the clinical usefulness of light was first recognized some 3,000 years ago when it was used to treat rickets , psoriasis , and other common ailments.1 more recently , the term photodynamic therapy ( pdt ) surfaced and has been used to describe the use of light to treat a variety of diseases , including cancer.25 specifically , over the last 100 years , great strides have been made in developing photodynamic therapies for use in the clinical setting .
the first component is the photosensitizer , the photosensitive material that targets diseased tissue , akin to chemotherapeutic agents .
the second component is the irradiation of the target area with light of a specific wavelength .
excitation of the photosensitive material to a triplet state in the presence of oxygen results in triplet 
triplet annihilation , producing cytotoxic reactive oxygen species ( ros ) that destroy cellular components , thus triggering cell death .
therefore , the success of photodynamic therapeutics depends largely on the photosensitizer concentration throughout the tissue , adequate intracellular oxygen concentration , and proper source of excitation .
the discovery of this process was extremely promising in providing a more targeted , regulated method of cancer treatment .
however , one of the main drawbacks of this method of pdt is that it is oxygen dependent .
although cancer cells have abnormally high vascularization , intracellular oxygen concentration is low due to poor morphology of the vessels and abnormally high metabolic and oxygen consumption rates , which are characteristics of cancerous tissues.2 without oxygen available to interact with the photosensitizer , ros can not be formed and the therapeutic method becomes relatively ineffective or far less effective than intended .
in addressing the aforementioned issue , we recently took an alternative approach in attempting to trigger cell death by employing a photoresponsive agent with low dark cytotoxicity . rather than producing ros as the main  damaging  component
, we used a non - toxic sulfonium - based photoacid generator ( pag ) to cause intracellular ph imbalance.6 upon proper light excitation , the pag lowers the ph in the moderately ph - sensitive intracellular environment ; the acidic environment thus created leads to the malfunction and destruction of many cellular components , likely including key enzymes .
this process is considered an oxygen - independent pdt ( oi - pdt ) and , essentially , relies on two main components , the sulfonium salt distribution in the affected area and a proper source of excitation , rather than the three components mentioned earlier .
in essence , reducing the number of variables in the therapeutic process development improves the chances of success in material design and subsequent treatment .
given the hydrophobic nature of pags prepared in our preliminary study,6 we opted to introduce a polyethylene glycol ( peg ) unit that can confer hydrophilicity to the pdt agent while maintaining its photo - induced cytotoxicity .
along the same line , we prepared highly monodispersed silica nanoparticles ( sinps ) functionalized with amines that served as handles to covalently bond pag molecules via an amide linkage .
the incorporation of the pag into a nanomaterial delivery formulation is also intended to improve delivery properties of the proposed pdt agent as nanomaterials have emerged as a promising solution to the issues of cellular specificity , time - controlled delivery , and aqueous solubility.7,8 herein , we report the design , synthesis ( scheme 1 and supplementary material ) , and investigation of a new pag and its incorporation on sinps ( sinp  pag9 ) , which offers better solubility in aqueous media , greater efficacy than water - soluble pegylated pag ( peg  pag9 ) , and low dark cytotoxicity .
pag9 and sinp  pag9 were designed having similar aa core structure to the one reported in our initial work , where the two acceptor units ( a ) , no2 and sph2 , flank a fluorenyl - stilbene -spacer . using the c(9 ) of fluorene 1 to attach a handle for further functionalization , a propanoic acid unit was added as depicted in scheme 1 .
initially , treatment of 1 with one equivalent of n - buli at 78c generated a carbanion , stabilized by resonance through the fluorene -system ; this was followed by careful addition of ethyl bromide to afford the monoalkylated intermediate 2 .
bi - substituted fluorene 3 was obtained through a triton b - assisted michael addition of 2 with acrylonitrile in satisfactory yield ( 77% ) .
although later stages of the synthesis that use aqueous acids have resulted in the hydrolysis of the nitrile moiety , such acidic hydrolysis was incomplete and often resulted in low yields and multiple side products .
therefore , we opted to hydrolyze the nitrile group using aqueous naoh to afford the carboxylic acid derivative 4 in good yield .
subsequent intermediates 59 were prepared following our reported procedure to afford pag 9 in ~61% yield over four steps .
this compound was conjugated to an amine - terminated peg moiety by preparing the benzotriazole intermediate in situ , followed by the addition of the amine - terminated peg to afford peg 
sinps having amine appendages sinp(nh2)n ( preparation detailed in the supplementary material ) were treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide condensation agent , followed by the addition of an excess of pag 9 ; the good solubility of pag 9 in water allowed the dialysis of the resulting sinp 
surface modification of sinp(nh2)n by addition of pag 9 molecules was first assessed using a zetasizer nano system ( malvern instruments , malvern , uk ) , where such addition resulted in particle size increase from an average of 4 nm to 9 nm ( supplementary material ) .
in addition , the nuclear magnetic resonance ( nmr ) spectrum of sinp  pag9 in d2o ( supplementary material ) showed the presence of peaks in the aromatic region that corresponded to pag 9 , hence verifying the successful functionalizing of the sinps with the pdt ( pag ) molecules .
next , the photophysical properties of peg  pag9 and sinp  pag9 were examined to ensure that the magnitude of photoacid generation is preserved after modification .
initially , the absorption profiles of peg  pag9 and sinp  pag9 were obtained in water , where both show a similar absorption band to previously reported pl-127-encapsulated pag , with an absorption max of 384 nm .
pag9 was maintained at ~0.4 ( supplementary material ) , suggesting that the aqueous environment has little effect on the relevant photophysical properties of peg 
furthermore , the ph drop inside cell lysosomes was estimated by recording the changes in the absorption intensity of rhodamine b ( rhb ) base . conceptually , it is safe to assume that the number of protons generated by peg 
pag9 upon exposure is the same as the number of rhb base molecules converted to rhb+ , which is visualized as an increase in the absorption peak at ca .
extrapolation of the calibration curve ( figure 1b ) suggests that 50 m of peg 
pag9 would result in an increase of [ h]lysosomal by 9.110 m after a radiation dose of 10 min ; consequently , the lysosomal ph would be reduced by at least 0.3 ph units to ~4.4 ( supplementary material).9
in order to evaluate the intrinsic toxicity of peg  pag9 and sinp  pag9 , cell viability assays with celltiter 96 aqueos one solution reagent ( promega corporation , fitchburg , wi , usa ) were performed in the dark ( dark viability ) to prevent the production of acid and determine suitable concentration for effective pdt ( supplementary material ) . in this
regard , hct-116 cells ( human colorectal carcinoma ) were incubated with various concentrations of peg  pag9 and sinp 
pag9 has very low dark cytotoxicity , even at 100 m dose concentration ( figure 2a ) .
pag9 loading was doubled from 10 m to 20 m ( figure 2b ) . in order to further assess the cytotoxicity of pdt agents , post - exposure viability assays were performed using 50 m and 10 m of peg  pag9 and sinp  pag9 , respectively . as can be seen in figure 3 , exposure of hct-116 cultures incubated with peg 
such a drastic decrease in cell viability can be the result of pag distribution within the lysosomes and endosomes .
mechanistically , photoexcitation of pag 9 molecules that are densely grafted on sinp(nh2)n generates a highly localized dose of protonated species , and , thus , a greater localized ph drop .
consequently , a localized surge in the protonated species concentration creates an acidic microenvironment that could override mechanisms by which cells tend to regulate intracellular [ h ] , resulting in severe cytoplasmic acidification and inducing cell death.10 although this report is largely qualitative , the error bars of the cell viability in figures 2 and 3 provide some insight into the statistical significance of the results .
pag9 exhibits high cytotoxicity at very low concentration and short exposure time . in order to determine the localization of peg 
pag9 in hct 116 cells , lysotracker green ( thermo fisher scientific , waltham , ma , usa ) , a commercial dye that localizes in lysosomes after cell uptake , was used along with peg  pag9 .
pag9 was collected inside cells ( figure 4a  c ) , showing a good uptake efficiency of peg 
overlay image ( figure 4d ) exhibited good colocalization between peg  pag9 and lysotracker green , which indicated that peg 
next , we verified the reduced lysosomal ph by employing lysosensor green as a suitable intracellular ph sensor.6 since the fluorescence quantum yield of lysosensor green increases in increasingly acidic environments,11 fluorescence intensity as a function of exposure time was studied , which showed a drop in intralysosomal ph following irradiation in hct-116 cells incubated with peg  pag9 ( figure 5 and supplementary material ) .
as demonstrated in our previous study,6 time - lapsed micrographs ( figure 6 ) of hct-116 cells incubated with peg  pag9 and sinp 
pag9 ( figures 6 and 7 , respectively ) show significant cell swelling , loss of cell adhesion that is followed by a 
figure 8 shows a simplified jablonski diagram summarizing the process of photoacid generation by either one - photon or two - photon ( 2p ) excitation .
we demonstrated that sulfonium salts can be used to selectively induce cell death by photoexcitation .
pags induced necrotic cell death by creating a ph imbalance in the hct-116 cells via generation of photoacid within the lysosomes .
higher efficacy was achieved when these pag molecules were conjugated to silica - based nanoparticles , resulting in a higher cytotoxic effect . ultimately , near - infrared ( ir )
2p excitation of the water - soluble pag should afford deep tissue excitation and pdt .
thus , light - guided acid generation could be considered an attractive tool for treatment of hypoxic tumors and other diseases .
currently , our efforts are focused on evaluating the prepared pags in vivo and for 2p excitation in the near - ir spectral range .
 photodynamic therapy ( pdt ) processes involving the production of singlet oxygen face the issue of oxygen concentration dependency . despite high oxygen delivery , a variety of properties related to metabolism and vascular morphology in cancer cells result in hypoxic environments , resulting in limited effectiveness of such therapies . 
 an alternative oxygen - independent agent whose cell cytotoxicity can be remotely controlled by light may allow access to treatment of hypoxic tumors . toward that end , we developed and tested both polyethylene glycol ( peg)-functionalized and hydrophilic silica nanoparticle ( sinp)-enriched photoacid generator ( pag ) as a nontraditional pdt agent to effectively induce necrotic cell death in hct-116 cells . 
 already known for applications in lithography and cationic polymerization , our developed oxygen - independent pdt , whether free or highly monodispersed on sinps , generates acid when a one - photon ( 1p ) or two - photon ( 2p ) excitation source is used , thus potentially permitting deep tissue treatment . 
 our study shows that when conjugated to sinps with protruding amine functionalities ( sinp  pag9 ) , such atypical pdt agents can be effectively delivered into hct-116 cells and compartmentalize exclusively in lysosomes and endosomes . 
 loss of cell adhesion and cell swelling are detected when an excitation source is applied , suggesting that sinp  
 pag9 , when excited via near - infrared 2p absorption ( a subject of future investigation ) , can be used as a delivery system to selectively induce cell death in oxygen - deprived optically thick tissue . 
Introduction
Approach
Materials and methods
Results
Conclusion