daniel L. Vega ¹^,², Patrick Lodge ¹and Juan L. Vivero-Escoto ¹^,²^,

*

Received: 30 November 2015; Accepted: 28 December 2015; Published: 31 December 2015

Academic Editor: Michael R. Hamblin

1

Department of Chemistry, University of North Carolina at Charlotte, Charlotte, NC 28223, USA;

dvega1@uncc.edu (D.L.V.); plodge@uncc.edu (P.L.)

The Center for Biomedical Engineering and Science, University of North Carolina at Charlotte, Charlotte,

2

NC 28223, USA

*

Correspondence: jviveroe@uncc.edu; Tel.: +1-704-687-5239; Fax: +1-704-687-0960

Abstract: The development of stimulus-responsive photosensitizer delivery systems that carry a

high payload of photosensitizers is of great importance in photodynamic therapy. In this study,

redox-responsive polysilsesquioxane nanoparticles (PSilQNPs) built by a reverse microemulsion

approach using 5,10,15,20-tetrakis(carboxyphenyl) porphyrin (TCPP) silane derivatives as building

blocks, were successfully fabricated. The structural properties of TCPP-PSilQNPs were characterized

by dynamic light scattering (DLS)/ζ-potential, scanning electron microscopy (SEM) and

thermogravimetric analysis (TGA). The photophysical properties were determined by UV-vis and

ﬂuorescence spectroscopy. The quantity of singlet oxygen generated in solution was measured

using 1,3-diphenylisobenzofuran. The redox-responsive release of TCPP molecules was successfully

demonstrated in solution in the presence of a reducing agent. The internalization of TCPP-PSilQNPs

in cancer cells was investigated using laser scanning confocal microscopy. Phototoxicity experiments

in vitro showed that the redox-responsive TCPP-PSilQNPs exhibited an improved phototherapeutic

effect on cervical cancer cells compared to a non-responsive TCPP-PSilQNP control material.

Keywords: photodynamic therapy; photosensitizer delivery; porphyrin; stimulus-responsive

materials; polysilsesquioxane nanoparticles; cancer therapy

1

. Introduction

Photodynamic therapy (PDT) is an innovative minimally invasive therapy that has great potential

to selectively destroy malignant cells while sparing normal cells [1–5]. PDT is currently approved

for the treatment of various types of cancers, including lung, head and neck, esophageal and cervical

cancers. PDT uses photosensitizer (PS) agents that will localize, ideally, in a speciﬁc tumor tissue, at

which point irradiation with light of the appropriate wavelength will activate the PS. Upon activation

1

with light, the PS molecule interacts with molecular oxygen to generate singlet oxygen ( O ) and

2

reactive oxygen species (ROS), leading to the destruction of cancer cells through apoptosis or through

necrosis [

approach has been limited. Several reasons can account for that such as the poor penetration of light

in tissue and its dependence on the presence of oxygen [ ]. In addition, there are several limitations

associated speciﬁcally to the PS agents such as the development of non-speciﬁc skin phototoxicity,

poor water solubility and inefﬁcient delivery to tumor tissues [ 11]. Therefore, novel delivery systems

are necessary to improve the speciﬁcity and enhance the phototherapeutic efﬁcacy of PDT.

2,3,6,7]. Despite the favorable advantages of PDT, the clinical application of this therapeutic

2,8

9–

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www.mdpi.com/journal/ijms

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Nanoparticle-based PS delivery platforms have emerged as alternative approaches to overcome

some of the delivery issues of PSs. Nanoparticles offer several advantages as PS delivery systems: they

can carry large payloads of PS molecules; their surfaces and compositions can be tailored to develop

multifunctional systems; and, due to their sizes in the nanoscale regime, these materials are known to

accumulate at tumor sites by the so-called enhanced permeability and retention (EPR) effect [12

Several groups have already demonstrated that PS-loaded nanocarriers could enhance the tumor

target speciﬁcity and therapeutic efﬁcacy in cancer treatment [19 22]. In addition, nanoparticulate

approaches have been used for combination therapy including PDT [23 26]. Hayashi and coworkers

–18].

–

recently reported on the synthesis of an iodinated silica/porphyrin hybrid nanoparticle. This platform

was successfully applied for the PDT/Photothermal therapy (PTT) combination treatment of multiple

myeloma in vivo [25]. Despite the encouraging results using nanoparticle-based PS delivery systems,

there are two main problems that prevent nanoparticles from reaching their highest potentials as PS

1

carrier platforms. One issue is the potential trapping of the produced oxidative species ( O and

2

ROS) inside the nanoparticle due to the presence of the nanocarrier’s matrix, which slows down or

completely prevents the out-diffusion of the generated oxidative species [16]. Moreover, another

hurdle is the self-quenching of PSs encapsulated inside the nanoparticles, which occurs because of

their spatial proximity [27]. This effect is enhanced in PS delivery platforms that contain large number

of PSs [28–30]. Both limitations would largely reduce the phototoxic effect of PSs against cancer

cells. One of the strategies that has been explored to overcome these issues is the development of

stimuli-responsive nanoparticle-based platforms that can degrade upon speciﬁc conditions such as

low pH, highly reducing environments, etc. These materials increase the phototherapeutic efﬁcacy in

tumor tissues after the material has dissociated inside cancer cells [29–33]. Our group and others have

explored the use of disulﬁde bonds to develop redox-responsive PS delivery systems. The introduction

of disulﬁde bonds enables the PS delivery nanocarrier to release its payloads efﬁciently in intracellular

reductive environments [34–38]. Huh and coworkers reported on the synthesis and application

in vitro and in vivo of the PDT agent pheophorbide A (PheoA) conjugated with glycol chitosan (GC)

polymer via reducible disulﬁde linkages [34]. The developed polymer self-assembled forming core-shell

spherical nanoparticles (CNPs) (PheoA-ss-CNPs) about 200 nm in diameter. The photoactivity and

therapeutic efﬁcacy of this platform was compared with non-reducible NPs (PheoA-CNPs) in vitro

The reducible NPs showed rapid cellular uptake and signiﬁcantly higher phototoxicity than the

non-reducible NPs due to the dissociation of NPs in the intracellular reductive environment. The in vivo

imaging results showed that the reducible NPs selectively accumulated to the tumor site through

the EPR effect. The results of in vivo therapeutic efﬁcacy studies in tumor-bearing mice showed

that a signiﬁcantly decreased tumor volume was observed for PDT with PheoA-ss-CNPs. Durand

and coworkers reported on the development of biodegradable two-photon PDT medical devices

using disulﬁde linkers. In this work, bridged silsesquioxane (BS) NPs were used as platforms to

incorporate disulﬁde bridges, two-photon electron donor (diamino diphenylbutadiene, 2PS) agents or

zinc-5,10,15,20-tetra(propargyloxyphenyl) porphyrins (POR) [35]. The BSNPs had a high loading of

.

2

PS (28 wt %) and POR (10–14 wt %). Moreover, these NPs were degraded in the presence of a reducing

agent (2 mM mercaptoethanol). The photo imaging and therapeutic properties of this platform was

successfully evaluated in vitro using breast cancer MCF-7 cells. Our group has also reported on the

synthesis, characterization and in vitro application of redox-responsive nanoparticles containing the

protoporphyrin-IX (PpIX) molecule as a PS agent (RR-PpIX-PSilQNPs) [37]. This platform showed the

redox-responsive release capabilities of PSs in the presence of a reducing agent. Moreover, phototoxic

evaluation of RR-PpIX-PSilQNPs in HeLa cells showed higher phototoxicity than that of a control

sample (C-PpIX-PSilQNPs) that did not contain disulﬁde bonds in the network. We hypothesized

that the enhancement in the phototherapeutic effect for RR-PpIX-PSilQNPs was due to selective

release of PpIX molecules after internalization in cancer cells. This hypothesis was later corroborated

by confocal microscopy using a double-labeled core-shell nanoparticulate approach [38]. In this

study, we report on the synthesis, characterization and in vitro application of a redox-responsive

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PSilQ platform containing tetrakis(carboxy)phenyl porphyrin (TCPP) (Scheme 1). Two building block

molecules based on TCPP, one control (C-TCPP) and one redox-responsive (RR-TCPP) derivatives

(Scheme 2), were synthesized in multi-step reactions. The RR-TCPP ligand incorporates a disulﬁde

bond that is cleaved under reducing conditions such as those found inside of cancer cells. Both TCPP

derivatives include triethoxysilane groups, which, after condensation in a reverse microemulsion

reaction, afforded the PSilQNPs. The structural properties of these TCPP-based PSilQNPs showed

that PSilQNPs were synthesized with sizes of 50–70 nm in diameter and high contents of TCPP, on the

order of 120–150 µmol per g of PSilQNPs. Moreover, we have shown that once the RR-TCPP-PSilQNPs

have been internalized in the cells, the redox-responsive PSilQ platform increases phototoxicity in

comparison to the C-TCPP-PSilQNPs material.

Scheme 1. Schematic representation of the redox-responsive porphyrin-based polysilsesquioxane

nanoparticle (PSilQNP) platform developed in this work. The framework of the nanoparticle is made

of tetrakis(carboxyphenyl) porphyrin (TCPP)-based monomers, which contains a disulﬁde bridge and

silica bonds as connecting units.

Scheme 2. Two TCPP-based monomers are synthesized in this work, control TCPP (C-TCPP) (left

)

and redox-responsive TCPP (RR-TCPP) (right). Both molecules contain triethoxysilane groups that

can be polymerized to afford PSilQNPs and carboxylic acid moieties that can be used for further

functionalization. Moreover, RR-TCPP has disulﬁde bonds that are cleaved under high reducing

conditions, such as those found in cancer cells.

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2

. Results and Discussion

2

.1. Synthesis and Characterization of Redox-Responsive Tetrakis(Carboxyphenyl) Porphyrin (RR-TCPP) and

Control Tetrakis(Carboxyphenyl) Porphyrin(C-TCPP) Silane Derivatives

To fabricate the TCPP-PSilQNPs developed in this work, two novel TCPP silane

derivatives were synthesized and characterized (Schemes 2 and 3). First, the synthesis of

5

,10,15,20-tetrakis(4-carbomethoxyphenyl) porphyrin (TCM PP;

1

) was carried out through the reaction

4

˝

of benzaldehyde and pyrrole in propionic acid at 150 C. TCM PP then underwent hydrolysis

4

under basic conditions in tetrahydrofuran (THF)/Ethanol (EtOH) to afford TCPP (

2). A distinct

´

1

change in the stretching vibration of the carbonyl group from the methyl ester (1720 cm ) to

´

1

the carbonyl corresponding to the carboxylic acid (1694 cm ), along with the disappearance of

1

13

the methyl group in H- and C-NMR demonstrated the successful synthesis of TCPP. The next

synthetic step was the conjugation of TCPP with N-hydroxysuccinimide (NHS) in the presence of

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to afford TCPP-succinimide ester (SE) (3).

The TCPP-SE derivative includes a succinimide ester, which is an excellent leaving group for the

nucleophilic acyl substitution with amines to afford the corresponding amides. The synthesized

TCPP-SE molecule showed a diagnostic stretching vibration in IR corresponding to the ester and

´

1

succinimide groups (1736, 1770 and 1803 cm ). In addition, the appearance of the ethylene groups

1

13

of the succinimide in H- and C-NMR provided further evidence for the successful synthesis of

TCPP-SE. To afford the C-TCPP silane derivative, was reacted with serine in dimethylsulfoxide

DMSO) followed by aqueous work-up in acidic conditions to afford the amino acid form of TCPP,

3

(

TCPP-Serine (4). The amine group of serine is a stronger nucleophile than the alcohol group, which

allowed the exclusive synthesis of the amide bond, but not of the ester derivative. The disappearance

1

of the succinimide peaks from NHS and the appearance of serine peaks in IR and H-NMR conﬁrmed

the synthesis of TCPP-Serine. It is important to point out that compounds 1, 2, 3 and 4 were

also conﬁrmed with matrix-assisted laser desorption/ionization-time of ﬂight (MALDI-TOF) mass

spectrometry (see Experimental Section). Lastly, the C-TCPP silane derivative was synthesized

by reacting TCPP-Serine with triethoxysilane propyl isocyanate (TES-PI) under N atmosphere in

2

anhydrous dimethylformamide (DMF) for 22 h. This was followed by aqueous work-up in acidic

conditions to afford C-TCPP (5). The stretching vibrations in the IR spectrum for the carbonyl group

´ ´1 ´1

1

(

1706 cm ), along with the appearance of the Si–C (1233 cm ) and Si–O (1016 cm ), are indications

of the successful synthesis of C-TCPP.

The synthesis of the RR-TCPP silane derivative was carried out following the steps depicted in

Scheme 3. First, TCPP-SE reacted with pyridyl disulﬁde cysteamine (PDSCA; 10) and Et N in DMF

3

˝

1

at 80 C to afford TCPP-PDSCA (

6

). H-NMR conﬁrmed the synthesis of TCPP-PDSCA, the peaks

in the H-NMR for the succinimide group are no longer present; moreover, the aromatic protons

corresponding to the pyridine group are observed. TCPP-PDSCA is further reacted through a disulﬁde

1

˝

exchange reaction with cysteine in DMF at 60 C to obtain TCPP-Cysteine (

7

). The disappearance of

1

the peaks in H-NMR corresponding to pyridine and the appearance of the protons associated with

cysteine suggested that the disulﬁde reaction was successful. Finally, TCPP-Cysteine reacted with

TES-PI in anhydrous DMF under N atmosphere to afford RR-TCPP (

8). The stretching vibrations in

2

the IR spectrum provide evidence for the synthesis of RR-TCPP. The IR shift for the carbonyl group

´

1

´1

(

1714 cm ) along with the appearance of the Si–C (1222 cm ) and Si–O (1019 cm ) are indicative of

the successful synthesis of RR-TCPP.

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Scheme 3. Schematic representation of the synthesis of C-TCPP and RR-TCPP silane derivatives.

For simpliﬁcation, the tetrakis(phenyl) porphyrin is represented as the R group. See details of the

experimental conditions for each of the reactions in the Experimental Section. The synthesis of

5

,10,15,20-tetrakis(4-carbomethoxyphenyl) porphyrin (TCM PP) (

1

) is carried out by the condensation

under basic conditions afforded TCPP ( );

can be activated toward acyl nucleophilic substitution by the formation of the ester

bond with N-hydroxysuccinimide (NHS) ( ); ( ) For the synthesis of C-TCPP, compound underwent

an acyl nucleophilic substitution with serine to produce TCPP-Serine ( ); ( ) Finally, reacted with

triethoxysilane propyl isocyanate (TES-PI) to obtain C-TCPP ( ); ( ) To produce RR-TCPP, compound

also underwent acyl nucleophilic substitution with pyridyl disulﬁde cysteamine (PDSCA) (10) to form

TCPP-PDSCA ( ); ( ) Compound is synthesized by the disulﬁde exchange reaction between and

4

of pyrrole and benzaldehyde; (

a) Hydrolysis of

1

2

(

b

) Compound

2

3

c

3

4

d

4

5

e

3

6

f

7

6

cysteine; (g) Lastly, RR-TCPP (8) is produced by reacting 7 with TES-PI.

2

.2. Singlet Oxygen Generation of TCPP-Serine (4) and TCPP-EtSH (9)

1

PDT is dependent on the presence of molecular oxygen. This suggests that O generated by

2

the photosensitization of molecular triplet oxygen is the principal toxic species formed during PDT.

Therefore, the generation of singlet oxygen is extremely crucial to the success of PDT, and one of the

1

ﬁrst tests performed on new PSs is to probe their abilities for O generation [39]. The photophysical

2

1

properties of porphyrins, such as quantum yields, lifetimes and O generation, are mainly affected

2

by core modiﬁcations with the incorporation of transition metals and/or the replacement of one or

more of the porphyrin pyrrolic nitrogens with other heteroatoms [40,41]. However, modiﬁcations

on the meso phenyl rings with heavy atoms in molecules like tetraphenylporphyrin have also shown

1

enhancement in the generation of O [42]. To evaluate whether the chemical modiﬁcations of TCPP

2

1

(

2

) with serine and cysteamine cause an effect on the O generation, we measured the amount of

2

1

O produced by TCPP-Serine (4) and TCPP-EtSH (9). TCPP-EtSH is the PS agent produced after

2

RR-TCPP is reduced in the presence of a reducing agent (see insert in Figure 1); the Experimental

1

Section shows the details for the synthesis of

9

. The O production is measured by using a singlet

2

oxygen chemical probe (1,3-diphenylisobenzofuran, DPBF). DPBF is a singlet oxygen scavenger that

reacts in a Diels–Alder [[4 + 2]-cycloaddition with the singlet oxygen generated by the excited PS.

1

DPBF usually absorbs light at 419 nm; however, after the reaction with O the resulting product

2

does not absorb light at that wavelength [43]. Samples of 2, 4 and 9 were prepared in DMF (2.5 µM)

Int. J. Mol. Sci. 2016, 17, 56

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2

together with DPBF (5

µ

M). The samples were illuminated using white (400–700 nm; 41 mW/cm ) or

2

red (630–700 nm; 89 mW/cm ) light at different times. The data show that there is an increased in the

1

generation of O by

4 and 9 as compared with 2 after irradiation with white light. Nevertheless, there

2

were no statistically signiﬁcant differences between

were irradiated with red light, a slightly difference in the generation of O was observed following the

4

and

9

(Figure 1). When the TCPP derivatives

1

2

1

trend of

9

>

4

>

2

(Figure 1). The most important conclusion from the O generation data for the goal

2

1

of this work is that there was not a dramatic reduction in the O production after the functionalization

2

of TCPP molecule. Additional experiments, which are out of the scope of this work, need to be done

1

to ﬁnd out whether the differences in O generation from compounds

4

and

9

are due to solubility

2

and/or electronic effects associated with the chemical modiﬁcations of 2.

1

Figure 1. Determination of O production by compounds

2

(

blue),

4

(red) and

9 (green) after

2

irradiation with white (left) and red (right) light. Insert: Chemical structure of

9

. Error bars represent

the standard deviation of three independent experiments.

2

.3. Synthesis and Structural Characterization of RR-TCPP-PSilQ and C-TCPP-PSilQ Nanoparticles

The PSilQNPs in this work were synthesized by following a reverse microemulsion method

composed of a quaternary system. Reverse phase microemulsions consist of water droplets in the

nanoscale regimen, which are stabilized by a surfactant and/or co-surfactant in an organic phase [44].

The quaternary system consists of triton X-100, 1-hexanol, cyclohexane and C-TCPP or RR-TCPP, which

are used as surfactant, co-surfactant, organic solvent and silica precursor, respectively. To synthesize

the TCPP-based PSilQNPs, the silica precursor is dissolved in water in the presence of a base (NH OH)

4

to accelerate the polymerization reaction. Previous experience in our group with porphyrin-based silica

precursors has shown several challenges to dissolve these silica precursors in aqueous solutions [37].

However, in the case of C-TCPP and RR-TCPP molecules, the presence of carboxylic acid groups

facilitates this step because they can be deprotonated under basic conditions affording carboxylates,

which are more soluble in aqueous solutions. The solution containing the TCPP silane derivative

is later added to the organic phase, which is composed of triton X-100, 1-hexanol and cyclohexane.

The reverse microemulsion reaction is carried out for 24 h at room temperature (RT). After that,

the TCPP-based PSilQNPs are obtained by centrifugation after the material has been crashed down

with EtOH. The structural properties of these PSilQNPs were characterized by DLS,

and TGA (Figure 2 and Table 1). The DLS showed that the hydrodynamic diameter of C-TCPP- and

RR-TCPP-PSilQNPs is 183.8 10.5 and 144.3 15.0 nm, respectively. The hydrodynamic diameter is

around two times bigger than what it is observed by SEM. The SEM micrographs showed diameters

for C-TCPP- and RR-TCPP-PSilQNPs of 60.1 9.2 and 57.5 7.7 nm (see Figure 2 and Figure S1),

ζ-potential, SEM

˘

respectively. The difference between the hydrodynamic diameter and the particle size found by

SEM may be due to the inﬂuence of the solvent, the aggregation of the PSilQNPs and/or their

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ability to swell after adsorption of water molecules, hydrogel-like behavior [45]. However, it is

important to point out that the colloidal stability of this TCPP-PSilQNPs has increased dramatically

with what we have reported before for porphyrin-based PSilQNPs [37]. The improvement in the

colloidal stability is most likely due to the presence of the carboxylate groups on the surface of

the nanoparticles. The

ζ

-potential for C-TCPP and RR-TCPP-PSilQNPs in PBS (1 mM, pH 7.4) was

-potential measurements conﬁrmed that the

´

39.7 ˘ 2.8 and ´44.5 ˘ 2.5 mV, respectively. The

ζ

surface of the PSilQNPs is negatively charged, as mentioned above, due to the presence of carboxylates

groups on the surface of the nanoparticles. The amount of aromatic organic content by TGA for

C-TCPP and RR-TCPP PSilQNPs was determined to be 10.1 and 11.3%wt., respectively. These values

˝

were determined by using the weight lost between 350 and 800 C, which is the region where TCPP

losses more than 95%wt. of its organic content (Figure S2). Based on this data the amount of TCPP

loaded to C-TCPP and RR-TCPP-PSilQNPs was calculated as 127.7 and 142.0

respectively. Nevertheless, the values obtained through UV-visible spectroscopy for the loading of

TCPP were 80.3 and 89.3 mol per g of PSilQNPs for C-TCPP and RR-TCPP-PSilQNPs, respectively.

µmol per g of PSilQNPs,

µ

The difference can be accounted by the aggregation of TCPP molecules inside PSilQNPs, which

prevents the absorption of light as compared with individual units in solution.

(

a)

(

b)

(

c)

Figure 2. Structural properties of C-TCPP- and RR-TCPP-PSilQNPs. (

of C-TCPP- (green) and RR-TCPP-PSilQNPs (red); ( ) SEM image of C-TCPP-PSilQNPs (green circles

show the diameter of individual nanoparticles); Scale bar = 200 nm; and ( ) Thermogravimetric analysis

TGA) plot of C-TCPP- (green) and RR-TCPP-PSilQNPs (red).

a) Dynamic light scattering plot

b

c

(

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Table 1. Structural properties of control tetrakis(Carboxyphenyl) porphyrin (C-TCPP)- and redox-responsive

tetrakis(carboxyphenyl) porphyrin (RR-TCPP)-polysilsesquioxane nanoparticles (PSilQNPs).

Diameter (nm) *

ζ-Potential (mV) *

n = 3

Aromatic

Content (%)

Loading of TCPP

Sample

PDI

n = 3

(µmol/g)

C-TCPP-PSilQNPs

RR-TCPP-PSilQNPs

183.8 ˘ 10.5

0.39

0.33

´39.7 ˘ 2.8

10.1

11.3

127.7

142.0

144.3 ˘ 15.0

´44.5 ˘ 2.5

*

Data measured in phosphate buffer solution (1 mM; pH 7.4)/Concentration of PSilQNPs = 0.1 mg/mL;

PDI = Polydispersity index.

2

.4. Photophysical and Photochemical Properties of C-TCPP- and RR-TCPP-PSilQ Nanoparticles

UV-vis spectroscopy showed the successful encapsulation of TCPP in the C-TCPP and RR-TCPP

PSilQNP framework as shown by the Soret band at 420 nm and the Q bands at 518, 552, 592 and

6

4

48 nm (Figure 3). These bands are similar to the parent TCPP molecule; which are Soret band at

19 nm and the Q bands at 515, 551, 590 and 646 nm. Fluorescence spectroscopy measurements show

that the emission spectra of both the C-TCPP- and RR-TCPP-PSilQ materials is also similar to TCPP

without any signiﬁcant spectral shifts (Figure 3). These results suggest that TCPP was successfully

incorporated into PSilQNPs without major inﬂuence in the photophysical properties of the parent

1

porphyrin. In addition, the production of O by C-TCPP- and RR-TCPP-PSilQNPs was determined

2

1

using the singlet oxygen probe DPBF as described in Section 2.2. Interestingly, the amount of O

2

generated by the nanoparticles has been dramatically reduced, even though they have the same

concentration of TCPP molecules as the experiment depicted in Section 2.2 (Figure 3). To obtain

1

meaningful values from the O test, we had to increase the irradiation time for both white and red

2

light. This clearly indicates that the TCPP molecules incorporated in the framework of the PSilQNPs

do not generate singlet oxygen efﬁciently [34,37].

(

a)

(b)

(

c)

(d)

Figure 3. Photophysical and photochemical properties of C-TCPP- and RR-TCPP-PSilQNPs. (

spectroscopy of (blue), C-TCPP- (green) and RR-TCPP-PSilQNPs (red); and ﬂuorescence spectroscopy

) of (blue), C-TCPP- (green) and RR-TCPP-PSilQNPs (red). Determination of O production

a) UV-vis

2

1

(

b

2

by C-TCPP- (green) and RR-TCPP-PSilQNPs (red) after irradiation with white (

Error bars represent the standard deviation of three independent experiments.

c) and red (d) light.

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2

.5. Stimuli-Responsive Properties of RR-TCPP-PSilQ Nanoparticles

The RR-TCPP-PSilQNPs were designed to be stable under normal physiological conditions, but

they can be readily dissociated to release the TCPP-EtSH ( ) molecules upon the reductive cleavage

of the disulﬁde bonds by reducing agents such as dithiothreitol (DTT), such as glutathione and

cysteine [34 37]. To evaluate the degradation ability of RR-TCPP-PSilQNPs under high reducing

conditions, we measured the release of in solution in the presence and the absence of a reducing

9

,

9

agent. The release experiment revealed that RR-TCPP-PSilQNPs are stable in the absence of reducing

agents (ﬁrst 9 h), with only 10% or less background released (Figure 4). However, after the addition of a

DTT solution (10 mM), TCPP-EtSH molecules were immediately released from the RR-TCPP-PSilQNPs

reaching 25% release in the ﬁrst hour and a half-life (t₁_/₂) of approximately 23 h. In this experiment,

more than 80% of the TCPP-EtSH molecules were released after 59 h of incubation with DTT. By

contrast, in our control experiment, the RR-TCPP-PSilQNPs that are not incubated with DTT solution

showed less than 18% release after 96 h of incubation. The total amount released after 96 h in the

presence of DTT was 40.9 µmol TCPP-EtSH per g of RR-TCPP-PSilQNPs. The material was completely

degraded after eight days of incubation in the presence of DTT agent (data not shown).

Figure 4. Release proﬁle of TCPP-EtSH from RR-TCPP-PSilQNPs incubated in the presence (red circles

)

and the absence (blue circles) of the reducing agent dithiothreitol. Dithiothreitol (DTT) was added at

time equal 9 h.

2

.6. In Vitro Phototoxicity of C-TCPP- and RR-TCPP-PSilQ Nanoparticles

The phototocytotoxicity of C-TCPP- and RR-TCPP-PSilQNPs in human cervical cancer (HeLa)

cells was investigated by the MTS assay. HeLa cells were inoculated at different concentrations for 24 h

2

with each material and then irradiated with red light (630–700 nm; 89 mW/cm ) for 20 min. The “dark”

cytotoxicity, samples not irradiated with light, was also determined at the same concentrations of

PSilQNPs as the control experiment. Figure 5 shows the cell survival of HeLa cells that have been

incubated for 24 h after light irradiation. The cytotoxicity of the samples in absence of light showed that

both PSilQNPs are non-cytotoxic at the concentrations evaluated in this experiment. Nevertheless, the

cell viability decreased in the presence of both C-TCPP- and RR-TCPP-PSilQNPs after light exposure.

Of note, the decrease in cell survival is more noticeable with RR-TCPP-PSilQNPs as an indication of the

capability of this material to transport and deliver PS agents in a more efﬁcient way. Based on previous

works from the literature, we hypothesized that TCPP-EtSH molecules are released in monomeric

form under intracellular reducing conditions and without any loss of photoactivity [34

,37,38]. The half

maximal inhibitory concentration (IC ) for RR-TCPP-PSilQNPs after irradiation with red light is

50

around 0.1 µM. The internalization of RR-TCPP-PSilQNPs in HeLa cells was conﬁrmed by laser

scanning confocal microscopy (Figure S3). Overall, the in vitro data show that the RR-TCPP-PSilQNPs

1

can efﬁciently transport and deliver the TCPP-EtSH molecules, thereby avoiding O trapping in

2

the nanoparticle framework and self-quenching. As a result, the phototoxic effect on HeLa cells has

been improved.

Int. J. Mol. Sci. 2016, 17, 56

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Figure 5. Phototoxicity of C-TCPP-PSilQNPs (green) and RR-TCPP-PSilQNPs (red) in the absence of

light, and C-TCPP-PSilQNPs (blue) and RR-TCPP-PSilQNPs (orange) after light exposure (630–700 nm;

2

8

9 mW/cm ; 20 min). Error bars represent the standard deviation of three independent experiments

with six repetitions each.

3

. Experimental Section

3

.1. Materials and Methods

All of the reagents were purchased from Aldrich and used without further puriﬁcation.

Thermogravimetric analysis (TGA) was determined using a Mettler Toledo TGA/SDTA851 instrument

Mettler-Toledo AG Analytical, Schwersenbach, Switzerland) with a platinum pan and a heating rate

(

˝

of 1.0 C/min from 25.0 to 800.0 C under a nitrogen atmosphere. The sample was held at 800.0 C for

h to make sure that all the organic material had been calcined. A Raith 150 Field Emission Scanning

3

Electron Microscope (SEM) (Raith America Inc., New York, NY, USA) was utilized to measure the

particle size and shape of the materials. Nanoparticle samples were suspended in methanol in

preparation for the SEM. Dynamic light scattering (DLS) and ζ-potential measurements were carried

out using a Malvern Instrument Zetasizer Nano (red laser 633 nm) (Malvern Instrument Ltd., Malvern,

UK). The amount of TCPP molecules loaded into the PSilQNPs was quantiﬁed by UV-vis spectroscopy

(

Varian, Cary 300 Bio UV/vis spectrometer) (Varian, Sidney, Australia). The photophysical properties

of TCPP-based PSilQNPs were determined using UV-vis and ﬂuorescence (Varian, Cary Eclipse

ﬂuorescence spectrometer) (Varian, Sidney, Australia).

3

.2. Synthesis of 5,10,15,20-Tetrakis(carbomethoxy)phenyl Porphyrin (TCM PP) (1)

4

To synthesize

1, 2.294 g of methyl-4-formyl benzoate (14.0 mmol) was added to propionic acid

˝

(

150.0 mL), this solution was stirred and heated at 151 C. Then, 970 µL (14.0 mmol) of pyrrole was

added and the solution was allowed to reﬂux for 1 h. The product was puriﬁed by washing with

cold methanol and ﬁltered to obtain deep purple crystals. The product was dried under high vacuum

´

1 1

and stored at room temperature. Yield: 651 mg, 22.0%. IR: 1720 cm ; H-NMR: (300 MHz; CDCl ):

3

δ

, ppm (s, 12H, 4.1), (d, 8H, 8.3), (d, 8H, 8.4), (s, 8H, 8.8); ¹³C-NMR: (300 MHz; CDCl₃):

δ_C, ppm

H

5

2.60, 119.51, 128.11, 129.87, 131.21, 134.64, 146.72, 167.35 (ester); Calculated mass for 1: 846.90 g/mol;

+

MS (MALDI positive ion): m/z 847.34 [M + 1] , 848.67 [M + 2] , 849.38 [M + 3] .

3

.3. Synthesis of Tetrakis(carboxy)phenyl Porphyrin (TCPP) (2)

To synthesize , 500 mg of (590 mol) was added to a mixture of THF:EtOH (30 mL; 1:1 v/v)

2

1

µ

˝

containing 4 mL of KOH (2 M). The mixture was stirred at 70 C for 24 h. The product was obtained

by rota-evaporating the solvent mixture and dissolving in 300 mL of water followed by the addition of

850 µL HCl (37%/v) to afford precipitation of 2. The dark blue crystals were ﬁltered, dried under high

Int. J. Mol. Sci. 2016, 17, 56

11 of 16

vacuum and stored at room temperature. Yield: 452 mg, 96.8%. IR: 1694 cm ¹; ¹H-NMR: (300 MHz,

d₆-DMSO): _C, ppm 119.84,

, ppm (q, 16H, 8.26–8.39), (s, 8H, 8.85); ¹³C-NMR: (500 MHz, d -DMSO):

: 790.79 g/mol; MS

´

δ

H

δ

6

1

28.43, 129.95, 131.00, 132.16, 134.99, 145.95 167.97 (acid); Calculated mass for

2

´

1

´1

cm ,

(

MALDI): m/z [M], 790.9; Absorbance (DMF): Soret band (λmax = 419 nm,

ε

= 399,000 M

¨

2

r = 0.9989).

3

.4. Synthesis of Succinimide Ester of TCPP (TCPP-SE) (3)

To synthesize , 455 mg of (575 mol) was combined with 993 mg NHS (8.6 mmol), 422 mg

dimethylaminopyridine (DMAP) (3.5 mmol) and 1.1 g EDC (5.7 mmol) in a mixture of dichloromethane

DCM):DMSO (110 mL; 1.75:1 v/v). This solution was stirred for 2 h in an ice bath. After that,

3

2

µ

(

the mixture was removed from the ice bath and stirred for another 48 h at room temperature.

The succinimide ester TCPP-based molecule was obtained by precipitation in aqueous solution

containing 20% EtOH. The solid was washed several times with the same ethanolic solution and dried

˝

´1

using a lyophilizer. The ﬁnal product was stored at

´

20 C. Yield: 651 mg, 96%. IR: 1736 cm (ester),

770 cm (NHS), 1803 cm (NHS); H-NMR: (300 MHz, d -DMSO): _H, ppm (s, broad, 16H, 3.00),

_C, ppm 26.15 (methylene), 162.64

: 1175.13 g/mol; MS (MALDI positive ion): m/z 1176.04

´

1

´1

1

δ

6

13

(

[

q, 16H, 8.51–8.58), (s, 8H, 8.95); C-NMR: (500 MHz, d -DMSO): δ

6

ester), 171.01 (NHS). Calculated mass for

M + 1] , 1177.42 [M + 2] , 1178.56 [M + 3] .

3

+

3

.5. Synthesis of TCPP Serine Derivative (TCPP-Serine) (4)

To synthesize , 300 mg of (255 mol) was combined with 203 mg of L-serine hydrochloride

1.9 mmol) and 437 L N,N-Diisopropylethylamine (DIPEA) (2.5 mmol) in DMSO (25 mL). The serine

was ﬁrst dissolved in water (3.75 mL) before adding to DMSO. The mixture was stirred for 48 h at

4

3

µ

(

µ

˝

1

00 C. After that, the serine derivative was puriﬁed by precipitation in aqueous solution containing

2

5% EtOH followed by the addition of 180

µ

L HCl (37%/v). The blue crystals were washed several

˝

times with the same solution and dried using a lyophilizer. The ﬁnal product was stored at

Yield: 243 mg, 84.0%. IR: 1634 cm (amide); H-NMR: (300 MHz; d -DMSO):

3

´

20 C.

δ , ppm (d, 8H,

H

´

1

6

.92–3.94), (m, 4H, 4.63–4.69); ¹³C-NMR: (300 MHz; d -DMSO):

δ

= 56.41, 61.86 (aliphatic carbons),

6

1

66.93 (amide), 172. 57 (acid); Calculate mass for

4: C H N O , 1139.10 g/mol; MS (MALDI):

60

50

8

16

´

1

´1

2

m/z [M] 1139.10; Absorbance (DMF): Soret band (λmax = 419 nm, ε = 324,600 M ¨ cm , r = 0.9994).

3

.6. Synthesis of Control TCPP Silane Derivative (C-TCPP) (5)

To synthesize C-TCPP ( ), 170 mg of (149

-(triethoxysilyl)propyl isocyanate (TES-PI) (626 mol) and 183 µ

5

4

µ

mol) was combined with 155

µL of

3

L triethylamine (Et N) in anhydrous

3

N,N-Dimethylformamide (DMF) (10 mL) and stirred for 2 h in an ice bath under N atmosphere.

2

The mixture was removed from the ice bath and stirred at room temperature for another 20 h under

N atmosphere. The control ligand was obtained by precipitation in 80 mL of water followed by

2

the addition of 150

µ

L of HCl (37%/v). The black powder was washed several times with the same

˝

ethanolic solution and dried using a lyophilizer. The ﬁnal product was stored at

´20 C. Yield 219 mg,

´

1

´1

6

9.0%. IR: 1016 cm (Si–O), 1233 cm (Si–C), 1706 cm (carbamate).

3

.7. Synthesis of TCPP-Pyridine Disulﬁde Cysteamine (TCPP-PDSCA) (6)

To synthesize

6, compound 3 (314 mg, 267 mmol) was combined with 10 (386 mg, 1.7 mol) and

˝

Et N (292 L, 2.1 mmol) in DMSO (6.5 mL) and stirred at 80 C for 3 days. The product was puriﬁed

µ

3

by precipitation in aqueous solution containing 20% EtOH. The brown powder was washed several

times with the same ethanolic solution and dried using a lyophilizer. The ﬁnal product was stored

˝

´1

1

at

´

20 C. Yield: 195 mg, 50.0%. IR: 1605 cm (aromatic), 1638 cm (amide); H-NMR: (300 MHz;

_H, ppm (t, 8H, 2.91–3.21), (m, 8H, 3.62–3.89), (t, 4H, 7.24–7.32), (d, 4H, 7.58–7.65), (t, 4H,

.76–7.85), (q, 16H, 8.16–8.39), (d, 4H, 8.46–8.50), (s, 8H, 9.09).

d -DMSO):

δ

6

7

Int. J. Mol. Sci. 2016, 17, 56

12 of 16

3

.8. Synthesis of TCPP-Cysteine Disulﬁde (TCPP-Cysteine) (7)

To synthesize , compound (152 mg, 104 mol) was combined with L-Cysteine hydrochloride

128 mg, 729 mol) in DMF (5.2 mL). The solution was stirred at 60 C for 48 h. The cysteine derivative

was puriﬁed by precipitation in aqueous solution containing 25% EtOH followed by the addition

7

6

µ

˝

(

µ

HCl (180

µ

L, 37%/v). The reddish-brown material was washed several times with the same ethanolic

˝

solution and dried using a lyophilizer. The ﬁnal product was stored at

´20 C. Yield: 124 mg, 80.0%.

1

H-NMR: (300 MHz; d -DMSO):

δ , ppm (m, 8H, 2.98–3.11), (m, 8H, 3.52–3.61), (m, 8H, 3.68–3.87), (m,

H

6

2

H, 4.28–4.41), (m, 2H, 4.63–4.71), (m, 16H, 8.12–8.41), (s, 8H, 8.86).

3

.9. Synthesis of Redox-Responsive TCPP Silane Derivative (RR-TCPP) (8)

To synthesize RR-TCPP, compound

7 (153 mg, 61 µmol) was combined with TES-PI (106 µL,

428

µ

mol) and Et N (125 L, 895 mol) in anhydrous DMF (11 mL). The solution was stirred in an

µ

3

ice bath for 2 h under N conditions. The mixture was removed from the ice bath and stirred at

2

room temperature for another 20 h still under N atmosphere. The redox-responsive ligand was

2

puriﬁed by precipitation in H O (60 mL) followed by the addition HCl (150

µL, 37%/v). The black

2

powder was washed several times with the same aqueous solution and dried using a lyophilizer.

˝ ´1 ´1

20 C. Yield: 152 mg, 65.0%. IR: 1019 cm (Si–O), 1222 cm (Si–C),

The ﬁnal product was stored at

´

1

714 cm (carbamide).

3

.10. Synthesis of TCPP-Ethyl Thiol (TCPP-EtSH) by Reduction of TCPP-PDSCA with DL-Dithiothreitol

(

DTT) (9)

To synthesize

9, compound 6 (66.9 mg, 46 µmol) was combined with DTT (127 mg, 823 µmol)

in DMF (6.5 mL). The solution was stirred at room temperature for 24 h. The thiol derivative was

puriﬁed by precipitation in aqueous solution containing 25% EtOH followed by the addition HCl

(

60

µ

L, 37%/v). The brown solid was washed several times with the same solution and dried using a

˝

lyophilizer. The ﬁnal product was stored at

´20 C. Yield: 31.5 mg, 67.0%. The successful synthesis

of TCPP-EtSH was conﬁrmed by the disappearance of the aromatic protons for the pyridine in the

1

H-NMR. H-NMR: (300 MHz; d -DMSO): δ_H, ppm (q, 8H, 2.74–2.85), (m, 8H, 3.54–3.65), (m, 16H,

6

8

.21–8.42), (s, 8H, 8.86).

3

.11. Synthesis of 2 Pyridyl Disulﬁde Cysteamine (PDSCA) (10)

To synthesize PDSCA, cysteamine hydrochloride (1.132 g, 9.96 mmol) was dissolved in MeOH

1

(

10 mL) and added dropwise to a mixture of 2,2 -dipyridyl disulﬁde (4.4062 g, 20 mmol) and acetic

acid (800 L, 99%/v) in MeOH (20 mL) over 30 min. The mixture was stirred at room temperature for

4 h. The compound was puriﬁed by rotatory evaporation of MeOH followed by precipitation with

diethyl ether. The white crystals were dried under high vacuum and stored at room temperature. Yield:

µ

2

´

.86 g, 84.0%. IR: 1608 cm (aromatic); H-NMR: (300 MHz; d -DMSO): δ , ppm (m, 4H, 2.98–3.18),

6 H

t, 1H, 7.27–7.33), (d, 1H, 7.73–7.78), (t, 1H, 7.81–7.88), (s, 3H, 8.16–8.28), (d, 1H, 8.49–8.53); C-NMR:

1

13

(

300 MHz; d -DMSO):

aromatic carbons).

δ

_C, ppm 35.30, 38.21 (aliphatic carbons), 120.60, 122.21, 138.49, 150. 40, 158.59

6

1

3

.12. Singlet Oxygen ( O ) Determination for TCPP (2), TCPP-Serine (4) and TCPP-EtSH (9)

2

1

To measure the amount of O generated by 2, 4, and 9, 40 µL of DPBF from a stock solution

2

(

8 mM, DMF) were dissolved in 4 mL of a DMF solution of photosensitizer (2.5

µ

M). The solution

2

was irradiated with white light (400–700 nm, 41 mW/cm ) at different times (20, 40 and 60 s).

The absorbance at 419 nm of these solutions was measured using a UV-vis spectrophotometer after

illumination. Moreover, control experiments were run in the absence of light. In addition, experiments

2

were carried out using red light (630–700 nm, 89 mW/cm ) following the same protocol. All the

Int. J. Mol. Sci. 2016, 17, 56

13 of 16

experiments were run by triplicate. The decrease from the original amount of DPBF was used to

1

calculate the concentration of O produced.

2

3

.13. Synthesis of C-TCPP- and RR-TCPP-PSilQ Nanoparticles

The synthesis of PSilQNPs was carried out through a reverse-microemulsion method. An organic

phase was prepared mixing cyclohexane (7.5 mL), 1-hexanol (1.6 mL) and Trition X-100 (1.9 mL). At the

same time, an aqueous solution containing C-TCPP (8 mg), NH OH (4 mL) and H O (4 mL) was

4

2

prepared and immediately added to the organic phase solution dropwise. The mixture was allowed

to stir at room temperature for 24 h. After that, the C-TCPP-PSilQNPs were obtained by crashing

down the material after addition of EtOH (40 mL). The material was separated from the solution by

centrifugation and washed twice with EtOH to get rid of any starting reagents. The ﬁnal product was

stored in EtOH. RR-TCPP-PSilQNPs were fabricated using the same protocol.

1

3

.14. Singlet Oxygen ( O ) Determination for C-TCPP- and RR-TCPP-PSilQ Nanoparticles

2

1

To measure the amount of O generated by C-TCPP- and RR-TCPP-PSilQNPs, 40

µL of DPBF

2

from a stock solution (8 mM, DMF) were dissolved in 4 mL of a DMF dispersion of PSilQNPs containing

the equivalent amount of 2.5

µ

M of TCPP. The solution was irradiated with white light (400–700 nm,

2

41 mW/cm ) at different times (60 and 120 s). The absorbance at 419 nm of these solutions was

measured using a UV-vis spectrophotometer after illumination. Moreover, control experiments were

run in the absence of light. In addition, experiments were carried out using red light (630–700 nm,

2

8

9 mW/cm ) following the same protocol. All the experiments were run by triplicate. The decrease

1

from the original amount of DPBF was used to calculate the concentration of O produced.

2

3

.15. Photophysical Characterization of C-TCPP- and RR-TCPP-PSilQ Nanoparticles

A Cary 300 Bio UV/vis (Varian, Sidney, Australia) and a Cary Eclipse ﬂuorescence spectrometers

(Varian, Sidney, Australia) were used to determine the absorption and ﬂuorescence emission of

C-TCPP- and RR-TCPP-PSilQNPs, respectively. The nanoparticles were redispersed in DMF with a

concentration of 0.5 mg/mL. TCPP (4 µM) dissolved in DMF was used as control sample.

3

.16. Release Proﬁle of TCPP-EtSH from RR-TCPP-PSilQNPs under High Reducing Environment

To determine the release of TCPP-EtSH compound under simulated reducing conditions, the

reducing agent dithiothreitol (DTT) was used. The RR-TCPP-PSilQNPs were washed several times (at

least ﬁve) with DMF to eliminate any physisorbed porphyrin. The nanoparticles were redispersed in

1

0 mL of DMF with a concentration of 0.35 mg/mL. Then, the dispersion was stirred for 9 h total under

N atmosphere to determine the amount of background TCPP-EtSH. After that, DTT was dissolved in

2

the dispersion to get a ﬁnal concentration of DTT of 10 mM. Aliquots were taken at certain intervals of

time and the absorption was measured to determine the amount of TCPP-EtSH molecules released.

A similar procedure was followed for the control RR-TCPP-PSilQNPs that were only stirred in DMF

(no addition of DTT).

3

.17. In Vitro Phototoxicity of C-TCPP- and RR-TCPP-PSilQ Nanoparticles in Human Cervical Cancer

(

HeLa) Cells

4

HeLa cells were seeded at a density of 1

in 100 L of RPMI-1640 cell media for 24 h at 37 C. Cells were then inoculated with C-TCPP- and

RR-TCPP-PSilQNPs (0.01, 0.05, 0.1, 0.5 and 1.0 M of TCPP) for 24 h in cell media, followed by PBS

ˆ

10 cells/mL in a 96-well cell plates and incubated

˝

µ

washing steps, and then further incubated in PBS for light exposure. Samples were exposed to a

2

LumaCare LC122 light source (630–700 nm; 89 mW/cm ) for 20 min. After irradiation, the cells were

incubated in cell media for another 24 h and the cell survival was tested by the MTS assay (CellTiter

®

9

6 AQueous Assay, Promega, Madison, WI, USA). The absorbance was measured at a wavelength of

Int. J. Mol. Sci. 2016, 17, 56

14 of 16

4

50 nm in plate reader Multiskan FC. Cell viability percentage was calculated based on the absorbance

measured relative to that of control culture cells.

4

. Conclusions

We have developed a redox-responsive TCPP-PSilQNP platform for the transport and delivery of

porphyrin-based photosensitizers with improved phototherapeutic effect toward human cervical cancer

cells. TCPP-PSilQNPs are stable under simulated physiological conditions and exhibited a high content of

PSs, 120–150 µmol of TCPP per g of PSilQNPs. The redox-responsive properties of the RR-TCPP-PSilQNPs

were tested in solution using DTT as reducing agent. The phototoxic efficacy of these nanoparticles was

evaluated in vitro using HeLa cells under light exposure by the MTS assay. RR-TCPP-PSilQNPs showed a

higher phototoxicity than the control C-TCPP-PSilQNPs. Presumably, because of the efficient transport and

intracellular release of TCPP-EtSH molecules. Moreover, TCPP-PSilQNPs contain carboxylic acid groups

that can be further functionalized with polymers such as poly(ethylene glycol) and targeting agents to

improve their targeting ability and therapeutic efficacy. TCPP-PSilQNP platform is a promising strategy in

the fabrication of versatile photosensitizer nanocarriers with stimulus-responsive properties for oncological

photodynamic therapy. Nevertheless, to move this PSilQNP system toward clinical applications, there

are still several barriers that need to be overcome such as evaluating its efficacy, pharmacokinetics

and biodistribution in animal models; its scalability and reproducibility following good manufacturing

practices (GMP); and its biocompatibility and efficacy in clinical trials. Our group is currently testing the

performance of this platform in animal models.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/

1

7/1/56/s1.

Acknowledgments: The authors would like to thank the University of North Carolina at Charlotte (start-up,

the Faculty Research Grant program and the Junior Faculty Development Award) and the Nanoscale Science

program for ﬁnancial support. The authors would also like to thank The Department of Biological Sciences at

UNC-Charlotte for allowing us to use their confocal microscopy facilities. Patrick Lodge was supported by the

National Science Foundation Research Experiences for Undergraduates (NSF REU) Site program in partnership

with the Awards to Stimulate and Support Undergraduate Research Experiences (ASSURE) program of the

Department of Defense (DoD) under NSF Grant No. CHE 1156867. We are grateful for Richard Jew for critical

reading of the manuscript and helpful suggestions.

Author Contributions: Daniel L. Vega and Patrick Lodge performed the synthesis and characterization of the

TCPP monomers and PSilQ materials. Juan L. Vivero-Escoto carried out the in vitro experiments. Daniel L. Vega

and Juan L. Vivero-Escoto conceived and designed the experiments. Juan L. Vivero-Escoto wrote the paper.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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