navadeep shrivastava, kenneth hoyt, tayyaba hasan and

Research Article

Mina Guirguis, Chanda Bhandari, Junjie Li, Menitte Eroy, Sushant Prajapati, Ryan Margolis,Navadeep Shrivastava, Kenneth Hoyt, Tayyaba Hasan and Girgis Obaid*

Membrane composition is a functionaldeterminant of NIR-activable liposomes inorthotopic head and neck cancerhttps://doi.org/10.1515/nanoph-2021-0191Received April 26, 2021; accepted June 23, 2021;published online July 6, 2021

Abstract: Near-infrared (NIR)-activable liposomes contain-ing photosensitizer (PS)-lipid conjugates are emerging astunable, high-payload, and tumor-selective platforms forphotodynamic therapy (PDT)-based theranostics. Todate, theimpact that the membrane composition of a NIR-activableliposome (the chemical nature and subsequent conformationof PS-lipid conjugates) has on their in vitro and in vivo func-tionality has not been fully investigated.While their chemicalnature is critical, the resultant physical conformation dictatestheir interactions with the immediate biological environ-ments.Here,we evaluateNIR-activable liposomes containinglipid conjugates of the clinically-used PSs benzoporphyrinderivative (BPD; hydrophobic, membrane-inserting confor-mation) or IRDye 700DX (hydrophilic,membrane-protrudingconformation) anddemonstrate thatmembrane compositionis critical for their function as tumor-selective PDT-basedplatforms. The PS-lipid conformations were primarilydictated by the varying solubilities of the two PSs andassisted by their lipid conjugation sites. Conformation was

further validated by photophysical analysis and computa-tional predictions of PSmembrane partitioning (topologicalpolar surface area [tPSA], calculated octanol/water partition[cLogP], and apparent biomembrane permeability coeffi-cient [Papp]). Results show that the membrane-protrudinglipo-IRDye700DX exhibits 5-fold more efficient photody-namic generation of reactivemolecular species (RMS), 12-foldexpedited phototriggered burst release of entrap-ped agents,and 15-fold brighter fluorescence intensity as compared tothe membrane-inserting lipo-BPD-PC (phosphatidylcholineconjugate). Although the membrane-inserting lipo-BPD-PCexhibits less efficient photo-dynamic generation of RMS, itallows for more sustained phototriggered release, 10-foldgreater FaDu cancer cell phototoxicity, and 7.16-fold highertumor-selective delivery in orthotopic mouse FaDu head andneck tumors. These critical insights pave the path for therational design of emerging NIR-activable liposomes,whereby functional consequences of membrane composi-tion can be tailored toward a specific therapeutic purpose.

Keywords: head and neck cancer; nanomedicine; nearinfrared; photodynamic therapy; tumor delivery.

1 Introduction

Photonanomedicines (PNMs) are light-activable nanoscaledrug delivery systems that facilitate photodynamic therapy(PDT). PDT destroys disease tissue through photochemistry,a light-initiated reaction between a light activable mole-cule – the photosensitizer (PS) – and oftentimes molecularoxygen [1, 2]. Common lipid-based PNM platforms that arefrequently used for PDT-based regimens include liposomes,liposome bilayer coated nanoconstructs, and monolayerlipid-coated nanoconstructs. Visudyne™, a near-infrared(NIR)-activable liposome containing the PS benzoporphyrinderivative (BPD), was the first PNM to gain Food and DrugAdministration approval in 2000 for PDT of Age-relatedMacular Degeneration [3]. In recent years, NIR-activablePNMs, especially those based on nanometric liposomal

MinaGuirguis and ChandaBhandari contributed equally to this study.

*Corresponding author: Girgis Obaid, Department of Bioengineering,University of Texas at Dallas, Richardson 75080, Texas, USA,E-mail: [email protected]. https://orcid.org/0000-0002-9452-4467Mina Guirguis, Chanda Bhandari, Junjie Li, Menitte Eroy, SushantPrajapati, Ryan Margolis, Navadeep Shrivastava and Kenneth Hoyt,Department of Bioengineering, University of Texas at Dallas,Richardson 75080, Texas, USA. https://orcid.org/0000-0001-6056-6098 (M. Guirguis)Tayyaba Hasan, Wellman Center for Photomedicine, MassachusettsGeneral Hospital and Harvard Medical School, Boston 02114,Massachusetts, USA; andDivision of Health Sciences and Technology,Harvard University and Massachusetts Institute of Technology,Cambridge 02139, Massachusetts, USA. https://orcid.org/0000-0003-0871-6057

Nanophotonics 2021; 10(12): 3169–3185

Open Access. © 2021 Mina Guirguis et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0International License.

https://doi.org/10.1515/nanoph-2021-0191

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formulations, have seen an exponential rise in use as plat-form technologies that combine a PS for PDT and photo-triggered release of secondary therapeutics. Such secondary,and at times tertiary, therapeutics include small moleculeinhibitors [4], targeted biologics [5, 6], chemotherapeutics[7–12], and immunotherapeutic agents [13–16]. Salient fea-tures that are unique to NIR activation of liposomes include(1) rigorous control over the dose deposition of photody-namic reactive molecular species (RMS) and of entrappedsecondary therapeutics, in addition to (2) spatiotemporalcontrol over the induction of therapeutic combination regi-mens [1, 2].

In recent years, PS-lipid conjugates have emerged asversatile liposome membrane anchors with remarkableoptical, photochemical, and phototherapeutic properties,in addition to providing unique stability for PSs within theliposomal membranes [1, 11, 12, 17]. This stability is criticalfor keeping the PS molecule strongly associated with theliposome during in vivo circulation [11], for facilitatingtruemolecular specificity when targeting tumor-associatedreceptors [1, 18], and for nanoscale precision in targetingvarious organelles [19–21]. Molecular specificity forreceptor-targeted PDT can only truly be achieved when PSmolecules are stably anchored by lipids into the bilayer.We have previously demonstrated that BPD nonspecifi-cally and rapidly leaks out of liposomes into nontarget cells[18]. Conjugation of BPD to 16:0 lyso phosphatidylcholine(PC) and 20:0 lyso PC entirely prevents this nonspecific andpremature leakage from liposomal membranes [18]. In thecase of secondary and tertiary entrapped agents, this de-gree of stability within a liposome is also critical for themolecular specificity of co-delivery. Even if these agentsare released prematurelywithin the tumor interstitium, it isconceivable that a molecularly targeted liposome con-taining a stable lipid-conjugated PSwill sensitize the targetcells to the secondary agents nearby. However, this isspecific to the secondary or tertiary agents, their stabilitywithin liposomes, their mechanism of action, and themechanisms of their synergy with PDT [1].

Significant strides have been made in the therapeutic,diagnostic, and theranostic applications of NIR-activableconstructs that contain PS-lipid conjugates. Recent note-worthy examples include porphysomes comprised entirelyof self-assembled porphyrin-phospholipid conjugates andliposomes doped with porphyrin-phospholipid conjugatesentrapping chemotherapy developed by Zheng, Lovell andcolleagues [11, 12, 17]. The membrane composition (thechemical nature and resultant physical conformation) ofNIR-activable liposomeshas only beenstudied in the contextof their impact on the optical properties of the PS and onbiophysical self-assembly. Such compositions that lead to

membrane-protruding or membrane-inserting conforma-tions include chromophore molecules conjugated to theterminal end of phospholipid acyl chains [22], sn-1 or sn-2hydroxyl groups of lysophospholipids [17, 18, 21, 23], phos-phate headgroups [24, 25], hydroxyl groups of cholesterol[21], and to terminal hydrophilic functional groups of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol (DSPE-PEG) [21, 26, 27]. In our prior studies, we re-ported the first synthesis and characterization of a panel oflipid-based NIR-activable PNMs containing BPD conjugatesto 16:0 lyso PC, 20:0 lyso PC, cholesterol, and DSPE-mPEG2000-NH2 [21]. These constructs demonstrated a highdegree of tunability in their optical and photochemical ac-tivity [21].Wehavealso shown that 16:0 lysoPCand 20:0 lysoPC lipid conjugation of BPD resulted in an exclusively lyso-somal localization within cancer cells, thereby allowingmulti-organelle targeted PDT regimens when used in com-bination with unmodified BPD liposomes that target theendoplasmic reticulum and mitochondria [19–21]. This strat-egy has demonstrated superior outcomes in two-dimensionaland three-dimensional (3D) cancer models when using asingle-wavelength 690 nm irradiation protocol [19–21].

To date, studies have mostly dealt with PS-lipid conju-gates as passive spectators in the liposomeswhose functionsare to facilitate PDT, phototriggered agent release, or acombination of both. Considering the significance andclinical relevance of emerging NIR-activable liposomes, thisstudy explores the in vitro and in vivo functional impact thatmodulating the liposomal membrane composition has withrespect to the chemical nature and the subsequent physicalconformation of PS-lipid conjugates. The physical confor-mation of the PS-lipid conjugate is a direct consequence ofits different chemical nature.While both thechemical natureand physical conformation of the PS-lipid conjugate arelikely to affect functionality in concert, the physical differ-ences can dictate a lot of their behavior, namely: (1) photo-chemical efficiency following self-assembly and variousdegrees of static quenching, (2) accessibility of RMS to thebiological milieu, (3) accessibility of probes to RMS thatreach beyond the bilayer, (4) intracellular uptake, (5) serumcorona size and composition, (6) tumor delivery, and (7)tumor selectivity, amongst others. In this study, we explorethe collective functional impact of liposome membranecomposition using in vitro and in vivo orthotopic humanhead and neck cancer models, in addition to fundamentalphotophysical and photochemical analyses.

While we have previously reported liposomes contain-ing DSPE-PEG-BPD, the hydrophobicity of BPD prevents itfrom assuming a truly membrane-protruding conformation.When included at quantities exceeding 0.15 mol%, the li-posomes aggregate immediately the following extrusion as

3170 M. Guirguis et al.: Membrane composition is a functional determinant

a result of hydrophobic interactions between the DSPE-PEG-BPD containing liposomes [21]. Even at 0.15 mol%DSPE-PEG-BPD, the liposomes are 50% larger than BPD-PCliposomes making them unsuitable as counterparts whenexploring the role of conformation also. The larger size islikely a result of membrane contortion as the BPD reinsertsinto the bilayer upon lipid film. The opposite is also true thata stronglyhydrophilic PSmolecule cannotbe stably insertedinto the bilayer just by conjugating it to PC. Even withamphiphilic PSmolecules such as indocyanine green (ICG),a truly membrane-protruding conformation is not possible.A salient study by Lajunen et al. has demonstrated that ICGcan be stably inserted into the bilayer when mixed duringlipid film preparation, and can also noncovalently be hid-den within the PEG brush if introduced to the liposomesafter self-assembly [28]. This important study explored thefundamental functional impact of the membrane localiza-tion of ICG i.e. entrapped in lipid bilayer versus being non-covalently inserted in the PEG brush. While the study diddemonstrate that the location of ICG resulted in markeddifferences in photochemistry, phototriggered calceinrelease, and PS stability, the study did not explore theimpact on phototoxicity, tumor kinetics, or pharmacoki-netics in vivo. Importantly, the study did not use a PS-lipidconjugate, and thus did not probe the functional impact ofboth the nature and conformation of a PS-lipid conjugate inthe membrane.

The physical conformation of a PS-lipid conjugate isprimarily a consequence of its chemical nature. Consideringthat a single PS molecule cannot simultaneously be stablyinserting into the bilayer and stably protruding away fromthe bilayer, it is imperative that two separate PS moleculesare used to conjugate to lipids. This is to achieve bothdiscrete membrane-inserting and membrane-protrudingconformations, given that the differences in the chemicalnature between the two are understood and accounted for.As such, here we report the synthesis of a novel membrane-protruding DSPE-mPEG2000-NH2 conjugate of IRDye700DX,a hydrophilic clinical PS in phase III clinical trials for pho-toimmunotherapy of head and neck cancer (Clinical-Trials.gov Identifier: NCT03769506; accessed Mar 2021).Liposomes containing the membrane-protruding DSPE-mPEG2000-IRDye700DX are contrasted with liposomescontaining the membrane-inserting 20:0 lyso PC-BPD con-jugate (Figure 1). Themembrane-protruding andmembrane-inserting conformations are validated by computationalpredictions of their topological polar surface area (tPSA;ChemDraw 18.0), calculated octanol/water partitions(cLogP; ChemDraw 18.0), and apparent biomembranepermeability coefficients (Papp), as well as photophysicalanalysis. The two membrane compositions are evaluated in

terms of their impact on photochemical efficiency, cellularuptake, subcellular localization, PDT efficacy, photo-triggered and passive agent release kinetics, in vivo phar-macokinetics, and tumor-selective delivery in orthotopicFaDu head and neck cancer murine xenografts. To the bestof our knowledge, this is the first demonstration of how themembrane composition of NIR-activable liposomes con-taining PS-lipid conjugates is a key determinant of theirfunctionality as photodynamic and phototriggerable plat-forms for tumor-selective multi-agent therapy. As such, ourresults enable the rational design ofNIR-activable liposomesby introducing membrane composition (both chemical na-ture and subsequent physical conformation of PS-lipidconjugates) as a critical component that can be selected in apurpose-specific manner for optimal therapeutic andtheranostic functionality. Furthermore, combinations ofbothmembrane compositionswedemonstrate here couldberationally integrated into a single construct to capitalize onthe powerful properties of both.

2 Results

2.1 Fabrication and characterization ofNIR-activable liposomes

In this study, liposomes were fabricated as a prototypicallipid-based PNM platform, which comprised 0.5 mol% ofeither a novelmembrane-protrudingDSPE-PEG2000-NH2 lipidconjugate of IRDye700DXor amembrane-inserting 20:0 lyso-PC lipid conjugate of BPDwhichwe have previously reported(Figure 1) [18, 21]. Themembrane-protruding andmembrane-inserting conformations of IRDye 700DX and BPD, respec-tively, are primarily dictated by the inherent chemicaldifferences between the two molecules. The membrane-protruding and membrane-inserting conformations are thenconferred by conjugating IRDye 700DX to the hydrophilicmembrane-protruding terminal amine of DSPE-PEG2000-NH2

and by conjugating BPD to the membrane-inserting sn-2 hy-droxyl group of 20:0 lyso PC, respectively. Computationalpredictions of the membrane conformation of the two PSmolecules were simulated and validated by their topologicalpolar surface area (tPSA; ChemDraw 18.0), calculated octa-nol/water partitions (cLogP; ChemDraw 18.0), and apparentbiomembrane permeability coefficients (Papp). Papp wassimulated using the pkCSM model that predicts small-molecule pharmacokinetic properties using graph-basedsignatures [29]. Simplified molecular-input line-entry sys-tem sequences of IRDye 700DX and BPD were obtained fromPubChem (https://pubchem.ncbi.nlm.nih.gov) and wereused for the pkCSM model. Computational simulations

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predicted that DSPE-PEG-IRDye700DXwas in factmembraneprotruding, with a high topological polar surface area (tPSA)of 471.72, a low cLogP of −14.2 and a low biomembranepermeability coefficient of Papp = 0.071 × 10−6 cm/s. Compu-tational simulations also predicted that the 20:0 Lyso PC-BPD(abbreviated to BPD-PC) was in fact membrane-inserting,with a 2.9-fold lower tPSA of 164.75, a higher cLogP of 5.85and a 3.5-fold higher biomembrane permeability coefficientof Papp = 0.249 × 10−6 cm/s (Figure 1).

The absorption spectra of each PS-lipid conjugate andrespective liposomal formulations are shown in Figure 2Aand B. Lipidated BPD-PC exhibited an identical absor-bance when free in dimethyl sulfoxide (DMSO) and whenformulated into liposomes in phosphate buffered saline(PBS) (Figure 2A). However, DSPE-PEG-IRDye700DXexhibited a 4 nm red shift in the Q-band absorbancemaximum when formulated into liposomes in PBS(Figure 2B). The full absorbance spectra showing the Soretand Q-bands of lipo-IRDye700DX and lipo-BPD-PC areshown in Figure 2C. The Q-band absorbance and emission(Exc 680 nm) spectra of lipo-IRDye700DX and lipo-BPD-PCare shown in Figure 2D. As expected, the Q-band emissionmaximum of BPD-PC was unchanged following lipidconjugation (Figure 2E). However, the Q-band emissionmaximum of DSPE-PEG-IRDye700DX was red-shifted by2 nm upon liposomal formulation (Figure 2F), whichcorresponded to the slight red-shifting observed in theQ-band absorbance maximum. While the conjugation of

20:0 lyso PC conjugation to BPD had no impact on its fluo-rescence intensity in DMSO, conjugation of IRDye700DX toDSPE-PEG2000-NH2 enhanced its fluorescence emission by15.1% in DMSO (Figure S1A and B and Table 1). However,upon liposomal formulation, the intensities of the fluores-cence emission of the PS-lipid conjugates changed signifi-cantly as a result of varying degrees of static quenching,a phenomenon that is influenced by their membrane-protruding and membrane-inserting conformations (Figure3A and B). The membrane-protruding lipo-IRDye700DXexhibits a moderate 25% fluorescence quenching uponformulation, whereas membrane-inserting lipo-BPD-PC ex-hibits a 3-fold greater quenching at 74.2% upon formulation(Figure 3A and Table 1). Thismarked difference is likely dueto the tight packing of membrane-inserting lipo-BPD-PC,which is unlike the less-dense PS packing in themembrane-protruding lipo-IRDye700DX. The Q-band emission spectraof lipo-IRDye700DXand lipo-BPD-PC followingexcitationofthe Soret bands (Exc 354 nm) are shown in Figure S1C.

The only clinically used form of IRDye700DX isthe clinical photoimmunoconjugate Cetuximab (Cet)-IRDye700DX. As such, Cet-IRDye700DX, as well as free PSmolecules have been used as controls in this study. Lipo-IRDye700DX and Cet-IRDye700DX have identical absor-bance and emission profiles; however, Cet-IRDye700DXexhibits 3-fold greater quenching of fluorescence emissionlikely due to increased static quenching when conjugatedto the antibody (Figure S1D–F) [30].

Figure 1: Graphical representation ofliposomal photonanomedicines formulatedwith membrane-protruding DSPE-PEG-IRDye 700DX or membrane-inserting 20:0lyso-PC-benzoporphyrin derivative(BPD-PC). Topological polar surface area(tPSA) and calculated octanol/water parti-tions (cLogP) of the PS molecules weredetermined using ChemDraw 18.0. Theapparent biomembrane permeability co-efficients (Papp) were simulated usingpkCSM computational modeling [29].(* depicts values ×10−6 cm/s).


The hydrodynamic diameters of the lipo-IRDye700DXand lipo-BPD-PC were found to be 144.3 and 138.1 nm,respectively (Table 2). Matching of hydrodynamic di-ameters is critical for eliminating size-based differences incellular uptake, cellular phototoxicity, tumor delivery, tu-mor tissue selectivity, and tumor penetration. The ζ -po-tential of the lipo-IRDye700DX was 5.85 mV less than thatof the lipo-BPD-PC, which could be attributed to the 6sulfonates per IRDye700DX molecule at the surface of thelipo-IRDye700DX (Table 2). Although this difference isalmost negligible, it is taken into consideration throughoutthe study. While the nature of the two PSs is the mostpronounced chemical difference between the membrane-protruding lipo-IRDye700DX and membrane-insertinglipo-BPD-PC, the conformation of the PS-lipid conjugatesremains to be the most pronounced physical difference

Figure 2: Normalized absorbance spectra of BPD-PC in free form and in liposomal form (A) and of DSPE-PEG-IRDye700DX in free form and inliposomal form (B). (C) Normalized full absorbance spectra of both liposomes reveal the Soret and Q-bands of both PSs. (D) Normalizedabsorbance and emission spectra (Exc680 nm) of both liposomes at the red-NIR region. Normalized fluorescence emission spectra of BPD-PC infree form and in liposomal form (E) and of DSPE-PEG-IRDye700DX in free form and in liposomal form (F) using Exc630 nm to reveal the fullemission band profiles and to simulate the excitation of the in vivo fluorescence imaging system.

Table : Fluorescence emission properties of each PS in its nativeform, when conjugated to its respective lipid, and when formulatedinto liposomes.

Sample Solvent Emission wave-length maxima

(nm)

% Change in fluo-rescence emission

IRDyeDX DMSO N/ADSPE-PEG-IRDyeDX

DMSO +.% (upon lipidconjugation)

lipo-IRDyeDX

PBS −.% (uponformulation)

BPD DMSO N/ABPD-PC DMSO % (upon lipid

conjugation)lipo-BPD-PC PBS −.% (upon

formulation)


between the two. It is also important to note that lipo-IRDye700DX exhibits a 15.3-fold greater fluorescence in-tensity than lipo-BPD-PC in PBS (Figure 3A and B), which hasimplications in theranostic tumor imaging. This is alsoconsistent in serum (Figure S2). While naturally, the two PSsare inherently different in their photochemical efficiency andsinglet oxygen quantum yields, upon 690 nm light-emittingdiode (LED) illumination in DMSO, unformulated BPD-PCappears tobeonly 25%less efficient thanunformulatedDSPE-PEG-IRDye 700DX, as demonstrated by decay in absorbanceof the singlet oxygen probe anthracene dipropionic acid(ADPA; Figure 3C). In addition to the fact that both BPD andIRDye 700DX are activatable by the same wavelength of NIRlight, this small difference in their photochemical efficiencymakes the two sensitizers appropriate for the comparison ofthe two conformations given that differences in their chemicalnature are taken into consideration. Their chemical naturesmust be different in order to allow the two liposomes toexhibit either a truly membrane-protruding conformation ormembrane-inserting conformation. These chemical differ-ences are taken into consideration throughout the study andare further discussed in the respective results sections.

2.2 Reactive molecular species generation

We have previously shown that fluorescence quenching oflipidated PSmolecules upon formulation into liposomes and

Figure 3: Raw fluorescence emission spectra of DSPE-PEG IRDye700DX and lipo-IRDye700DX (A) and of BPD-PC and lipo-BPD-PC (B) usingExc630 nm to reveal the full emission band profiles and to simulate the excitation of the in vivo fluorescence imaging system. (C) Singlet oxygenproduction by the unformulated lipid conjugates of IRDye700DX and BPD in DMSO detected by a decrease in absorbance of the colorimetricsinglet oxygenprobe, anthracenedipropionic acid (ADPA;maximumpeak centered at 378nm). BPD-PCproduces 25% less singlet oxygen thanDSPE-PEG IRDye700DX.

Table : ζ-potentials and hydrodynamic diameters of lipo-IRDyeDX and lipo-BPD-PC in PBS.

lipo-IRDyeDX lipo-BPD-PC

ζ-Potential (mV) −. ± . −. ± .Hydrodynamic diameter (nm) . ± . . ± .

(Values are mean ± standard deviation).

micelles directly correlatedwith a reduction in singlet oxygengeneration [21]. As such, we evaluated the additional impactthat conformation has on the photochemical efficiencies ofthe membrane-protruding lipo-IRDye700DX and membrane-inserting lipo-BPD-PC given the aforementioned 25% differ-encebetween the two.Photochemical efficiencywasassessedusing three optochemical techniques. Singlet oxygen pro-duction following690nmirradiationwasmeasuredusing thecolorimetric probe anthracene dipropionic acid (ADPA)(photobleaches upon oxidation) as described earlier and us-ing the fluorometric probe Singlet Oxygen Sensor Green(SOSG). Furthermore, photochemical production of hydroxylradicals and peroxynitrite radicals was measured using thefluorometric probe hydroxyphenyl fluorescein (HPF). Thesemeasurements were all performed while taking into consid-eration that the efficiency of singlet oxygen production byDSPE-PEG-IRDye700DX is inherently ca. 25% greater thanthat of BPD-PC. Results reveal that themembrane-protrudinglipo-IRDye700DX is consistently more efficient thanmembrane-inserting lipo-BPD-PC at the photogeneration ofsinglet oxygen, hydroxyl radicals, and peroxynitrite radicalsupon 690 nmLEDphotoexcitation (Figure 4).With regards tothe photogeneration of singlet oxygen asmeasured byADPA,lipo-IRDye700DX is 2.3-fold more efficient than lipo-BPD-PC(Figure 4A and B). This difference exceeds the inherent ca.25% (1.25-fold) greater efficiency in singlet oxygenproductionof DSPE-PEG-IRDye700DX. This suggests that the lowerfluorescence quenching seen in the membrane-protrudinglipo-IRDye700DX is largely responsible for its superior effi-ciency in singlet oxygen production. When using SOSG, thesinglet oxygen production of lipo-IRDye700DX is found to be4.9-fold more efficient than for lipo-BPD-PC (Figure 4C andD). Similarly, the hydroxyl radical and peroxynitrite radicalphotogeneration was found to be 4.2-fold more efficient forthe membrane-protruding lipo-IRDye700DX, as compared to


the membrane-inserting lipo-BPD-PC (Figure 4E and F). It islikely that the lower photochemical efficiencies observed inthe membrane-inserting lipo-BPD-PC predominantly also aresult of conformational differences between the two lipo-somes. It is conceivable that the probes ADPA, SOSG, andHPF are also less accessible to the membrane-insertedBPD-PC than to the membrane protruding DSPE-PEG-IRDye700DX, hence detecting less RMS. The opposite mayalso be true that the RMS are not capable of traveling farenough away from the membrane to be detectable by theprobesADPA, SOSG, andHPF. Thiswould also be true for theaccessibility ofRMS to the immediatebiological environment.In either case, the PS-lipid conjugate conformation here ap-pears to be the predominant factor influencing their photo-chemical efficiencies.

As controls, the photochemical efficiency of freeIRDye700DX and the clinical photoimmunoconjugate Cet-IRDye700DX were also measured (Figure S3). While thephotochemical efficiency of free IRDye700DX was margin-ally greater than that of lipo-IRDye700DX, lipo-IRDye700DXwas significantly more efficient than the clinical photo-immunoconjugate Cet-IRDye700DX. As compared to Cet-IRDye700DX, this superior photochemical efficiency seen inthe lipo-IRDye700DX can also be attributed to the signifi-cant 76.43% fluorescence quenching observed in Cet-IRDye700DX (Figure S1).

2.3 Passive and phototriggered calceinrelease

The purpose of photochemistry for NIR-activable liposomesand PNM platforms, in general, is two-fold: (1) to mediatephotodynamic cancer cell killing, and (2) for phototriggereddrug release to spatiotemporally control induction of com-bination regimens. As such, we evaluated the efficiency ofphototriggered release of a hydrophilic drug surrogate,namely, calcein disodium salt, encapsulated in the core oflipo-IRDye700DX and lipo-BPD-PC at a self-quenching con-centration of 100 mM. Dequenching of calcein fluorescencewas used as a prototypical indicator of agent released fromthe liposomes. While both liposomes ultimately releasedcomparable levels of calcein upon irradiation with a total of50 J cm−2 (2 μM; 40% of entrapped calcein), the rate ofphototriggered release of calcein from lipo-IRDye700DX(k = 0.201/J cm−2) was 11.92-fold greater than that of the lipo-BPD-PC (k = 0.0169/J cm−2) (Figure 5A). The membrane-protruding lipo-IRDye700DX elicited a burst-release of cal-cein, while the membrane-inserting lipo-BPD-PC mediated asteadier, sustained phototriggered release of calcein. Inaddition,membrane composition also influenced thepassive

release of calcein at 37 °C over the span of 24 h. Resultsshow that lipo-BPD-PC passively releases calcein at a ratethat is 13.2-fold slower than lipo-IRDye700DX in the dark(Figure 5B). By 24 h, 3 μM calcein (60%) was passivelyreleased from the membrane-protruding lipo-IRDye700DX,whereas only 0.23 μM calcein (4.5%) was passively releasedfrom themembrane-inserting lipo-BPD-PC. As such, it can beconcluded that the presence of membrane-inserting BPD-PCsignificantly reduces the liposomal membrane permeabilityof entrapped molecules. A similar trend has been previouslyobserved, whereby liposome membrane permeability wasreduced by more than 10-fold when a membrane-insertingporphyrin-lipid conjugate was included in the bilayer [31]. Areduction in membrane permeability by the membrane-inserting BPD-PC is also likely to decrease the susceptibilityof the membrane to phototriggered release [31]. These find-ings suggest that the membrane-protruding lipo-IRDye700DX, is better suited as a rapid burst release platform forentrapped agents, whereas the membrane-inserting lipo-BPD-PC, is better suited for applications whereby controlledand sustained agent release is required. Interestingly, theresults also suggest that membrane insertion of PSs is notnecessary for phototriggered agent release from liposomes,as the nearby localization of the membrane-protrudingIRDye700DX is sufficient and in fact superior for photo-triggered release. This is consistent with the key study byLajunen et al. which showed that ICG noncovalently asso-ciatedwith the PEG coating of a liposomewas still capable ofinducing phototriggered calcein release [28]. Furthermore, itappears that release kinetics are likely to be dictated by acombination of the photochemical efficiency of the systemand by passivemembrane permeability, which appears to besignificantly reduced by the presence of the membrane-inserting BPD-PC lipid conjugate (Figure 5B).

2.4 Cellular uptake of liposomes and PSvariants

Cellular interaction and uptake are oftentimes key de-terminants for the efficacy of liposomes as PDT agents andas nanoplatforms for combination therapy. As such, weexplored the impact of membrane composition of themembrane-protruding lipo-IRDye700DX and membrane-inserting lipo-BPD-PC in FaDuhumanhead andneck cancercells. It was found that no significant difference existedbetween the cellular uptake of lipo-IRDye700DX and lipo-BPD-PC, suggesting that the small difference in ζ -potentialalso had no impact on internalization (Figure 6). Interest-ingly, while there was no significant difference in uptakebetween the free IRDye700DXand the lipo-IRdye700DX, the


Figure 4: (A and B) singlet oxygen generationby the membrane-protruding lipo-IRDye700DX and membrane-inserting lipo-BPD-PC in PBS measured by the decreasingabsorbance of the colorimetric singlet oxy-gen probe anthracene dipropionic acid(ADPA). (C and D) singlet oxygen generationby the two formulations measured by theincreasing fluorescence emission of singletoxygen sensor green (SOSG). (E and F) hy-droxyl radical and peroxynitrite anion gen-eration by the two formulations measuredby the increasing fluorescence emission ofhydroxyphenyl fluorescein (HPF). (Data aremean ± standard error; statistical signifi-cance was calculated using a two-tailedt-test, ***: P ≤ 0.0002, ****: P ≤ 0.0001).

Figure 5: Phototriggered (A) and passive (B) calcein release from lipo-IRDye700DX and lipo-BPD-PC. Phototriggered calcein release wasperformed using 690 nm light at an irradiance of 17.86mWcm−2 (Data aremean ± standard error; statistical significance was calculated usingone-way ANOVA with a Tukey post-test, ***: P < 0.001; ****: P < 0.0001).


Cet-IRDye700DX clinical photoimmunoconjugate controlwas internalized more efficiently (Figure S4). This can beexplained by the receptor-mediated endocytosis throughtargeting of epidermal growth factor receptor (EGFR) byCetuximab. Owing to its small molecular weight andamphiphilic nature, free BPD was the most efficientlyinternalized agent tested. These results thus suggest thatboth the membrane-protruding lipo-IRDye700DX andmembrane-inserting lipo-BPD-PC present themselves asequivalent intracellular drug delivery systems.

2.5 Intracellular localization

Although the degree of FaDu cell uptakewas equivalent forboth liposomes, we further explored whether the con-structs were localized in different organelles. Our previousfindings have shown that various iterations of lipo-BPD-PCstrictly localize within endolysosomes [18–20]. This is notatypical, as endocytosis of liposomes leading to theirpresence in endolysosomal compartments has beenwidelyreported for various types of liposomes [32–34]. In thisstudy, we incubated FaDu cells with both liposomes for24 h, and the cells were counter-stained with Lyso-Tracker™ Green DND-26 prior to imaging with confocalmicroscopy. Microscopy images (Figure 7) revealed thatboth lipo-BPD-PC and lipo-IRDye700DX appear as punctawithin cells, which is typical of sequestration in endoly-sosomal compartments. This was further confirmed by the

partial colocalization of both lipo-BPD-PC and lipo-IRDye700DX with LysoTracker™ Green DND-26. Imagesof lipo-BPD-PC and lipo-IRDye700DX were processes withthe corresponding the LysoTracker™ Green DND-26 im-ages to isolate pixels that were co-localized, therebydemonstrating the liposomes that were present within theendolysosomal compartments (yellow, Figure 7). Theseresults confirm that both liposome compositions result inthe same localizationwithin endolysosomal compartmentsin FaDu cells, and are internalized with the same efficiencyas demonstrated earlier. This is not unexpected consid-ering that the sizes of both liposomes arematched, and alsosuggests that the membrane composition along with thesmall difference in ζ -potential have no impact on theirintracellular trafficking.

2.6 Photodynamic therapy

As we have previously shown that photochemical effi-ciency alone is not prognostic of in vitro photodynamicefficacy of lipid-based PNMs (liposomes as well as mi-celles) [21], we further investigated the role of lipo-IRDye700DX and lipo-BPD-PC membrane composition onFaDu cell phototoxicity. Despite being 2-5-fold less effec-tive at generating RMS, lipo-BPD-PCwas significantlymoreeffective than lipo-IRDye700DX at photodestruction ofFaDu cells (Figure 8). At 500 nM PS equivalent, lipo-BPD-PC (LD50 = 1.1 J cm−2) was 9.9-fold more phototoxicthan lipo-IRDye700DX (LD50 = 10.9 J cm−2). At 2000 nM PSequivalent, lipo-BPD-PC was also significantly moreeffective than lipo-IRDye700DX at cell killing, as the LD50

of lipo-IRDye700DX was 3.25 J cm−2, while the LD50 of lipo-BPD-PCwas too low to be accurately determined (Figure 8Eand F). Interestingly, the superior phototoxicity of the lipo-BPD-PC cannot be attributed to a difference in the effi-ciency of cellular internalization, subcellular localization,or to its photochemical efficiency, which remains 2-5-foldless efficient than lipo-IRDye700DX. It is unclear at thisstage what the cause for this enhanced phototoxicity is,yet it is most plausible that the radical-based photo-degradation products of lipo-BPD-PC have a greater impacton cell viability than those generated by the photo-irradiation of lipo-IRDye700DX. This hypothesis will bethe focus of key future mechanistic studies. Of all agentstested, the Cet-IRDye700DX control was the most cytotoxic(Figure S5). Its cytotoxicity, however, was greatly influ-enced by its dark toxicity, which can be attributed toEGFR downregulation and subsequent induction ofapoptosis [35].

IRDye

700D

XBPD

lipo-I

RDye70

0DX

lipo-B

PD-PC

0.0001

0.001

0.01

0.1

Qua

ntity

of P

hoto

sens

itizer

Equi

vale

nt/C

ell (

pmol

es)

Cellular Uptake of Photosensitizersand Liposomes

ns

ns

Figure 6: Comparison of FaDu cell uptake of lipo-IRDye700DX andlipo-BPD-PC, as well as free IRDye700DX and free BPD after 24 hincubation. (Data are mean ± standard error; statistical significancewas calculated using one-way ANOVA with a Tukey post-test,****: P ≤ 0.0001).


2.7 In vivo tumor kinetics andpharmacokinetics

Finally, the impact of membrane composition on in vivopharmacokinetics and tumor penetrating behavior wasstudied in an orthotopic murine model of FaDu human headandneck cancer. Swiss nu/numice bearing FaDu tumors thatwere implanted by a transcervical injection through the floorof the mouth were injected intravenously with 6.95 nmol PSequivalent of the membrane-protruding lipo-IRDye700DX ormembrane-inserting lipo-BPD-PC. Interestingly, tumor accu-mulationwas visiblymore efficient at 24 h for themembrane-inserting lipo-BPD-PC than for the membrane-protrudinglipo-IRDye700DX (Figure 9A). At 24 h, the tumor selectivity ofthe lipo-BPD-PCwas also significantly higher than that of thelipo-IRDye700DX (Figure 9B). Using semi-quantitative fluo-rescence-based analysis of liposome tumor kinetics, tumoraccumulation of the lipo-BPD-PCwas found to be higher thanthat of the lipo-IRDye700DX at all time points within 24 hfollowing administration (Figure 9C). Furthermore, bio-distribution analysis of tumor uptake revealed that tumor

uptake of lipo-BPD-PC was 7.16-fold greater than the lipo-IRDye700DX at 24 h following administration (Figure 9D). Itwas also found that tissue uptake of lipo-BPD-PC was higherthan that of lipo-IRDye700DX at 24 h following administra-tion in all other organs assessed.

Although tumor kinetics and in vivo pharmacokineticswere markedly altered by the membrane composition, itwas interesting to observe that it had no impactwhatsoeveron tumor penetration. At the core of the tumors, both thelipo-IRDye700DX and the lipo-BPD-PC accumulated at ca.80%with respect to the regions of highest accumulation atthe tumor periphery (Figure 10). This is likely due to the factthat the size of the two constructs is matched, which re-mains to be a major determining factor in the degree oftumor tissue penetration. It is also worth noting that thesmall difference in ζ -potential between the two liposomesdid not influence the degree of tumor penetration.

The pronounced differences in orthotopic FaDu tu-mor kinetics and biodistribution observed between themembrane-protruding lipo-IRDye700DX and membrane-inserting lipo-BPD-PC reveal an unprecedented impact that

Figure 7: Confocal microscopy images of FaDu cells at 60×magnification after a 24 h incubationwith lipo-IRDye700DX (top, red), lipo-BPD-PC(bottom, red), and LysoTracker™ Green DND-26 (middle, green). The liposomes co-localized with the lysosomes are presented in theprocessed logical AND operator images (right, yellow, processed on ImageJ). (Scale bars are 50 µm).


Figure 8: Percent metabolic activity of FaDu cells as determined by the MTT assay performed 48 h following PDT with lipo-IRDye700DX, lipo-BPD-PC, and free PS controls.Concentrations were at 5 nM (A), 50 nM (B), 500 nM (C), and 2000 nM (D) PS equivalent and irradiation at 690 nm light was performed at anirradiance of 27.7 mW cm−2 up to a fluence of 10 J cm−2. The percent metabolic activity at a fluence of 2.5 J cm−2 with a PS equivalent of 500 nM(E) and 2000 nM (F) is also presented. (Data are mean ± standard error; statistical significance was calculated using one-way ANOVA, ****:P ≤ 0.0001).


Figure 9: (A) Representative FLI/µCT images of orthotopic FaDu tumors 24 h following intravenous administration of lipo-BPD-PC and lipo-IRDye700DX. (B) Tumor selectivity of lipo-BPD-PC and lipo-IRDye700DX 24 h following intravenous administration. (C) Longitudinal fluores-cence imaging of lipo-IRDye700DX and lipo-BPD-PC in orthotopic FaDu tumors to assess tumor kinetics. (D) Semi-quantitative fluorescence-based analysis of liposome biodistribution 24 h following intravenous administration. (Data are mean ± standard error; n ≥ 4 mice per arm;statistical significancewas calculated using one-wayANOVAwith a Tukeypost-test, *:P ≤0.0.5, **:P ≤0.005, ***:P ≤0.0005, ****:P ≤0.0001).

Figure 10: (A) Three-Dimensional projections of tumor penetration of lipo-IRDye700DX and lipo-BPD-PC in orthoptic FaDu tumors 24 h afteradministration. The x- and y-axis correspond to spatial dimensions (inches) and the z-axis corresponds to the fluorescence intensities of thelipo-IRDye700DX and lipo-BPD-PC (R.F.U). (B) Quantitation of tumor penetration at the tumor core reveals no difference between lipo-IRDye700DX and lipo-BPD-PC. (Data are mean ± standard error; n ≥ 4 tumors per arm, statistical significance was calculated using one-wayANOVA with a Tukey post-test).


membrane composition has on liposomes as NIR-activabletheranostic drug delivery platforms. How such a markeddifference in liposome in vivo behavior is influenced purelyby membrane composition remains to be the focal point offuture studies. Undoubtedly, a deeper understanding iswarranted of the molecular constituents and physical at-tributes of the protein coronas that form on themembrane-protruding and membrane-inserting liposomes upon con-tact with serum. These future studies into the proteincorona, in addition to the careful and detailed function-specific selection of membrane composition, will inevi-tably prove to be important for the further advancement ofmultifunctional liposomes.

3 Conclusions

NIR-activable liposomes are emerging as tunable platformsfor PDT-based anticancer combination regimens. Lipidconjugates of PS molecules serve as the photodynamicagents for phototoxicity as well as phototriggered releaseof secondary and tertiary therapeutics. As a result of dif-ferences in their chemical nature and lipid conjugationsites, these PS-lipid conjugates take on various conforma-tions at the liposomal membrane, with the PSs being eithermembrane-protruding or membrane-inserting. While bothmembrane-protruding and membrane-inserting confor-mations have been reported, the collective functional roleof liposome membrane composition on their in vitro and invivo behavior is yet to be fully explored. To the best of ourknowledge, this study is the first to report that PS-lipidconjugates are not simply passive spectators that facilitatePDT and phototriggered agent release, but their chemicalnature and subsequent physical conformation are in fact akey functional determinant of their in vitro and in vivobehavior. Results in this study show that membranecomposition plays central role in photochemistry, photo-dynamic efficacy, phototriggered agent release kinetics,and orthotopic tumor delivery and selectivity. As such, ourfindings here propose that liposomes containing amembrane-protruding DSPE-PEG-IRDye700DX PS-lipidconjugate are a superior platform for rapid photo-triggered agent release. Conversely, liposomes containinga membrane-inserting BPD-PC PS-lipid conjugate are abetter-suited platform for sustained phototriggered agentrelease and efficient photodynamic tumor cell destruction.Furthermore, membrane-inserting lipo-BPD-PC providessignificantly greater tumor delivery and therefore presentsitself as a superior platform for tumor delivery of combi-nation agents including the PS-lipid conjugate itself.However, owing to its 12-fold enhanced phototriggered

release kinetics and 15-fold brighter fluorescence emission,the membrane-protruding lipo-IRDye700DX is a superiorplatform for phototriggered drug release in the vascularlumen whereby delivery into the tumor interstitium maynot be necessary, and as theranostic platforms for opticaltumor imaging and image-guided phototherapy.

The mechanisms underlying the unprecedented func-tional impact of liposome membrane composition are un-deniably multifactorial and revolve around differences inthe chemical nature of the PSmolecules, in addition to theirconsequent differences in conformation. As we discussed,tumor delivery and tumor tissue selectivity of liposomes areimpacted by theprotein corona that spontaneously formsonliposomes, as is also the case for nanomedicines in general.Future studies, likely revolving around proteomics, willhelp identify the differences between the composition ofthe protein coronas that form on the membrane-insertinglipo-BPD-PC and on the membrane-protruding lipo-IRDye700DX. These findings may also aid in further advancingnanotechnology-oriented drug delivery strategies wherebymembrane composition of optical and nonoptical nano-medicines alike can be manipulated to tune pharmacoki-netics. However, the photochemical and phototherapeuticconsequences of liposomal membrane composition aresignificantly more complex. The presence of a membrane-inserting BPD-PC appears to reduce the rate of photo-triggered calcein release by 12-fold and passive calceinrelease by 13-fold. Studies have previously explored thebiochemical mechanisms of phototriggered agent release.While some studies have focused on the role of oxidizableunsaturated membrane phospholipids in facilitating pho-totriggered drug release [36], others have explored the roleof PS degradation products in triggering membranepermeability upon photoexcitation [37]. Future studies intothe photodegradation products of the membrane-insertingBPD-PC and the membrane-protruding DSPE-PEG-IRDye700DX, their reactivities with membrane constituents, andtheir various phototoxicity profiles are therefore also war-ranted. With regards to phototoxicity, the radical-basedphotodegradation products of the individual PS-lipidconjugates are likely to provide insights into why themembrane-inserting lipo-BPD-PC exhibits such a markedincrease in phototoxicity even though it is less efficient atgenerating RMS. This is also intriguing considering that nodifferences in cellular uptake efficiencies or subcellularlocalization exist between the two liposomal membranecompositions. It does, however, highlight the significance ofthe differences in the chemical nature of the two PS-lipidconjugates that give rise to the distinct conformations.

Overall, the findings of this study provide vital insightsthat can guide the application-specific and rational design


and engineering of NIR-activable liposomes, wherebyemerging combination therapies integrated into suchplatforms can be tailored to best suit the needs of thespecific therapeutic modalities at hand. As such, thepowerful attributes of NIR-activable liposomes and otherlipid-based PNMs can be translated tomaximal therapeuticbenefits to patients with head and neck cancer, as well aspatients with other cancer and noncancer indicationswhereby phototriggered combination therapy can prove tobe pivotal. Furthermore, the rational combination of bothcompositions into the same construct can be leveraged tocapitalize on the powerful attributes of the respectivecompositions.

4 Methods

4.1 Synthesis of DSPE-PEG-IRDye700DX and BPD-PClipid conjugates

DSPE-PEG-IRDye700DX was synthesized using an adaptation of ourpreviously published protocols [21, 38]. Briefly, 0.5 mg IRDye700DX-NHS (LI-COR)was dissolved in 100 µl anhydrousDMSO tomake a 5mg/ml solution. The IRDye700DX-NHS solution was added to dryDSPE-PEG2000-NH2 (Avanti) at a molar ratio of 1:1. This solution wasstirred for 48 h at 2500 rotations per minute (RPM) at room temperaturein the dark. The DSPE-PEG-IRDye700DX was purified by diluting thecrude reaction mixture with 900 µl methanol and running through aSephadex LH20 column (Cytivia) pre-equilibrated with methanol. Pu-rified DSPE-PEG-IRDye700DX was stored at −20 °C in the dark inmethanol.

The 20:0 lyso PC-benzoporphyrin derivative conjugate (BPD-PC)was synthesized as we previously described [18–21, 39]. Briefly,1-arachidoyl-2-hydroxy-sn-glycero-3-phosphocholine (20:0 Lyso PC,Avanti), BPD (US Pharmacopeia), EDC (Sigma), 4-(Dimethylamino)pyridine (DMAP) (Sigma), and N,N-Diisopropylethylamine (DIPEA)(Sigma) were mixed at molar ratios of 1:5:50:25:60, respectively, in5 ml of dichloromethane (Fischer Scientific, high-performance liquidchromatography [HPLC] grade) and stirred at 2500 RPM for 72 h atroom temperature. BPD-PC was purified using preparatory thin-layerchromatography and extracted in a 2 : 1 dichloromethane/methanolmixture. The extracted BPD-PC was finally filtered through a 0.22 µmpolytetrafluoroethylene (PTFE) filter and stored at −20 °C in the dark inchloroform.

4.2 Synthesis of liposomes

For the synthesis of lipo-IRDye700DX, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (Avanti), cholesterol (Avanti), DSPE-mPEG2000

(Avanti), and DSPE-PEG-IRDye700DX were mixed in chloroform at amolar ratio of 0.67, 0.30, 0.025, and 0.005, respectively. For the syn-thesisof lipo-BPD-PC, the lipidsDPPC, cholesterol, DSPE-mPEG2000, and20:0 lyso-PC-BPD were mixed in chloroform at a molar ratio of 0.665,0.30, 0.03, and 0.005, respectively. Liposomes were prepared using aconventional thin-filmhydration-extrusionmethod, byhydration inPBS

and extrusion through 100 nm polycarbonate membranes (Whatman)[18–21, 39].

4.3 Characterization of liposomes

The hydrodynamic diameters, polydispersity indices, and ζ -potentialsof the lipo-IRDye700DX and lipo-BPD-PC were performed by dynamiclight scattering using the Zetasizer Pro (Malvern). 2 µL of each lipo-some were mixed with 1 mL of PBS, and the hydrodynamic diametersand polydispersity indices were measured in triplicate. ζ -potentialswere measured in triplicate using 5 µL of each liposome mixed with1 mL of 0.9% NaCl solution in MiliQ H2O.

To calculate the concentrations of DSPE-PEG-IRDye700DX andBPD-PC equivalents in the respective formulations, lipo-IRDye700DXand lipo-BPD-PC were diluted in DMSO and the absorbance wasmeasured (Thermo Evolution 350 Spectrophotometer), using themolar extinction coefficient, ε687 = 34,895M−1 cm−1 for BPD-PC [21] andε687 = 210,000 M−1 cm−1 (LI-COR) for DSPE-PEG-IRDye700DX. Addi-tional absorption and emission spectra were measured in a TecanSpark plate reader.

4.4 Quantification of photogenerated RMS

Singlet oxygen was measured using the colorimetric probe anthra-cene-9,10-dipropionic acid (ADPA; Fisher Scientific) and with thefluorogenic probe Singlet Oxygen Sensor Green (SOSG; Fisher Scien-tific). For the ADPA singlet oxygen measurement assay, samples wereprepared at 5 μM PS equivalent concentrations either in PBS (for li-posomes, free IRDye700DX, and Cet-IRDye700DX) or in DMSO (forDSPE-PEG-IRDye700DX and BPD-PC). The samples in PBS wereplaced in 96 well plates in 100 µL aliquots, and 5 µL of ADPA (6 mMstock inmethanol)was added to each plate. The samewas repeated forthe samples in DMSO, although 10 µL of ADPA (6 mM stock in meth-anol) was used to account for the lower absorbance of ADPA in DMSO.The absorbance of all samples containing ADPA was measured from200 to 800 nm using a Tecan Spark Plate reader before and afterirradiation with 690 nm LED light. Samples were irradiated using anirradiance of 17.86 mW cm−2 up to a total fluence of 3 J cm−2, withabsorbance spectra being measured in 0.5 J cm−2 increments.

For the SOSG singlet oxygen measurement assay, samples wereprepared at 5 μM PS equivalent concentrations in PBS and placed in96 well plates in 100 µL aliquots. 10 µL of SOSG (50 µM stock) wereadded to each sample and fluorescence emission wasmeasured from500 to 600 nm using an excitation wavelength of 460 nm on theTecan Spark Plate reader. Samples were irradiated with 690 nm LEDlight using an irradiance of 17.86 mW cm−2 up to a total fluence of3 J cm−2, with fluorescence spectra being measured in 0.5 J cm−2

increments.Hydroxyl radicals and peroxynitrite radicals were measured us-

ing the fluorogenic probe hydroxyphenyl fluorescein (HPF, FisherScientific). Samples were prepared at 5 μM PS equivalent concentra-tions and placed in 96 well plates in 100 µL aliquots. 20 µL of HPF(200 µM stock) were added to each sample and fluorescence emissionwas measured from 500 to 600 nm using an excitation wavelength of460 nm on the Tecan Spark Plate reader. Samples were irradiatedwith690 nm LED light using an irradiance of 17.86 mW cm−2 up to a totalfluence of 3 J cm−2, with fluorescence spectra being measured in0.5 J cm−2 increments.


4.5 Passive and phototriggered calcein release

Lipo-IRDye700DX and lipo-BPD-PC were both prepared as describedearlier and hydrated with 100 mM calcein disodium salt (Sigma) dis-solved in PBS as an optical surrogate for water-soluble drug payloads.Calcein-loaded liposomes were purified from unentrapped calcein us-ing size exclusion chromatography by running on a Sepharose CL-48column (Sigma) equilibrated with PBS. To calculate the concentrationof calcein in each liposomal formulation, lipo-IRDye700DX and lipo-BPD-PC were diluted in DMSO, the absorbance was measured (ThermoEvolution 350 spectrophotometer), and the concentration was calcu-lated using themolar extinction coefficient, ε495 = 80,000M−1 cm−1 [40].To measure passive and phototriggered release, samples of each lipo-some were prepared at concentrations of 5 µM of calcein equivalent ofpurified lipo-IRDye700DX and lipo-BPD-PC. The fluorescence emissionof the liposomes was measured from 500 to 600 nm with an excitationwavelength of 460 nmusing a Tecan Spark Plate reader. For the passiverelease studies, liposomes were incubated in a 37 °C incubator, and thefluorescence dequenching of calcein as it escaped the liposomes wasmeasured at 1, 3, 6, and 24 h of incubation. For the phototriggeredrelease studies, the liposomeswere irradiated after purification at roomtemperature by a 690 nm LED system (BioLambda; 17.86 mW cm−2) in10 J cm−2 increments up to 50 J cm−2. The fluorescence dequenching ofcalcein as it escaped the liposomes was measured following each irra-diation fraction.

4.6 Cellular uptake of liposomes and PS variants

FaDu cells were seeded in 96-well plates at a density of 50,000 cells perwell in Dulbecco’s Modified Eagle Medium (DMEM). After 24 h, 250 nMPS equivalent of lipo-IRDye700DX and lipo-BPD-PC, as well as freeIRDye700DX, free BPD at Cet-IRDye700DX were added to cells andfurther incubated for another 24 h. Media was then aspirated, and cellswere washed with 200 µl of PBS three times. After washing, 100 µl ofDMSO was added to each well and the fluorescence emission wasmeasured at 670–800 nm, using an excitation wavelength of 630 nmusing a Tecan Spark Plate reader. Standard curves of each PS in theirrespective formats (i.e. free, liposomes, conjugates, etc.) were preparedin DMSO and used to calculate PS concentrations taken up by cells.

4.7 Confocal microscopy

FaDu cells were seeded in glass-bottom 96-well plates at a density of50,000 cells per well. After a 48 h incubation at 37 °C, lipo-IRDye700DX and lipo-BPD-PC were added to the cells at a 2000 nMPS equivalent and were further incubated for 24 h. Prior to imaging,the liposomeswere removed from the cells and freshmediawas addedcontaining either Hoechst 33342 or LysoTracker™ Green DND-26, asper the instructions of ThermoFisher Scientific. The Hoechst andLysoTracker™ were incubated with the cells for 30 min and the cellswere imaged using a Leica SP8 microscope with a 405 nm laser(Hoechst excitation), a 488 nm laser (LysoTracker™ excitation), a647 nm laser (lipo-IRDye700DX and lipo-BPD-PC excitation), and a60× oil immersion objective.

5 Photodynamic therapy

FaDu cells were seeded in transparent 96-well platesat a density of 1500 cells per well. After 24 h incubation at37 °C, lipo-IRDye700DX and lipo-BPD-PC, as well as freeIRDye700DX, free BPD at Cet-IRDye700DX were incubatedwith the cells for a further 24 h at 5, 50, 500, and 2000 nMPS equivalent. After incubation, cells were irradiated usingthe 690 nm BioLambda LED system with various fluences:0, 1, 2.5, 5, and 10 J cm−2 at a consistent irradiance of17.86 mW cm−2. After irradiation, cells were incubated for48 h then assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay. Theabsorbance of the MTT product formazan was measured at555 nm using a Tecan Spark Plate reader.

5.1 Biodistribution and tumor imaging inorthotopic head and neck tumors

7.5 × 105 FaDu cells in sterile PBS were implanted trans-cervically into the floor of the mouth of male Swiss NudeMice (20 g, 4–6 weeks old, Charles River) [41]. Tumordevelopment was monitored using ultrasound imaging(Vevo 3100 VisualSonics). Once tumors reached 5 mmin diameter, mice were intravenously injected with6.95 nmol PS equivalent of lipo-IRDye700DX and lipo-BPD-PC. Longitudinal fluorescence imaging of the lipo-somes in the tumors was performed using an LI-CORPEARL system. Tumors were also imaged using fluores-cence tomography/micro-computed tomography (FLT/µCT, MI Labs). At 24 h after administration, tumors andorgans were harvested, liposome content was quantifiedusing the LI-COR PEARL system and tumor penetrationwas imaged in bisected tumors using the LI-COR Odysseysystem. All images were corrected for autofluorescencebackground signals and for the inherent variability influorescence intensities of the two constructs in therespective imaging systems. Tumor accumulation wasquantified using the LI-COR PEARL software and tumorpenetration was quantified using the 3D surface plot toolon ImageJ (NIH).

Acknowledgments: Infrastructure support from Dr JyotiMisra and Dr David Schmidtke, and technical and editorialsupport from Jude Franklin, Drhuv Kapoor, and TaylorHinchliffe are gratefully acknowledged.


Author contribution: All the authors have acceptedresponsibility for the entire content of this submittedmanuscript and approved submission.Research funding: This work was supported by theNational Institutes of Health [R00CA215301 to G. O., andP01CA084203 and R01CA160998 to T. H.] and the CancerPrevention and Research Institute of Texas Award[RP180670 to K. H.].Conflict of interest statement: The authors declare noconflicts of interest regarding this article.

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Supplementary Material: The online version of this article offers sup-plementary material (https://doi.org/10.1515/nanoph-2021-0191).


https://doi.org/10.3390/jcm9082390

https://doi.org/10.1515/nanoph-2021-0191

navadeep shrivastava, kenneth hoyt, tayyaba hasan and

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