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Precision Nanomedicine

The official journal of CLINAM

About the Journal: Precision Nanomedicine (PRNANO) is a peer-reviewed, not-for-profit, international society journal to promote all practical, rational, and progressive aspects of theory and practice of nanomedicine, from basic research through translational and clinical aspects including commercialization.

PRNANO provides an open access forum with reliable content and quick turnaround time.

We invite authors to submit both original, as well as replication studies. Discussions of negative results are also welcome if they move the field forward.  Papers are published continuously then organized into quarterly issues.

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PRNANO is digitally distributed by a scientists-owned non-profit publisher: Andover House Inc, 138 River Rd, Andover, Massachusetts, 01810, USA.

 ISSN: 2639-9431 (online)

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Cellular Trafficking of Sn-2 Phosphatidylcholine Prodrugs Studied with Fluorescence Lifetime Imaging and Super-resolution Microscopy

Research Article

Dolonchampa Maji PhDa,b *, Jin Lu PhDc, Pinaki Sarder Phdd, Anne H. Schmieder MSe, Grace Cui MSe, Xiaoxia Yang BSe, Dipanjan Pan PhDf, Matthew D. Lew PhDc, Samuel Achilefu PhDa,b and Gregory M. Lanza MD PhDb,e,[1]

aOptical Radiology Lab, Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA

bDepartment of Biomedical Engineering, Washington University in St. Louis, MO 63130, USA

cDepartment of Electrical and Systems Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA

dDepartment of Pathology and Anatomical Sciences, Jacobs School of Medicine & Biomedical Sciences, University of Buffalo, Buffalo, NY 14203

eDivision of Cardiology, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA

fDepartment of Bioengineering, University of Illinois at Urbana-Champaign, Champaign, IL, USA

* Current address: Department of Neurological Surgery, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611

Submitted: July 18, 2018; Accepted: July 30, 2018; Posted August 3, 2018

Graphical abstract

Fluorescence lifetime imaging microscopy (FLIM) and single-molecule super-resolution microscopy (SRM) illustrate the intracellular fate of Sn-2 phosphatidylcholine prodrugs.


While the in vivo efficacy of Sn-2 phosphatidylcholine prodrugs incorporated into targeted, non-pegylated lipid-encapsulated nanoparticles was demonstrated in prior preclinical studies, the microscopic details of cell prodrug internalization and trafficking events are unknown. Classic fluorescence microscopy, fluorescence lifetime imaging microscopy, and single-molecule super-resolution microscopy were used to investigate the cellular handling of doxorubicin-prodrug and AlexaFluor-488-prodrug. Sn-2 phosphatidylcholine prodrugs delivered by hemifusion of nanoparticle and cell phospholipid membranes functioned as phosphatidylcholine mimics, circumventing the challenges of endosome sequestration and release. Phosphatidylcholine prodrugs in the outer cell membrane leaflet translocated to the inner membrane leaflet by ATP-dependent and ATP-independent mechanisms and distributed broadly within the cytosolic membranes over the next 12 h. A portion of the phosphatidylcholine prodrug populated vesicle membranes trafficked to the perinuclear Golgi/ER region, where the drug was enzymatically liberated and activated. Native doxorubicin entered the cells, passed rapidly to the nucleus, and bound to dsDNA, whereas DOX was first enzymatically liberated from DOX-prodrug within the cytosol, particularly in the perinuclear region, before binding nuclear dsDNA. Much of DOX-prodrug was initially retained within intracellular membranes. In vitro anti-proliferation effectiveness of the two drug delivery approaches was equivalent at 48 h, suggesting that residual intracellular DOX-prodrug may constitute a slow-release drug reservoir that enhances effectiveness. We have demonstrated that Sn-2 phosphatidylcholine prodrugs function as phosphatidylcholine mimics following reported pathways of phosphatidylcholine distribution and metabolism. Drug complexed to the Sn-2 fatty acid is enzymatically liberated and reactivated over many hours, which may enhance efficacy over time.


Phosphatidylcholine prodrugs, Nanomedicine, Fluorescence lifetime imaging microscopy, Super-resolution microscopy




  • AF488 AlexaFluor 488
  • API active pharmaceutical ingredient
  • αvβ3 alpha v beta 3 integrin
  • CFDD Contact Facilitated Drug Delivery
  • cPLA2 cytosolic phospholipase A2
  • DAPI 4',6-Diamidino-2-phenylindole, dihydrochloride
  • DMEM Dulbecco's Modified Eagle’s Medium
  • dsDNA double-stranded deoxyribonucleic acid
  • ER endoplasmic reticulum
  • FLIM fluorescence lifetime imaging microscopy
  • MC540 merocyanine 540
  • PAINT Point Accumulation for Imaging in Nanoscale Topography
  • Paz-PC 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine"
  • PC phosphatidylcholine
  • PD prodrug
  • PFOB perfluoro-1-bromooctane
  • PE phosphatidylethanolamine
  • PE-PTX phosphatidylethanolamine-paclitaxel
  • PLA phospholipase
  • SRM super-resolution microscopy
  • Sn-2 stereospecific numbering position 2
  • τamp amplitude average lifetime

Rationale and purpose

Sn-2 phosphatidylcholine prodrugs in non-pegylated lipid nanoparticle membranes are known to be resistant to premature release and metabolism in circulation. This approach using targeted drug delivery has been demonstrated to be effective in a variety of preclinical models. Cellular uptake of these compounds is hypothesized to be secondary to a hemifusion contact-facilitated drug delivery mechanism with cytosolic enzymatic drug release, which circumvents sequestration in the endosome-lysosome pathway. This research utilized advanced complementary optical imaging methods, including confocal fluorescence lifetime and super-resolution single molecule microscopy, to elucidate and compare PC prodrug cellular trafficking and metabolism with previously known paths of natural PC.


The concept of achieving Paul Erhlich’s inspired vision of a “magic bullet” to treat disease remains challenging in the context of nanomedicine. While specific reasons vary among nanosystems, in general, early systemic drug loss, low net drug delivery to targeted cells, and limited free drug intracellular bioavailability have been major barriers [1]. Very hydrophobic drugs, such as fumagillin, incorporated into the phosphatidylcholine-based surfactant membranes of αvβ3-targeted nanosized emulsion particles [2-7] provided anti-angiogenic benefits that were broadly demonstrated in vitro and in vivo by imaging, microscopy, and functional outcomes [3, 6-8]. Hydrophobic drugs dissolved into the lipid surfactant spontaneously transferred to targeted cell membranes through an irreversible hemifusion process referred to as contact-facilitated drug delivery (CFDD) [2, 4]. Despite in vivo effectiveness, the pharmacokinetics of the drug relative to the other lipid surfactant components and the perfluorocarbon (PFC) core were discrepant and showed marked premature fumagillin loss during circulation [9]. Lipid-anchored homing ligands and gadolinium chelates co-incorporated into the surfactant of PFC nanoparticles (NP) had similar pharmacokinetic clearance rates as a percentage of injected dose in rodents. Less hydrophobic drugs dissolved into the lipid surfactant, such as paclitaxel or doxorubicin, had rapid circulatory clearance rates compared to the PFC NP core and other membrane constituents. The very poor particle retention of these drugs accounted for their lack of efficacy in cancer models when studied in vivo [10].

In an earlier report, rhodamine-PE was employed as a fluorescent drug surrogate to illustrate and elucidate the CFDD mechanism. Rhodamine-PE delivered with integrin-targeted PFC NP integrated into the cell membrane upon binding and readily passed via lipid rafts into C-32 human melanoma cells at 37C [4]. In contradistinction to rhodamine-PE, paclitaxel conjugated to phosphatidylethanolamine failed to translocate from the outer to inner cell membrane leaflets. The concept of chemically coupling drugs to the Sn-2 fatty acid of phosphatidylcholine (PC) was envisioned as an alternative approach to facilitate nanoparticle drug retention during circulation, to protect chemically-sensitive compounds from interactions with blood constituents, and to transfer into the targeted cell via CFDD. In vivo studies demonstrated that the Sn-2 prodrug motif was efficacious for fumagillin, docetaxel, and recently cMYC-inhibitors [9-12].

While the concept of Sn-2 phospholipid prodrugs was never considered for ligand-targeted drug delivery in association with the CFDD hemifusion mechanism, precedence for Sn-2 phospholipid prodrugs was found to exist, initially reported by David Thompson et al. in the context of triggered drug release mechanisms [13]. Later, Andresen and colleagues [14-24] pursued an approach to deliver chemotherapeutics as Sn-2 prodrugs via untargeted liposomes, anticipating that phospholipases liberated locally by cancers would trigger local drug release. Their hypothesis was essentially incorrect. Physical-chemical modeling by this team revealed that non-pegylated liposomes were resistant to free water penetration and premature hydrolysis of the synthetic ether-lipid prodrugs. However, pegylation of liposomes, a design feature commonly employed to extend circulatory half-life, wicked water into the membrane creating access to lipases with resultant rapid premature drug release.

Phosphatidylcholine (PC) populates all cell and intracellular membranes in contrast to phosphatidylethanolamine or phosphatidylserine, which preferentially reside in the inner cell membrane leaflet and other specific organelles, such as the mitochondria [25, 26]. The Sn-2 PC prodrug motif was adopted as a PC mimic that would circumvent endosomal NP internalization and resultant losses by intercalation into the cell membrane and trafficking throughout the intracellular membranes until lipases, such as phospholipase A2, liberated a bioactive drug. However, this working hypothesis for the effectiveness of Sn-2 PC prodrug has never been elucidated.

In the present study, complementary microscopic imaging techniques were utilized to explore cell membrane uptake, cellular internalization and intracellular distribution of a doxorubicin Sn-2 PC prodrug or AlexaFluor™ Sn-2 PC prodrug. The objectives were to compare the biopotency of a phosphatidylcholine doxorubicin prodrug (DOX-PD) with free doxorubicin (DOX) and to utilize fluorescence lifetime imaging microscopy (FLIM) to compare the trafficking of DOX-PD versus free DOX from the outer cell membrane to the nucleus. To corroborate and extend the FLIM results, single-molecule super-resolution optical microscopy [27-29] was employed to map Sn-2 AlexaFluor 488 prodrug (AF488-PD) trajectories from the cell membrane uptake, outer to inner membrane leaflet transfer, and throughout the intracellular membranes.

Experimental design

The present study employed traditional fluorescence microscopy, confocal fluorescence lifetime imaging microscopy (FLIM), and single molecule super-resolution optical microscopy in a complementary fashion to track optically active Sn-2 phosphatidylcholine prodrug in normal and cancerous cells. The experimental protocols utilized a doxorubicin prodrug for fluorescence microscopy and confocal fluorescence lifetime imaging for assessments of the cell membrane, cytosol, and nucleus. A more photostable dye, AlexaFluor™ 488, was synthesized into an Sn-2 prodrug motif to detect single prodrug molecules in the presence of autofluorescence. This enhanced photostability extended the SRM tracking time and permitted better estimates of molecular migration patterns and prodrug membrane retention.

Fluorescence microscopy differentiated free DOX and DOX-PD distribution from the outer cell membrane, cytoplasm and nucleus. FLIM, which assessed distributions between local biochemical environments based on changes in excited-state lifetimes, offered qualitative discrimination between drug retained in cell membranes, enzymatically released into the cytosol, or in the nucleus bound to dsDNA. Super-resolution optical microscopy dynamically tracked single prodrug (AF488-PD) molecules across multiple focal planes, demonstrating spatial uptake, translocation and distribution within the cell until the dye was enzymatically liberated, after which rapid molecular motion beyond the confocal volume precluded detection. Collectively, these observations and measurements demonstrate the CFDD drug delivery mechanism of PC prodrugs and the avoidance of endosomal sequestration and degradation.

Materials and methods

Synthesis and characterization of doxorubicin and AlexaFluor 488 prodrugs

Doxorubicin Sn-2 prodrug conjugates (DOX-PD) were synthesized in our laboratory starting from commercially available doxorubicin (Sigma Aldrich, St. Louis, MO) and oxidized phospholipids (NOF America, White Plains, NY). In this approach, doxorubicin (0.22 mM) was dissolved in dimethylsulfoxide (DMSO) and triethylamine (1 mM). Added to this solution was (S)-2-(8-carboxyoctanoyloxy)-3-(palmitoyloxy)propyl 2-(trimethylammonio) ethyl phosphate (PAzPC, 0.2 mM, NOF America) dissolved in chloroform with cyclohexylcarbodiimide (1 mM). The reagent mixture was shaken overnight at room temperature (RT) then purified using silica gel column chromatography. The desired product DOX-PD was characterized using mass spectroscopy: Chemical Formula: C60H91N2O20P, ESI-MS M+H+ Cal. 1190.63, exp.1191.6 (Shimadzu LCMS 2010A, Kyoto, Japan) (Figure S1).

The AlexaFluor 488 Sn-2 phospholipid conjugate (AF488-PD) was synthesized from commercially available AlexaFluor 488 (Thermo Fisher Scientific, Waltham, MA) and oxidized phosphatidylcholine (NOF America, PAzPC). In this approach, AlexaFluor 488 cadaverine (1.56 µmoles in 1 mL dimethylformamide) was combined with excess PAzPC (6.6 µmole) dissolved in 2 mL chloroform to which triethylamine (50 µl) and N,N’-diisopropylcarbodiimide (50 µL) were added. The mixture was reacted for 2-3 hours. Separation with silica TLC (0.2M NH4Ac:MeOH:water - 20:100:200) revealed only the conjugated dye (RF 0); the free dye control was seen at RF ~ 0.8. The sample was dialyzed (MWCO 500-1000) then lyophilized. Purified AF488-PD was characterized using mass spectroscopy: final chemical formula was C74H113N7NaO23PS2+2H+, ESI-MS M+H+ Cal. 793, exp.793.85 (Shimadzu LCMS 2010A, Kyoto, Japan) (Figure S1).

Synthesis and characterization of αvβ3 DOX-PD PFOB nanoparticles

Nanoparticles (NP) were prepared as a microfluidized suspension of 20% (v/v) perfluoro-1-bromooctane (PFOB, Exfluor Research Corporation, TX), 2.0% (w/v) of a surfactant co-mixture and 1.7% (w/v) glycerin in Milli-Q® water. An αvβ3-integrin antagonist, a quinolone nonpeptide developed by Bristol-Myers Squibb Medical Imaging (US patent US 6511648 and related patents) was used for homing (a gift, Kereos, Inc., St. Louis, MO). The surfactant co-mixture included: approximately 97.6 mole% lecithin, 0.15 mole% of αvβ3-ligand-conjugated lipid and 2.28 mole% of DOX-PD (0.5 mM). The surfactant components were combined with the PFOB, buffer and glycerine and Milli-Q® water, then the mixture was homogenized at 20000 psi for 4 min. The NP were preserved under inert gas in sterile sealed vials until use. Nominal αvβ3-DOX-PD PFOB NP and size by dynamic light scattering was 260 nm, with a polydispersity of 0.14 and zeta potential of -7.4 mV. The surfactants of fluorescent PFOB NP incorporated 0.1 mol% rhodamine-phosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL) or 0.1 mol% AF488-PD in the lipid commixture at the expense of phosphatidylcholine.

Absorption and fluorescence spectroscopy

DOX and DOX-PD were used for absorption and fluorescence spectroscopy. The materials were diluted in Milli-Q® water. Absorption spectra were measured on a DU 640 spectrophotometer (Beckman-Coulter, Brea, CA). Fluorescence emission spectra were recorded on a FluoroLog 3 spectrofluorometer (Horiba Jobin Yvon, Edison, NJ) using 585 nm/600-700 nm as excitation/emission wavelength with 5 nm slits.

Cell culture

For fluorescence microscopy, 2F2B mouse endothelial cells (ATCC CRL2168, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM, no phenol red, Gibco, Thermo Fisher Scientific, Waltham, MA) with 4.5 g/L glucose and 10% heat inactivated fetal bovine serum.  Cells were plated on sterile 12 mm cover glasses (#1.5) in a 24 well plate at approximately 1x105 cells per well and incubated overnight at 37 °C in a 5% CO2 incubator. Doxorubicin (Sigma, St Louis, MO, USA) or αvβ3-DOX-PD PFOB NPs were added to the cells at a final concentration of 3 µM active drug in complete media.  After 1 h, the cells were washed three times in PBS and fixed in 4% paraformaldehyde solution for 30 min.  The coverslips were rinsed in PBS; the cell nuclei were stained blue with DAPI (4',6-diamidino-2-phenylindole, dihydrochloride); and samples were analyzed by fluorescence microscopy (Olympus BX61, Shinjuku, Tokyo, Japan).

For confocal fluorescence lifetime imaging microscopy, human C-32 melanoma cells (Washington University Tissue Culture Support Center, St. Louis, MO) were cultured in Minimal Essential Media containing Earle’s salts (no phenol red, Gibco, Thermo Fisher Scientific, Waltham, MA) and supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich, St. Louis, MO), 1% penicillin and 1% streptomycin (Gibco). The cells were trypsinized and plated on glass bottom dishes (35 mm, glass #1, MatTek Corporation, Ashland, MA) and grown overnight in phenol red-free media. The drug formulations were diluted in the culture media and cells were incubated for varying times. After incubation and washing, fluorescence lifetime imaging was performed on live cells in a humidified stage-top incubator with 5% CO2.

For super-resolution imaging, 2F2B mouse endothelial cells were cultured in DMEM with 10% fetal bovine serum (Gibco) plus 1% penicillin and 1% streptomycin (Gibco) and incubated at 37ºC with 5% CO2. 2F2B cells were trypsinized and plated on ozone-pretreated high-tolerance coverslips (No. 1.5, 170 ± 5 μm thickness, Azer Scientific, Morgantown, PA), and incubated for 48 h. The cells were further treated with 1.6 μg/mL AF488-PD at 37 ºC for 12 h, or 0.05 μg/mL AF488-PD at room temperature for 5 min before imaging. The 2F2B cells were also treated with 1.4 μM PE-rhodamine at 37 ºC for 1 h and rinsed with PBS for standard epifluorescence imaging.

Cytotoxicity assay

The retained cytotoxicity of DOX-PD was compared to native DOX in mouse endothelial cells. 2F2B cells were seeded on a 96 well plate (2000-5000 cells/well) in DMEM with 10% heat-inactivated fetal bovine serum plus 10 µM angiotensin II (Sigma-Aldrich, St. Louis, MO) then incubated at 37 ºC with 5% CO2. After 24 h, cells were incubated for 1 h with a) αvβ3-DOX-PD PFOB NP (0.3 µM DOX PD), b) equimolar equivalent of native doxorubicin HCl or c) αvβ3-No drug NP. After incubation the wells were washed three times with PBS and returned to the incubator for 48 h. Cell proliferation was measured using the MTT assay for cytotoxicity, which measures the activity of cellular enzymes that reduce the tetrazolium dye to the insoluble formazan (Invitrogen; Thermo-Fisher Scientific, Waltham, MA). Each treatment was replicated 6 times.

Fluorescence lifetime spectroscopy, imaging and data analysis

Confocal fluorescence lifetime spectroscopy and imaging was performed with a MicroTime 200 microscope (Picoquant, Berlin, Germany). For spectroscopy, fluorescence lifetimes of DOX (50 µM) and DOX-PD (50 µM) solutions in MilliQ® water (final DMSO 0.5%) were measured under a 100X oil immersion objective with 485 nm (15 au) laser excitation pulsed at 40 MHz. Decay data were collected through a 519 nm long pass filter and accumulated until peak photon counts reached 10,000. Almost identical conditions were applied for αvβ3-DOX-PD PFOB NPs (25 µM doxorubicin concentration) except the pulse rate was reduced to 20 MHz in order to capture its slower decay. Live cell imaging utilized 485 nm laser excitation (~1000 au) at 20 MHz on cells maintained in a stage top CO2 incubator for the period of imaging.

Lifetime decay data collected from spectroscopy and microscopic imaging were analyzed on the SymphoTime software (Picoquant, Berlin, Germany). All decays were fit using multi-exponential tail fit using the equation below:

Equation 1

Where: n = number of exponentials (n ≤ 2 for all cases depending on goodness of fit); A = amplitudes; = lifetimes; Bkgr = correction for background (after-pulsing, dark counts, environmental light).

Spectroscopy data were fit to mono- or bi-exponential decay fits (depending on goodness of fit). For cell images, raw decay data from all cellular pixels were fit to bi-exponential models and this method was used to generate the corresponding lifetime images.

For cell compartment specific analyses, nuclear, perinuclear and extra-perinuclear regions of interest (ROIs) were drawn to achieve 1000 or more counts to ensure effective bi-exponential decay analysis. Bi-exponential tail fit was used to fit decays from each ROI and individual components (lifetime and amplitude) were obtained. Good χ2 values (0.8-1.2) and random distribution of x-axis residuals were considered necessary for an accurate decay fit. Amplitude average fluorescence lifetimes (τamp) were calculated and reported using the following model and reported as average lifetime values.

Equation 2

Analyzed data was plotted using Prism (Graphpad, La Jolla, CA). Wherever required, appropriate statistical analysis was performed using Prism’s built-in functions and corresponding p values were reported.

Super-resolution imaging and data analysis

Single-molecule super-resolution imaging was used to image the binding and cellular uptake of individual prodrug molecules. First, images of individual fluorescent molecules with switchable bright-dark states (blinking) were captured, and their positions were measured by fitting each blinking event to a two-dimensional Gaussian function. A super-resolved image was then reconstructed by combining all positions from the stochastic blinking events of fluorescent molecules. To resolve the fluorescence signal from a single molecule, a bright fluorophore, AlexaFluor 488 (AF488), was used and conjugated to the phospholipid at the Sn-2 position as the prodrug analog for super-resolution imaging. AF488-PD was more photostable than DOX-PD and permitted detection of single copies of AF488-PD in the presence of autofluorescence, extending SRM tracking time and improving estimates of molecular migration patterns and prodrug membrane retention. Freely-diffusing AF488-PD in the extracellular buffer and in the cytosol were not captured, since their quickly-moving fluorescence signals were out of focus and/or averaged out by the detector. However, when AF488-PD temporarily docked onto the lipid membrane, its slowed diffusion was captured and localized from which a super-resolution image was reconstructed with spatial resolution beyond refraction limit. In this way, single-molecule super-resolution imaging precisely localized the prodrug molecules with single-molecule sensitivity.

Super-resolution imaging was performed on a home-built microscope equipped with a 100X oil immersion objective (Figure S2) [30, 31]. After AF488-PD incubation, the cells were washed with PBS to remove the excess prodrug in the media. Cells were first illuminated with a 488-nm laser (OBIS, Coherent) at a peak intensity of 0.12 kW∕cm2 at the sample. The images were filtered with a 523/610 dual-band bandpass filter (Semrock FF01-523/610) and acquired by a sCMOS camera (Hamamatsu, C11440-22CU) with a 50 ms exposure time. Next, merocyanine 540 (MC540, Invitrogen) was introduced to cell media to image the cell membranes via PAINT (Point Accumulation for Imaging in Nanoscale Topography) [32]. Similar to AF488-PD, localizations were only acquired when MC540 temporarily docked with the cell membranes and emitted bright fluorescence, thereby enabling the co-localization of prodrug molecules relative to cell membrane with nanoscale resolution. MC540 was excited with a 561-nm laser (Sapphire, Coherent) at a peak intensity of 2.7 kW∕cm2 at the sample, and images were acquired with a 50 ms exposure time. The single AF488-PD and MC540 molecules were identified and localized by two-dimensional Gaussian fitting using an ImageJ plug-in ThunderSTORM [33].

The super-resolution images of prodrug and cell membranes consist of the sum of all localizations from AF488-PD and MC540 respectively, binned as a 2D histogram with bin size 58.5×58.5 nm2. AF488-PD localizations over time were grouped into a trajectory when present in consecutive frames (up to a 3-frame gap) within a distance of 0.59 μm. The trajectories were plotted using custom software (MATLAB, MathWorks). No trajectory analysis was performed for the endothelial filopodia.


Spectroscopic characterization of synthesized prodrugs

Absorption and fluorescence spectra and decay characteristics of the materials were measured in a water solution. DOX and DOX-PD had similar absorption spectra (Figure 1A). The spectra for αvβ3-DOX-PD PFOB NP was obscured by light scattering.

Figure 1. Spectroscopic characterization of doxorubicin (DOX), doxorubicin prodrug (DOX PD), and αvβ3-integrin targeted prodrug nanoparticles (αvβ3-DOX-PD PFOB-NPs) in water: Absorption spectra of the materials (A). The absorption of doxorubicin prodrug in nanoparticle formulation is obscured by light scattering. Presence of doxorubicin is confirmed in the corresponding fluorescence emission (B). Fluorescence decay characteristics of the materials (C). DOX decay can fit to mono-exponential decay of 1.09 ± 0.02 ns. DOX-PD can fit to bi-exponential decay with τa = 1.21 ns (47%); τb = 0.69 ns (53%). DOX-PD NP can fit to bi-exponential decay with τa = 3.78 ns (22%); τb = 1.34 ns (78%). Data represent mean ± sd, n=3 measurements. IRF – Instrument response function.

All drug formulations showed similar fluorescence emission spectra, which corroborated the presence of doxorubicin in the nanoparticles (Figure 1B). The emission peak at ~556 nm for doxorubicin in the αvβ3-DOX PFOB NP was relatively more prominent. This observation may reflect the unique interactions of the doxorubicin molecules with the core PFOB molecules that penetrate to the particle-water interface [34].

As shown in Figure 1C, free DOX fluorescence fit a mono-exponential decay of 1.09 ± 0.02 ns (χ2 = 1.06), consistent with literature findings. DOX-PD decay best fit a bi-exponential decay model with nearly equal contributions from a fast lifetime component τa = 0.69 ± 0.06 ns (53%) and a slow lifetime component of τb = 1.21 ± 0.03 ns (47%), indicative of a heterogeneous system (χ2 = 1.007). The amplitude average lifetime (τamp) was calculated to be 0.96 ± 0.04 ns. Dynamic light scattering analysis of a DOX-PD in water revealed a mixed suspension of free prodrug molecules and spontaneously formed prodrug micelle aggregates (~1600 nm). Fluorescence lifetime of doxorubicin bound to biological molecules (lipid membranes, DNA) and while encapsulated in nanoparticles increases as the molecules are shielded from external fluorescence quenchers and by - stacking [35-38]. Hence, the slower lifetime component from DOX-PD suspension was attributed to the micellar fraction of the drug distribution where the anthracycline portion of DOX-PD was shielded from surrounding water molecules. The faster lifetime of 0.69 ns can be attributed to the free DOX-PD molecules in water. This value is slightly different than that of free DOX in water, reflecting the effect of drug conjugation with the lipid molecule. Fluorescence decay of αvβ3-DOX-PD PFOB NP fit a double exponential decay model with components τa = 3.78 ± 0.03 ns (22%) and τb = 1.34 ns (78%) (χ2 = 1.03). The majority of the αvβ3-DOX-PD PFOB NP had the faster fluorescence lifetime component (1.34 ns) similar to that of DOX-PD in the micellar fraction (1.21), indicating similar hydrophobic interactions. The slower lifetime fractional component (3.78 ns) is likely related to hydrophobic interactions of DOX-PD within the nanoparticle lipid surfactant intercalated between PFOB molecules.

Cellular uptake and cytotoxicity of αvβ3-DOX-PD PFOB NP

The cellular distribution of DOX presented as the free drug and delivered within nanoparticle surfactant in prodrug form was observed with fluorescence microscopy (Figure 2).