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Tailoring Porphyrin Conjugation for Nanoassembly-Driven Phototheranostic Properties Marta Overchuk, Mark Zheng, Maneesha A. Rajora, Danielle M. Charron, Juan Chen, and Gang Zheng ACS Nano, Just Accepted Manuscript • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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Tailoring Porphyrin Conjugation for Nanoassembly-Driven Phototheranostic Properties Marta Overchuk,1,2 Mark Zheng,1,3 Maneesha A. Rajora,1,2 Danielle M. Charron,1,2 Juan Chen,1 Gang Zheng,*1,2,4 1Princess

Margaret Cancer Centre, University Health Network, 101 College Street,

Toronto, Ontario, M5G 1L7, Canada. 2Institute

of Biomaterials and Biomedical Engineering, University of Toronto, 101 College

Street, Toronto, Ontario, M5G 1L7, Canada. 3Department

of Biology, University of Waterloo, 200 University Avenue W, Waterloo, ON,

N2L3 G1, Canada 4Department

of Medical Biophysics, University of Toronto, 101 College Street, Toronto,

Ontario, M5G 1L7, Canada *Corresponding Author: Dr. Gang Zheng, 101 College Street, Princess Margaret Cancer Research Tower 5-354, Toronto, Ontario, M5G 1L7, Canada, E-mail: [email protected]

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ABSTRACT Lipoprotein mimetic nanostructures, which consist of an amphiphilic lipid shell, a hydrophobic core, and an apolipoprotein mimetic peptide, serve as a versatile platform for the design of drug delivery vehicles as well as the investigation of supramolecular assemblies. Porphyrin incorporation into biomimetic lipoproteins allows one to take advantage of the inherent multimodal photophysical properties of porphyrin, yielding various fluorescence, photoacoustic as well as photodynamic agents. To facilitate their incorporation into a lipoprotein structure, porphyrins have been conjugated through a variety of strategies. However, the effects of the conjugate structure on the associated nanoparticle’s phototherapeutic properties warrants further investigation. Herein, we systemically investigated the effects of two widely utilized porphyrin conjugates – oleylamide and lipid, on biophotonic properties of their resultant porphyrinlipoprotein nanoparticles in vitro and in vivo. Specifically, we demonstrated that incorporation of the porphyrin moiety as an oleylamide conjugate leads to a highly stable J-aggregate with strong photoacoustic contrast, while incorporation as an ampiphilic lipid moiety into the lipid shell yields an effective fluorescent and photodynamic agent. The current study proposes a rational design strategy for next-generation lipoprotein-based phototheranostic agents, where nanoassembly-driven biophotonic and therapeutic properties can be tailored through the specific selection of porphyrin conjugate structures. KEYWORDS nanomedicine, lipoprotein mimetic, photodynamic therapy (PDT), photoacoustic imaging, fluorescence


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Native lipoproteins are well-designed nanoparticles that solubilize and transport hydrophobic molecules within the human body.1 More specifically, lipoproteins consist of a phospholipid shell and a hydrophobic core, formed by cholesteryl esters, that are stabilized by various apolipoproteins.2 The natural ability of lipoproteins to integrate molecules of varying hydrophobicity has inspired scientists to reverse engineer a range of lipoprotein-like agents for both biomedical applications and fundamental studies.3,4 From an application standpoint, their long plasma circulation time, non-immunogenicity, core/shell loading and receptor targeting capabilities make them promising delivery vehicles for small molecule drugs,6–8 therapeutic siRNA8–11 and imaging agents.12–16 From a fundamental standpoint, lipoprotein-like structures provide a unique way of studying supramolecular interactions, in particular the effects of nanostructures on dye-dye interactions, such as self-quenching and aggregation.17–20

Porphyrins, amongst other dyes, have generated significant interest within nanoscience due to their unique photophysical properties that enable both diagnostic and therapeutic modalities such as fluorescence or photoacoustic imaging and photodynamic or photothermal therapy respectively. It has been known for decades that during circulation porphyrins tend to incorporate into plasma lipoproteins, which partially explains porphyrins’ unique ability to accumulate in tumors, due to long circulation times of native lipoproteins and upregulated lipoprotein receptors in tumors.21 To facilitate porphyrin incorporation into a lipoprotein and thereby take advantage of their capacity for long circulation and receptor-targeted delivery, a variety of porphyrin conjugation strategies have been applied. Specifically, introduction of amphiphilicity to the porphyrin molecule through lipid conjugation enables porphyrin incorporation into the lipid shell of these lipoproteins.19,22 Our group has developed porphyrin-containing high density lipoprotein


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(HDL)-like nanoparticles in which amphiphilic pyropheophorbide-lipid was incorporated into the nanoparticle shell for activatable fluorescence imaging and photodynamic therapy (PDT).22,23 In an alternative approach, highly hydrophobic porphyrin-fatty acid esters were successfully loaded into the HDL hydrophobic core.24 Recently, Harmatys and co-authors synthesized a series of hydrophobic chlorin derivatives that were encapsulated into a biomimetic HDL core, wherein the spatial constraints created by an ApoA-1 mimetic peptide resulted in dye J-aggregation characterized by a strong bathochromic shift (654 nm to 715 nm), enabling photoacoustic imaging.25 These examples indicate that the approach towards porphyrin inclusion into lipoproteins (for example core versus shell) may influence the biophotonic properties of the associated nanostructures.

Despite an abundance of literature describing porphyrin incorporation into lipoproteins using a variety of conjugation approaches, the specific effects of the conjugate structure on the nanoparticle’s biophotonic and phototherapeutic properties remain unclear. Eager to answer this question using a systematic approach, we designed a pair of HDL-like porphyrin nanoconstructs that mirror each other in their composition and properties. We combined lipid- and oleylamideconjugates of two porphyrins with complementary photophysical properties: pyropheophorbide a (Pyro - a potent photosensitizer) and bacteriochlorophyll (BChl - a near-infrared fluorophore). In the first particle, B(P)-HDL, we combined amphiphilic BChl-lipid with hydrophobic Pyrooleylamide, while in the second nanoparticle, P(B)-HDL, we combined Pyro-lipid and BChloleylamide (Figure 1).


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These resulting nanoparticles demonstrated significant differences in their biophotonic properties and their optimal in vivo function, highlighting the importance of tailoring porphyrin-conjugate design towards a desired application. Specifically, we demonstrated that P(B)-HDL nanoparticles possessed superior photoacoustic imaging capabilities due to a more favorable absorbance maximum at 844 nm resulting from a highly stable J-aggregate of hydrophobic BChl-oleylamide. Alternatively, BChl-lipid-containing B(P)-HDL nanoparticles were more favorable for NIR fluorescence imaging due to their low fluorescence quenching efficiency. Moreover, we demonstrated differences in the photodynamic activities of particles incorporating Pyro-lipid or Pyro-oleylamide in vitro and in a subcutaneous KB mouse model. Specifically, Pyro-lipidcontaining P(B)-HDL nanoparticles in combination with laser treatment elicited a strong PDT response, while in comparison Pyro-oleylamide photodynamic activity was significantly suppressed due to highly stable aggregation and quenching within B(P)-HDL nanoparticles. The current study thus introduces a design strategy for next-generation lipoprotein-based phototheranostic agents, where structure-dependent optical and therapeutic properties can be tailored by the specific selection of dye-conjugate structures.


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Figure 1. Sample photographs and schematic representations of B(P)-HDL and P(B)-HDL nanoparticles. (A) Solutions of freshly prepared B-HDL, (P)-HDL and B(P)-HDL nanoparticles. (B) B(P)-HDL structure and properties: BChl-lipid produces NIR fluorescence; stable Pyro-oleylamide aggregation results in PDT quenching. (C) Solutions of freshly prepared P-HDL, (B)-HDL and P(B)-HDL nanoparticles. (D) P(B)-HDL structure and properties: Pyro-lipid can be easily dissociated upon cellular uptake resulting in potent PDT; stable BChl-oleylamide aggregation results in strong photoacoustic signal.


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RESULTS AND DISCUSSION B(P)-HDL and P(B)-HDL synthesis and characterization. The core/shell architecture of native lipoproteins naturally allows for the incorporation of various porphyrin conjugates into different nanoparticle subunits (shell or core). Our group has explored porphyrin loading into a lipoprotein shell by introducing amphiphilicity to the porphyrin molecule through lipid conjugation19,22 or into a lipoprotein core by enhancing porphyrin hydrophobicity through oleylamide25 or bis(oleate) conjugation.24 Synthetic HDL-like nanoparticles recapitulate the structure of native lipoproteins, therefore porphyrin conjugates of varying hydrophobicity can be easily incorporated into their structure.26

To systematically investigate the effects of porphyrin conjugate structure on nanostructuredependent imaging properties and photodynamic activity, we synthesized lipid- and oleylamideversions of two porphyrins with complementary photophysical properties: pyropheophorbide a (Pyro - a potent photosensitizer) and bacteriochlorophyll (BChl - a near-infrared fluorophore). Synthesis of BChl-lipid, BChl-oleylamide, Pyro-lipid and Pyro-oleylamide was confirmed by UPLC-MS analysis with identified ESI+ mass spectrometry and corresponding UV−vis absorption (Figure S1 and S2). Next, we incorporated these porphyrin-conjugates as pair combinations into HDL-like nanostructures to form B(P)-HDL (BChl-lipid + Pyro-oleylamide) and P(B)-HDL (Pyro-lipid + BChl-oleylamide). Four nanoparticle controls with individual conjugate incorporation were also synthesized: B-HDL and P-HDL nanoparticles containing Pyro-lipid conjugate and BChl-lipid conjugate respectively, and (B)-HDL and (P)-HDL nanoparticles synthesized respectively with BChl-oleylamide and Pyro-oleylamide.


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Briefly, nanoparticles were prepared by sonicating the hydrated lipid films consisting of the appropriate amounts of the host lipid with porphyrin derivatives (Table S1) and cholesteryl oleate (CO). After sonication, an apolipoprotein A1-mimetic peptide containing 18 amino-acids (R4F) was added to the resulting multilamellar vesicle suspension and incubated overnight to generate an -helical network that resulted in the formation of core/shell lipoprotein structures. The resulting nanoparticles were characterized by collecting their absorbance and fluorescence spectra in their intact and disrupted states, and by transmission electron microscopy (TEM; Figure 2 and Figures S3 and S4). The B(P)-HDL absorbance spectrum revealed that Pyrooleylamide underwent a head-to-tail ordered aggregation, resulting in exciton coupling (Jaggregation), as evidenced by the bathochromic shift of the Qy absorbance peak from 672 nm to 706 nm. BChl-lipid absorbance did not exhibit any changes likely due to its relatively low encapsulation amount (5% of the total lipid). Examination of the P(B)-HDL absorbance spectrum demonstrated a similar trend for the lipid- and oleylamideconjugates with almost complete J-aggregation of BChl-oleylamide (Qy aggregation = 844 nm vs. Qy monomer – 750 nm), and minimal absorption shift of the Pyro-lipid peak. Absorbance spectra of the control nanoparticles (Pyro-oleylamide only (P)-HDL and BChl-oleylamide only (B)-HDL) confirmed that the formation of J-aggregates occurred independently of the presence of porphyrin-lipid conjugates in the nanoparticles’ shells (Figure 2, Table S2 and Figure S3).

Next, we examined the fluorescence emission of Pyro and BChl in B(P)-HDL and P(B)-HDL. As shown in Figure 2 and Table S2, BChl-oleylamide exhibited similarly strong fluorescence quenching in both P(B)-HDL and (B)-HDL nanoparticles (79.75 ± 0.93% and 80.32 ± 3.99%, n=3) indicating self-aggregation within the nanoparticles. Pyro-lipid in the nanoparticle lipid


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shell demonstrated similar self-quenching in both P(B)-HDL (88.5 ± 6.5 %, n=3) and P-HDL (78.0 ± 11.4%, n=3). Pyro-oleylamide, on the other hand, underwent highly efficient fluorescence quenching in B(P)-HDL nanoparticles (98.89 ± 0.11%, n=3), while in the control (P)-HDL particles its quenching efficiency was only 83.34 ± 7.52% (n=3). This higher fluorescence quenching of Pyro-oleylamide in B(P)-HDL might be caused by the presence of energy transfer from Pyro-oleylamide to BChl-lipid in B(P)-HDL due to the existing overlap between the Pyro emission and BChl absorption,27 thus leading to an enhanced fluorescence quenching of Pyro-oleylamide versus that in (P)-HDL. BChl-lipid, in turn, exhibited low fluorescence quenching (64.03 ± 8.91%, n=3) in B(P)-HDL, which can be explained by relatively low incorporated density (5% of total lipid) of BChl-lipid in the amphiphilic shell.

Finally, nanoparticle morphology was examined with negative staining TEM (Figure 2 and Figure S4). Both B(P)-HDL and P(B)-HDL nanoparticles consisted of mixtures of discoidal and spherical populations of sub-100 nm size characteristic of both native HDLs and established pyropheophorbide and BChl-containing lipoproteins.28–30 Size distributions of B(P)-HDL and P(B)-HDL were obtained by ImageJ analysis of >400 particles across TEM images (Figure S5). The mean size of B(P)-HDL was 32 nm and that of P(B)-HDL was 25 nm, consistent with the literature describing lipoprotein mimetic nanoparticles.4,19 Control B-HDL and P-HDL nanoparticles consisted of monodisperse spherical structures with well-defined cholesteryl oleate cores. (B)-HDL and (P)-HDL nanoparticles morphologies, similarly to B(P)-HDL and P(B)HDL, were characterized as a heterogeneous mixture of discoidal and spherical species, demonstrating a clear effect of porphyrin-oleylamide aggregation on the nanostructure shape.


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Figure 2. Optical properties and morphology of B(P)-HDL and P(B)-HDL. Absorption spectra of intact (solid line) and disrupted with 5% Triton X-100 solution (dashed line) B(P)-HDL (A) and P(B)-HDL (B). (C and D) Pyro fluorescence spectra (excitation: 630 nm) of the intact (solid line) and disrupted (dashed line) B(P)-HDL (C) and P(B)-HDL (D). (E and F) BChl fluorescence spectra (excitation: 730 nm) of the intact (solid line) and disrupted (dashed line) B(P)-HDL (E) and P(B)-HDL (F). Fluorescence was normalized to the maximum fluorescence intensity corresponding to the disrupted sample. Transmission electron microscopy images of B(P)-HDL (G) and P(B)-HDL (H). Scale bar = 200 nm.


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Structure-dependent fluorescence and photoacoustic imaging. To evaluate nanostructureenabled imaging capabilities of B(P)-HDL and P(B)-HDL, we conducted hyperspectral fluorescence and photoacoustic imaging of thin tubing phantoms containing increasing concentrations of the intact and disrupted nanoparticles (Figure 3). First, we observed strong quenching of Pyro fluorescence in both B(P)-HDL and P(B)-HDL samples that was restored upon nanoparticle disruption with a detergent (Figure 3A and 3C, upper panel). Interestingly, we observed that BChl-lipid signal increased in a concentration-dependent manner in the intact B(P)-HDL samples. BChl-lipid fluorescence in the intact particle can be explained by low BChllipid density in the nanoparticle’s shell, resulting in its low quenching efficiency and “alwayson” near-infrared fluorescence (Figure 3A, lower panel). On the other hand, BChl-oleylamide fluorescence was not detected in 25 and 50 M P(B)-HDL samples, with only minor signal observed in the 100 M solution, confirming strong fluorescence quenching (Figure 3C, lower panel). Next, we evaluated photoacoustic signal generated by the highly-quenched and aggregated porphyrin-oleylamide conjugates in B(P)-HDL and P(B)-HDL (Figure 3B and D). In both cases, we observed concentration-dependent increase in photoacoustic signal at wavelengths that corresponded with the associated porphyrin-oleylamide aggregate Qy absorption peaks (706 nm for B(P)-HDL and 844 nm for P(B)-HDL).

Finally, we evaluated the optical stability of B(P)-HDL and P(B)-HDL nanoparticles in biologically-relevant conditions (Figure S5). Nanoparticles were incubated in PBS that contained 10 vol% and 50 vol% fetal bovine serum for 24 h at 37°C. Changes in their absorption were expressed as the ratio of aggregate Qy / monomer Qy peaks. Overall, both nanoparticles


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demonstrated high optical stability with only minor changes in the aggregate : monomer peak absorbance ratio.

Figure 3. Nanoassembly-enabled fluorescence and photoacoustic properties of B(P)-HDL and P(B)-HDL nanoparticles. Maestro CRi fluorescence imaging of thin tubing phantoms containing (A) B(P)-HDL and (B) P(B)HDL at the following concentrations: 1 – 25 μM in PBS, 2 – 50 μM in PBS, 3 – 100 μM in PBS, 4 – 100 μM in 10% TritonX-100 and 5 - 10% TritonX-100 alone. (B) and (D) - photoacoustic imaging of B(P)-HDL and P(B)-HDL nanoparticles respectively as a function of oleylamide-conjugated porphyrin concentration. PA spectral scans from 680 nm to 900 nm show peak signals at 710 nm for intact B(P)-HDL particles (B) and at 844 nm for intact P(B)HDL (D), with a concentration-dependent increase in signal intensity up to 100 μM.

These data demonstrate that the increase in porphyrin hydrophobicity via oleylamine conjugation facilitates highly stable J-aggregation, which is instrumental for the design of photoacoustic agents. Therefore, the NIR-absorbing BChl-oleylamide J-aggregation enabled highly efficient


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photoacoustic imaging, while BChl-lipid exhibited strong fluorescence due to the low packing density of BChl-lipid in the nanoparticle shell. B(P)-HDL and P(B)-HDL demonstrate SR-BI targeting and photodynamic activity in vitro. SR-BI receptor is a membrane glycoprotein that plays a key role in lipoprotein-mediated cholesterol trafficking.31 Malignant tissue transformation is often marked by an increase in cholesterol consumption, and therefore upregulated expression of SR-BI receptor has been reported for many cancers,32 including adrenal,11 breast,10,11 and prostate.10 HDL-like platforms, similarly to their natural lipoprotein counterparts, possess high SR-BI binding affinity that can be exploited for the targeted delivery of various therapeutics. To investigate if the incorporation of porphyrin conjugates into HDL-like structures affects this targeting capability, B(P)-HDL and P(B)-HDL nanoparticles were incubated with ldlA-7 (SR-BI-deficient) and ldl(mSR-BI) (SR-BI over-expressing) cells for 3, 6 and 24 h to examine the porphyrin uptake specificity. ldl(mSR-BI) cells demonstrated markedly stronger fluorescence compared to ldlA-7 cells (Figure 4A and B). Image-based quantification of Pyro and BChl fluorescence signals in cells incubated with B(P)HDL and P(B)-HDL revealed that Pyro and Bchl fluorescence in ldl(mSR-BI) cells was significantly higher at all time points (Figure S7) than in ldlA-7 cells, confirming targeting specificity of both B(P)-HDL and P(B)-HDL.

Next, we quantified the difference in cellular uptake of the Pyro photosensitizer in its amphiphilic (P(B)-HDL) and hydrophobic (B(P)-HDL) forms. LdlA-7 and ldl(mSR-BI) cells were incubated with B(P)-HDL and P(B)-HDL at a matched Pyro concentration of 5 μM. Cellassociated Pyro fluorescence was measured at 24 h post incubation (Figure S8). Both B(P)-HDL and P(B)-HDL demonstrated significantly higher uptake in SR-BI(+) ldl(mSR-BI) cells versus


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SR-BI-deficient ldlA-7 cells, which corroborates with the fluorescence microscopy data shown in Figure 4. Notably, Pyro fluorescence was higher in SR-BI-positive cells incubated with P(B)HDL versus B(P)-HDL. To further quantify the absolute amounts of cell-associated Pyro, cells were lysed with 5% TritonX-100 to measure completely unquenched Pyro fluorescence (Figure S8B). Interestingly, it was observed that the absolute Pyro uptake in the cells incubated with B(P)-HDL was over two times higher compared to uptake in cells treated with P(B)-HDL at the equivalent Pyro concentrations. These data demonstrate that Pyro-lipid is highly prone to disruption and fluorescence unquenching upon cellular uptake, while Pyro-oleylamide remains highly quenched despite being more effectively delivered into cells.

To further investigate the effect of Pyro-lipid and Pyro-oleylamide dissociation differences on Pyro photodynamic activity, we performed in vitro PDT with B(P)-HDL and P(B)-HDL in ldl(mSR-BI) and ldlA-7 cells (Figure 4C and D). The cells incubated with either nanoparticle alone (with a matched Pyro concentration of 1 M) or cells treated solely with a 671 nm laser at the highest light dose of 2.5 J ⋅cm-2 did not demonstrate any significant viability changes. Combination of nanoparticles with laser treatment resulted in potent PDT, evidenced by a decrease in cell viability. Notably, both B(P)-HDL and P(B)-HDL demonstrated stronger PDT activity in SR-BI-positive cells versus ldlA-7 cells, further confirming SR-BI-specific targeting capabilities of the designed nanoparticles. Moreover, P(B)-HDL demonstrated significantly stronger photodynamic activity compared to B(P)-HDL in both ldlA-7 and ldl(mSR-BI) cells, further confirming that Pyro-lipid is more prone to dissociation for activateable PDT.


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Figure 4. In vitro targeting and photodynamic activity of B(P)-HDL and P(B)-HDL. (A) B(P)-HDL and P(B)-HDL uptake in ldl(mSR-BI) (high SR-BI expressing) cells with fluorescence microscopy at 3, 6 and 24 h post incubation. (B) B(P)-HDL and P(B)-HDL uptake in ldlA-7 (low SR-BI expression) cells with fluorescence microscopy at 3, 6 and 24 h post incubation. Both B(P)-HDL and P(B)-HDL are more effectively uptaken by SR-BI(+) versus SR-BI(-) cells at each time point (2.5 M porphyrin-oleylamide concentration). Scale bar = 20 m. Fluorescence was pseudocolored in red for BChl, green for Pyro and blue for Hoechst 33342. (C) B(P)-HDL and P(B)-HDL photodynamic activity in ldl(mSR-BI) cells. (D) B(P)-HDL and P(B)-HDL photodynamic activity in ldlA7 cells. Cell viability was normalized to untreated cells and is presented as the average of three replicates ± standard error of mean. Significant differences (*p < 0.05, n = 3) were observed between B(P)-HDL and P(B)-HDL photodynamic activity, wherein significantly higher toxicity was observed in P(B)-HDL-treated cells versus cells treated with B(P)HDL particles at equivalent light dose and photosensitizer concentration.

B(P)-HDL and P(B)-HDL exhibit excellent targeting and strong tumor accumulation in vivo. After investigating B(P)-HDL and P(B)-HDL targeting in vitro we validated it in vivo in a


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dual subcutaneous KB (SR-BI-expressing) and HT1080 (SR-BI deficient) murine model (Figure 5). Mice bearing dual tumors were injected with 50 nmol of B(P)-HDL and P(B)-HDL (based on the amount of the oleylamide-conjugated dye) prior to whole-animal in vivo fluorescence imaging at 3, 6 and 24 h post nanoparticle administration. Both nanoparticles exhibited markedly stronger accumulation in the SR-BI-expressing tumor (Figure 5A and C) compared to the SR-BI deficient tumor, which further confirmed the SR-BI targeting specificity of the particles. Interestingly, B(P)-HDL nanoparticles exhibited strong fluorescence in the NIR region which corresponded to BChl signal, while Pyro-oleylamide fluorescence remained negligible at 24 h post injection. Overall, in vivo fluorescence imaging confirmed the fluorescence quenching trends observed in solution (Table S2) and in cell targeting studies (Figure 4). Specifically, Pyrooleylamide in B(P)-HDL remained highly quenched up to 24 h, resulting in negligible Pyro fluorescence in vivo, while the low quenching efficiency of BChl-lipid enabled observation of strong BChl fluorescence at the SR-BI (+) tumor P(B)-HDL nanoparticles, on the other hand, gave rise to strong fluorescence signal from both dyes (BChl-oleylamide and Pyro-lipid). The observed BChl-oleylamide fluorescence can be explained by its incomplete quenching (80.3 ± 3.9%, n = 3) as determined by studies in solution (Table S2).


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Figure 5. In vivo targeting and biodistribution of B(P)-HDL and P(B)-HDL. (A) Representative fluorescence images of mice bearing dual SR-BI-positive (red arrow) and SR-BI-negative (white arrow) tumors that were intravenously injected with either B(P)-HDL or (C) P(B)-HDL at 3, 6, and 24 h post-injection. Fluorescence ex vivo organ distribution of B(P)-HDL (B) and P(B)-HDL (D), including the SR-BI positive (KB) and SR-BI negative (HT1080) tumors.

Nanoparticle biodistribution was qualitatively studied via ex vivo fluorescence imaging 24 h post injection (Figure 5B and D). Strong nanoparticle liver accumulation was observed for both B(P)HDL and P(B)-HDL. The spleen as well as KB tumor exhibited lower nanoparticle accumulation compared to the liver, while the rest of the organs (HT1080 tumor, muscle, heart, lung, skin, kidney, stomach and intestine) exhibited low to negligible/unappreciable fluorescence.

Nanostructure-enabled multimodal imaging of bacteriochlorophyll conjugates in B(P)HDL and P(B)-HDL. To explore the potential of B(P)-HDL and P(B)-HDL for nanostructure-


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enabled multimodal imaging, we used a clinically-relevant orthotopic prostate cancer model. We hypothesized that B(P)-HDL nanoparticles would generate strong fluorescence signal at the tumour site due to low BChl-lipid quenching, while P(B)-HDL nanoparticles would exhibit strong photoacoustic signal due to stable BChl-oleylamide aggregation. Athymic nude mice bearing PC-3M-luc-C6 orthotopic prostate tumors (n = 3 for each group) were injected with B(P)-HDL (30 nmol per animal based on BChl-lipid) or P(B)-HDL (230 nmol per animal based on BChl-oleylamide) respectively (Figure 6). Different nanoparticle injected doses were selected due to the differences in sensitivity betweenfluorescence and photoacoustic imaging. Tumor localization was confirmed using bioluminescence imaging (Figure 6A). Strong BChl-lipid fluorescence was observed in the tumors of B(P)-HDL-injected mice at 24 h post nanoparticle administration (Figure 6A). Importantly, organs surrounding the tumor, such as healthy prostate, testes, seminal vesicles and the rectal wall, exhibited negligible fluorescence signal, demonstrating high selectivity of the nanoparticle accumulation. Such high tumor selectivity is essential for fluorescence-guided surgery as well as other tissue-spearing focal therapies. Organ biodistribution (Figure 6B) demonstrated strong nanoparticle liver accumulation, similarly to its biodistribution in the subcutaneous mouse model. Tissue fluorescence microscopy of the frozen tumor sections revealed the presence of both BChl-lipid and Pyro-oleylamide signals, further confirming nanoparticle tumor deposition.

Next, we conducted time-dependent photoacoustic imaging of orthotopic prostate tumor-bearing mice following P(B)-HDL injection. At the earlier time points (0.5 h, 1 h, 3 h and 6 h) we observed signal at 844 nm scattered around the lower abdominal area. At 24 h post nanoparticle administration, P(B)-HDL accumulation was observed in the tumor rim area, while the


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surrounding organs exhibited negligible signal. Finally, at 48 h post P(B)-HDL injection, we observed a loss of photoacoustic signal, indicating disruption of the BChl-oleylamide Jaggregates and partial nanoparticle clearance and degradation.

This proof-of-concept imaging study illustrates how the differences in porphyrin conjugate hydrophobicity and structure can affect biophotonic properties of the resulting nanoparticles.

Figure 6. Fluorescence and photoacoustic imaging of B(P)-HDL and P(B)-HDL in the orthotopic prostate cancer model. (A) Representative bioluminescence and fluorescence (BChl) images of a mouse bearing an orthotopic prostate tumor (n = 3) 24 h post intravenous B(P)-HDL nanoparticle injection as well as its excised organs: (1)


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healthy prostate, (2) testes, (3) tumor, (4) seminal vesicles and (5) rectal wall. (B) Ex vivo fluorescence (BChl) organ distribution of mice intravenously injected with B(P)-HDL nanoparticles. (C) Fluorescence microscopy of an orthotopic prostate tumor 24 h post B(P)-HDL nanoparticle intravenous injection. (D) Representative timedependent photoacoustic imaging in orthotopic prostate tumor-bearing mouse (n = 3) intravenously injected with P(B)-HDL nanoparticles.

Modulation of pyropheophorbide photodynamic activity by lipid and oleylamine conjugation. After characterizing the differences in Pyro-oleylamide (B(P)-HDL) and Pyro-lipid (P(B)-HDL) dissociation and photodynamic activity upon cellular uptake in vitro we compared their PDT activity in vivo in a subcutaneous dual KB tumor model. Mice were injected with saline, B(P)-HDL or P(B)-HDL (Pyro-oleylamide and Pyro-lipid injected amount was matched to 100 nmol per animal, n = 3 for each group). At 24 h post nanoparticle or saline injection, a tumor on one side was subjected to laser treatment at a dose of 100 J ⋅ cm-2, while the corresponding contralateral tumor served as a ‘dark’ control. After the laser treatment, animals were kept alive for 24 h to allow PDT effects to develop. Tumors treated with the combination of P(B)-HDL nanoparticles and laser exhibited swelling, indicative of common inflammatory postPDT effects, while tumors treated with either laser only or B(P)-HDL in combination with the laser did not exhibit any signs of inflammation. Animals were then humanely sacrificed, tumors were excised, formalin-fixed and subjected to H&E and Ki-67 staining. As displayed in Figure 7, only the tumors treated with the combination of P(B)-HDL nanoparticles and laser were observed to exhibit decreased Ki-67 staining as well as tissue structure disruption in H&E stained sections. Quantification of Ki-67 staining revealed significantly lower cell viability in tumors treated with P(B)-HDL and laser, while the rest of the treatment groups (Laser only, P(B)-HDL only, B(P)-HDL only and B(P)-HDL + Laser) demonstrated no difference in tumor


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cell viability compared to the control (Figure S9). Finally, no acute damage to major organs 24 h post PDT treatment administration was observed as confirmed by H&E staining (Figure S10).

Figure 7. Pyropheophorbide photodynamic activity modulation via lipid and oleylamine conjugation 24 h post nanoparticle administration. (A) H&E and Ki-67 staining of “dark control” KB tumors from mice injected with PBS, B(P)-HDL and P(B)-HDL; (B) H&E and Ki-67 staining of KB tumors from mice injected with PBS, B(P)-HDL and P(B)-HDL and treated with a 750 nm laser at a light dose of 100 J⋅cm-2. Scale bar = 50 m.

While our group has extensively investigated the incorporation of a single porphyrin conjugate into a lipoprotein-like structure, this current work provides a framework for the rational combination of porphyrin conjugates with varying optical and structural properties. Specifically,


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fluorescence, photoacoustic and PDT data in this study demonstrate several trends in the HDLlike nanoparticle properties that are highly dependent on the porphyrin-conjugate structure. First, porphyrin-lipid conjugation results in effective porphyrin incorporation into the lipid shell of the lipoprotein-like particle. These porphyrin-lipid conjugates are easily dissociated upon cellular uptake, which enables effective fluorescence imaging (B(P)-HDL) and photodynamic therapy (P(B)-HDL). On the other hand, the conjugation of a hydrophobic oleylamide moiety to the porphyrin structure results in highly stable J-aggregation and fluorescence quenching in both B(P)-HDL and P(B)-HDL. B(P)-HDL nanoparticles can be applied as a dual “always-on” NIR fluorescent and photoacoustic agent enabled by BChl-lipid fluorescence and Pyro-oleylamide Jaggregation respectively. Notably, despite highly established photosensitizing properties of pyropheophorbide, its oleylamide conjugate was found to be ineffective for PDT due its lack of dissociation upon cellular uptake. P(B)-HDL nanoparticles serve as an example of a rationallydesigned phototheranostic agent, where the unique properties of each porphyrin are enhanced by the select conjugate. While Pyro-lipid-containing HDL-like nanoparticles have been successfully applied for PDT in a variety of animal models,22 introduction of the NIR-absorbing BChloleylamide to the nanoparticle structure enables non-invasive tumor detection with photoacoustic imaging, providing a compelling theranostic scenario.


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CONCLUSIONS In the current study, we extensively investigated how two porphyrin conjugation strategies affect the resulting nanostructures’ phototheranostic properties. We demonstrated that hydrophobic porphyrin-oleylamide conjugates are instrumental for photoacoustic imaging due to their efficient fluorescence quenching and stable J-aggregation. Amphiphilic porphyrin-lipid conjugates, on the other hand, are more prone to disruption upon cellular uptake, enabling effective fluorescence imaging and potent photodynamic activity. Overall, we demonstrated how the rational combination of porphyrins with complementary photophysical properties as well as the selection of the appropriate conjugate structures can be applied for the design of tunable phototheranostic agents.


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MATERIALS AND METHODS Synthesis and characterization of porphyrin derivatives. Pyro-lipid, BChl-lipid and Pyrooleylamide were synthesized following protocols reported previously.34 BChl-oleylamide was synthesized using a similar protocol as described for Pyro-oleylamide previously.25 Briefly, 50 nmol of bacteriochlorophyll acid (prepared as described by Kozyrev and coauthors34) in anhydrous N,N-diméthylformamide (DMF, 5 mL) were added to N,N,N′,N′-Tetramethyl-O-(1Hbenzotriazol-1-yl)uronium hexafluorophosphate, (HBTU, 2.1 mol equiv.), oleylamine (1 mol equiv.) and N, N-diisopropylethylamine (DIPEA, 3% (v/v)). After stirring at room temperature under argon for 1 h, the reaction mixture was concentrated under reduced pressure. The crude product was purified by preparative thin layer chromatography to gain pure BChl-oleylamide verified by uPLC-MS assay with a purity >95% and confirmed by ESI-MS: [M]+: calculated 859.6, found 860.0. The chemical structure and uPLC-MS characterization of Pyro-lipid, BChllipid, Pyro-oleylamide, and Bchl-oleylamide are provided in the Supporting Information (Figure S1 and S2). Nanoparticle preparation. B(P)-HDL, P(B)-HDL and control nanoparticles (P-HDL, B-HDL, (P)-HDL and (B)-HDL) were prepared by a film hydration method as previously reported.22 Briefly, lipid films were generated by combining 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, Avanti Polar Lipids), cholesteryl oleate (CO, Sigma Aldrich) and different combinations of porphyrin derivatives (Pyro-oleylamide, Pyro-lipid, BChl-lipid and BChl-oleylamide) in 0.3 mL of chloroform (HPLC grade, Fisher) within a glass vial, in ratios summarized in Table S1. The chloroform was dried under a gentle stream of nitrogen gas for >1 h to generate a lipid film. The films were rehydrated with 1 mL of phosphate buffered saline (PBS, 10 mM phosphate, pH 7.4) and water bath-sonicated for 40 minutes until the solution turned clear. The solution was


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transferred to 15 mL centrifuge tubes and sonicated using a Bioruptor at low frequency (30 kHz) for 30 cycles (30 s on/30 s off) at 40 °C. The resulting solution was transferred to a 1.5 mL Eppendorf tube and 2 mg of R4F peptide dissolved in 0.5 mL of PBS (4 mg⋅ml-1) was added in a drop-wise manner to the lipid suspension. The resulting particle solution was allowed to equilibrate overnight at 4°C. On the following day, the solution was centrifuged at 12, 000 rpm for 20 min at 4°C to remove any unincorporated dye and filtered through a 0.1 μm syringe filter (Millex®, Sigma-Aldrich).

Nanoparticle characterization. Size and morphology of the freshly prepared nanoparticles were evaluated with transmission electron microscopy using a FEI Tecnai 20 electron microscope (Nanoscale Biomedical Imaging facility, Peter Gilgan Centre for Research and Learning, Toronto). Carbon grids were charged for 30 s, nanoparticles were adsorbed to the grid, and samples were imaged with 2% uranyl acetate negative stain. Average particle sizes were obtained from the measurement of a minimum of 400 particles over six representative fields of view using ImageJ.

Absorption and fluorescence of intact (in PBS) and disrupted (in PBS containing 5% of TritonX100) nanoparticles was collected on a UV-Vis spectrophotometer Cary 50 (Agilent, Mississauga, ON) and Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon, NJ). Fluorescence spectra of intact and disrupted nanoparticle were collected (Pyro excitation: 630 nm; emission: 650-800 nm; BChl excitation: 730 nm; emission: 750-850 nm, slit width 5 nm) and the quenching efficiencies were calculated using the following equation, where Fintact and Fdisrupted represent the integration of fluorescence of the intact and disrupted sample, normalized by the sample optical density at the excitation wavelength.


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% Quenching efficiency = (1 −

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𝐹 𝑖𝑛𝑡𝑎𝑐𝑡 𝐹 𝑑𝑖𝑠𝑟𝑢𝑝𝑡𝑒𝑑)

x 100

B(P)-HDL and P(B)-HDL stability at 37C was evaluated in PBS, 10% and 50% fetal bovine serum (FBS; WISENT Inc.) using a CLARIOstar microplate reader (BMG LABTECH). Changes in ratios of the monomer and J-aggregation peaks of Pyro-oleylamide (B(P)-HDL) and BChloleylamide (P(B)-HDL) were expressed as a ratio of the absorbance at the corresponding monomer and aggregation wavelengths (Table S2).

Photoacoustic spectral measurements and fluorescence imaging in solution. For photoacoustic spectral measurements and fluorescence imaging in solution, intact B(P)-HDL and P(B)-HDL particles were prepared in PBS at concentrations of 25, 50, and 100 μM (based on the concentration of the oleylamide-porphyrin conjugate). Samples were injected into polyethylene tubes (0.381 mm inner dimeter, 1.092 mm outer diameter; BD INTRAMEDIC, VWR) that were immobilized in a custom-built plastic holder (FUJIFILM VisualSonics). The phantoms were imaged first with a CRi Maestro imaging system (Caliper Life Sciences, Waltham, MA). Pyro signal was collected using the red filter set (excitation: 616–661 nm bandpass; emission: 675 nm longpass) with a manually set 700 nm emission cut-off to prevent BChl signal interference. For BChl fluorescence detection, the NIR filter set was used (excitation: 684–729 nm bandpass; emission: 745 nm longpass). The exposure time was set to 250 ms. Following fluorescence imaging, the tubes were submerged in still water for optical coupling. 2D photoacoustic spectral images were collected using the Vevo 2100 LAZR system (Visualsonics, Fuji-Film, ON) equipped with a 21 MHz transducer (LZ250) and the following settings: focal depth, 10 mm; 2D gain, 45 dB; time gain compensation, constant


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with depth; persistence, off; spectral range, 680–900 nm; data interval, 1 nm. Area-averaged PA signal inside the polyethylene tube cross-section was plotted against the laser wavelength and sample concentration. Disrupted nanoparticles (10% Triton X-100 in PBS) were simultaneously imaged at the highest concentration of 100 μM.

Fluorescence microscopy and cell uptake studies. Chinese hamster ovary (CHO) ldl(mSR-BI) and ldlA- 7 cells were gifts from Dr. Monty Krieger (Massachusetts Institute of Technology, Cambridge, MA). Human KB, HT1080 and PC-3M-luc-C6 cells were purchased from ATCC. ldlA-A7 cells were cultured in Hams F-12 medium (Gibco) supplemented with penicillin– streptomycin (1 v/v%), FBS (5 v/v%) and L-glutamine (2 mM). ldl(mSR-BI) cells were cultured under similar conditions as ldlA-7 with the addition of 300 ug/ml of G418 Geneticin. PC-3M-lucC6, KB and HT1080 were cultured in EMEM medium (Gibco) supplemented with FBS (10 v/v%) and penicillin–streptomycin (1 v/v%). All cell cultures were maintained in a 37°C humidified incubator under 5% CO2.

For fluorescence microscopy experiments, ldl(mSR-BI) and ldlA-7 cells were seeded into 8-well coverglass-bottom chambers (Nunc Lab-Tek, Sigma–Aldrich, Rochester, NY) at a cell-seeding density of 2 × 104 cells per well. After 24 h of incubation, medium was replaced with serum-free medium containing B(P)-HDL or P(B)-HDL at a concentration of 5 μM based on the oleylamideconjugated porphyrin and incubated for 3, 6 and 24 h. 20 min prior to imaging, cells were washed twice with sterile PBS (Gibco) and incubated with the serum-free media containing 1 μg⋅mL-1of the nuclear stain Hoechst 33342 (ThermoFisher). Fluorescence imaging was performed on an Olympus IX73 inverted microscope using a 60× magnification objective. Nuclear stain was


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detected using a DAPI filter (excitation: 387∕11 nm bandpass; emission 447∕60 nm), Pyro was excited at 628/40 nm (Emission: 692/40 nm) and BChl was excited at 708/75 (Emission: 788/20 nm). Exposure time was kept consistent between the time points; image background was removed using ImageJ software. Mean fluorescence intensity of Pyro and BChl from at least 3 fields of view was background-corrected and normalized to mean fluorescence intensity in ldl(mSR-BI) cells after 24 h of incubation with the corresponding nanoparticle.

Relative Pyro-lipid and Pyro-oleylamide uptake efficiency in ldl(mSR-BI) and ldlA-7 cells was studied by measuring cell-associated Pyro fluorescence after incubation with B(P)-HDL and P(B)HDL. Cells were seeded into 96-well plates (COSTAR) at a density of 1.5 × 104 cells per well. After 24 h, cell media was replaced with F-12 media containing B(P)-HDL or P(B)-HDL at 5 μM Pyro concentration and incubated for another 24 h. Cells were washed 3 times with sterile PBS and cell-associated Pyro fluorescence was measured with a CLARIOstar microplate plate reader (BMG LABTECH) (excitation: 410/8 nm, emission: 671/8 nm, gain = 2500) using well-scan mode, where fluorescence from each well was collected from 30 separate points. After these measurements, cells were lysed with 5% TritonX-100 and fluorescence of the Pyro-lipid and Pyrooleylamide monomers was measured using the same imaging protocol.

In vitro PDT. In vitro PDT experiments were conducted using ldl(mSR-BI) and ldlA-7 cells. Cells were seeded at a density of 1.5 × 104 cells per well into black 96-well plates (COSTAR). The treatment and control groups were as follows: (1) no laser and no particle control, (2) nanoparticle treatment alone (B(P)-HDL or P(B)-HDL at 1 μM Pyro concentration), (3) laser treatment (2.5 J cm-2) alone, (4) Nanoparticles + laser treatment at 0.5, 1 and 2.5 J cm-2. 24 h after the cell seeding,


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medium was replaced with treatment medium consisting of B(P)-HDL or P(B)-HDL at 1 μM Pyro in F-12 medium (with L-glutamine; Gibco). After a 24 h incubation period, cells were washed with PBS three times and wells were replenished with fresh medium. PDT was performed at 671 nm using a free-space laser (LaserGlow Technologies), with a 25 mW power output, and 0.7 cm diameter spot size. Light doses of 0.5, 1 and 2.5 J cm-2 were administered through laser irradiation for 10, 20 and 50 s respectively. Following laser treatment, cells were incubated for an additional 24 h, after which medium was replaced with that containing 0.5 mg mL alamarBlue® (Invitrogen). Cells were incubated for 3 h, after which fluorescence emission was collected using a CLARIOstar microplate reader (BMG LABTECH) (excitation: 540/8 nm and emission: 590/8 nm). Percent cell viability for each experiment was determined by normalizing the averaged blank-corrected absorbance values of 4 replicate treatment wells against blank-corrected absorbance values of wells administered medium alone.

Xenograft mouse models. All animal studies were approved and conducted in compliance with the University Health Network Animal Resources Centre guidelines.

For the generation of subcutaneous tumor xenografts, athymic male nude mice under general anesthesia (2 vol% isoflurane in oxygen) were inoculated with 2.5 × 106 ldl(mSR-BI) or ldlA-7 cells in a 1:1 mixture of sterile saline and Matrigel (total volume = 60 μL) into the right or left flank. Tumor growth was monitored using electronic calipers and animals were administered nanoparticles once the tumors reached 0.7 – 1 cm in the largest dimension). An orthotopic prostate tumor model was generated as described elsewhere.28 Briefly, 2.5 × 106 PC-3M-luc-C6 (Caliper®) cells in 20 μL of saline were injected to the dorsal prostate lobe using a 28-gauge needle is aseptic


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conditions, animals were sutured back and administered 0.1 mg ⋅ kg-1 of bupenorphine in saline solution (0.1 ml) for analgesia. Orthotopic prostate tumor growth was monitored by ultrasound imaging (Vevo 2200 photoacoustic scanner (Visualsonics, Fuji-Film, ON).

In vivo fluorescence imaging. For the SR-BI targeting studies in vivo, animals bearing ldlA-7 (left flank) and ldl(mSR-BI) (right flank) subcutaneous tumors (n = 3) were injected intravenously with 50 nmol (based on the oleylamine-conjugated porphyrin) of B(P)-HDL or P(B)-HDL in 0.2 mL of saline and imaged at 3, 6 and 24 h post injection with a CRi Maestro imaging system (Caliper Life Sciences, Waltham, MA). For the detection of Pyro fluorescence, the red filter set was used (excitation: 635 nm (616 to 661 nm); emission: 675-nm longpass) with a manually set 700 nm emission cut-off to prevent BChl signal interference. For BChl fluorescence detection the NIR filter set was used (excitation: 704 nm (BP 684 – 729 nm); emission: 745 nm – LP). After 24 h, animals were humanely sacrificed by cervical dislocation under surgical plane isofluorane anesthesia. Major organs (including heart, spleen, lungs, liver, kidneys, adrenal, stomach, small intestine, large intestine, skin, and muscle) were excised, rinsed in saline and imaged with a CRi Maestro fluorescence imaging system under the settings described above. Integration time for all hyperspectral fluorescence images was 1000 ms.

Photoacoustic imaging. Photoacoustic imaging was performed in athymic nude mice bearing orthotopic PC-3M-luc-C6 prostate tumors (n = 3). Mice were injected via the tail vein with 230 nmol of P(B)-HDL (based on the amount of BChl-oleylamide) in 0.2 mL of sterile PBS. During imaging, animals were anesthetized with 2% v/v isoflurane in oxygen. Ultrasound gel was applied to the lower abdominal area and imaging was performed using the Vevo 2100 LAZR system


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(Visualsonics, Fuji-Film, ON) equipped with a 21 MHz transducer (LZ250) and the following settings: focal depth, 10 mm; 2D gain, 45 dB; time-gated-control, constant with depth; persistence, 4. 2D spectral (680–900 nm) and 3D scans of the tumor area were captured at predetermined intervals. Spectral imaging cubes were processed in MATLAB using in-house scripts. First, the PA values were smoothed using a 3D Gaussian filter with a standard deviation of 1. Next, the smoothed images were spectrally unmixed by non-negative linear least squares to identify oxyhemoglobin, deoxyhemoglobin, and P(B)-HDL signals. The absorption spectra of oxyhemoglobin and deoxyhemoglobin from https://omlc.org/spectra/hemoglobin/ were used for unmixing. The PA spectrum of P(B)-HDL was measured in a tubing phantom and the peak at 824 nm was fit to a Gaussian curve and used as the P(B)-HDL signal for unmixing. The unmixed P(B)HDL PA signals were overlaid on the simultaneously acquired ultrasound B-mode images. 3D data was reconstructed using the Vevo instrument software. The PA signal intensity at 875 nm was taken as endogenous background as it is relatively invariant to oxygenation status and was subtracted from the P(B)-HDL signal at 844 nm.

In vivo PDT. Mice bearing dual subcutaneous KB tumors were randomly divided into three groups: 1) saline, 2) B(P)-HDL, 3) P(B)-HDL (n = 3). Animals in group 2 and group 3 were intravenously injected with 100 nmol (based on Pyro-oleylamide or Pyro-lipid amount) of B(P)HDL and P(B)-HDL respectively. B(P)-HDL and P(B)-HDL uptake was monitored by in vivo fluorescence imaging at 24 h post injection, followed by tumor laser treatment. Tumors on the right flank were treated with light, while tumors on the left flank were shielded from light and served as a dark control. A single PDT light dose of 100 J⋅cm-2 (0.63 cm2 laser spot area; fluence rate 50 mW⋅cm-2) was delivered by a 671 nm laser (DPSS LaserGlow Technologies). At 24 h after


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the PDT treatment, animals were humanely sacrificed by cervical dislocation under surgical plane anesthesia. Tumors and organs (including heart, spleen, lungs, liver, kidneys, adrenal, stomach, small intestine, large intestine, skin, and muscle) were collected and fixed in 10% buffered formalin for histological analysis. Organs from three additional healthy mice were used as a no laser, no nanoparticle control. Organs and tumors were paraffin-embedded and sliced into 5 μm sections and stained with H&E. Tumors were additionally stained with Ki-67 proliferation marker to assess PDT treatment efficacy. Quantification of Ki-67-positive areas in tumors from the control, laser only, B(P)-HDL, B(P)-HDL + laser, P(B)-HDL and P(B)-HDL + laser groups 24 hours post PDT treatment was performed in ImageJ.

Statistical analysis. Independent samples t-test was used to determine statistical significance between means of two groups (equal variances not assumed). P-values less than 0.05 were considered significant.

ASSOCIATED CONTENT The Supporting Information contains information on: B(P)-HDL, P(B)-HDL and control nanoparticles composition; their absorption spectra; fluorescence quenching; chemical structures of Pyro-lipid, BChl-lipid, Pyro-oleylamide and BChl-oleylamide; chemical characterization of these porphyrin conjugates; optical stability of B(P)-HDL and P(B)-HDL; Pyro-oleylamide and Pyro-lipid cell uptake in SR-BI-positive and SR-BI-negative cells; quantification of Ki-67 staining in tumors 24 h post PDT and representative H&E-stained sections of major mouse organs 24 hours post PDT treatment.


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AUTHOR INFORMATION M.O. and M.Z. performed nanoparticle synthesis, cell culture, animal model induction, fluorescence microscopy, in vitro and in vivo PDT. M.A.R. performed TEM imaging on the nanoparticles and TEM image analysis. M.A.R. and M.O. performed the cell uptake study. D.M.C. performed photoacoustic imaging of phantoms and animals and analysis of photoacoustic data. M.O. wrote the manuscript and prepared figures. J.C. synthesized porphyrin conjugates used in this study. J.C., M.A.R. and D.M.C. contributed to manuscript writing and editing. G.Z., J.C. and M.O. conceived the original idea and performed experimental planning. The authors declare no competing financial interests.

ACKNOWLEDGMENTS The authors would like to acknowledge Lili Ding, Dr. Wenlei Jiang, Deborah Scollard, Teesha Komal and Dr. Warren Folz for their technical assistance as well as Dr. Kara Harmatys and Adam Koebel for valuable feedback regarding manuscript preparation. This work was funded by the Natural Sciences and Engineering Research Council of Canada (#386613), the Terry Fox Research Institute (PPG#1075), the Canadian Institute of Health Research (Foundation Grant #154326), Vanier Canada Graduate Scholarship, the Canada Foundation for Innovation (#21765), and the Princess Margaret Cancer Foundation.


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hv

Photoacoustic imaging

NIR fluorescence

1O 2

1O 2

ROS generation

O2


O2

ROS generation quenched

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