Hybrid Liquid Crystal Nanocarriers for Enhanced Zinc Phthalocyanine

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Hybrid Liquid Crystal Nanocarriers for Enhanced Zinc Phthalocyanine-Mediated Photodynamic Therapy Okhil Kumar Nag, Jawad Naciri, Jeffrey S. Erickson, Eunkeu Oh, and James B. Delehanty Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00374 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 12, 2018

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Bioconjugate Chemistry

Hybrid Liquid Crystal Nanocarriers for Enhanced Zinc PhthalocyanineMediated Photodynamic Therapy Okhil K. Nag1, Jawad Naciri1, Jeffrey S. Erickson1, Eunkeu Oh2,3, and James B. Delehanty1*

1

Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Code 6900,

4555 Overlook Ave. SW, Washington, DC, 20375, United States 2

Optical Sciences Division, Naval Research Laboratory, Code 5600, 4555 Overlook Ave. SW,

Washington, DC, 20375, United States 3

KeyW Corporation, Hanover, MD 21076 United States

*

Address correspondence to [email protected].

1 ACS Paragon Plus Environment

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ABSTRACT Current challenges in photodynamic therapy (PDT) include both the targeted delivery of the photosensitizer (PS) to the desired cellular location and the maintenance of PS efficacy. Zinc phthalocyanine (ZnPc), a macrocyclic porphyrin and a potent PS for PDT, undergoes photoexcitation to generate reactive singlet oxygen that kills cells efficiently, particularly when delivered to the plasma membrane. Like other commonly-employed PS, ZnPc is highly hydrophobic and prone to self-aggregation in aqueous biological media. Further, it lacks innate subcellular targeting specificity. Cumulatively, these attributes pose significant challenges for its delivery via traditional systemic drug delivery modalities. Here, we report the development and characterization of a liquid crystal nanoparticle (LCNP)-based formulation for the encapsulation and targeted tethering of ZnPc to the plasma membrane bilayer. ZnPc was co-loaded with the organic fluorophore, perylene (PY), in the hydrophobic polymeric matrix of the LCNP core. PY facilitated the fluorescence-based tracking of the LCNP carrier while also serving as a Förster resonance energy transfer (FRET) donor to the ZnPc acceptor. This configuration availed efficient singlet oxygen generation via enhanced excitation of ZnPc from multiple surrounding PY energy donors. When excited in a FRET configuration, cuvette-based assays revealed that singlet oxygen generation from the ZnPc was ~1.8-fold greater and kinetically 12 times faster compared to when the ZnPc was excited directly. The specific tethering of the LCNPs to the plasma membrane of HEK 293T/17 and HeLa cells was achieved by surface functionalization of the NPs with PEGylated cholesterol. In HeLa cells, LCNPs co-loaded with PY and ZnPc, when photoexcited in a FRET configuration, mediated 70% greater cell killing compared to LCNPs containing ZnPc alone (direct excitation of ZnPc). This was attributed to a significant increase of the oxidative stress in the cells during the PDT. Overall, this work details the ability of the LCNP


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platform to facilitate (1) the specific tethering of the PY-ZnPc FRET pair to the plasma membrane and (2) the FRET-mediated, augmented singlet oxygen generation for enhanced PDT relative to the direct excitation of ZnPc alone. INTRODUCTION Photodynamic therapy (PDT) is a clinically-approved method for tumor ablation and is one of the more promising emerging modalities for the treatment of certain cancers (e.g., esophageal and non-small cell lung cancers). PDT systems are composed of two elements: (1) a PDT drug (also known as a photosensitizer (PS)) and (2) incident excitation light for irradiating the PS.1, 2 During treatment, the photoexcited PS reacts with oxygen in the tissue environment and produces reactive oxygen species (ROS), such as singlet oxygen (1O2), that kill cells in the vicinity of the PS.2 Most PS molecules (e.g., phorphyrin, chlorin, prophycene) are macrocyclic compounds which suffer from poor water solubility and a tendency to aggregate via π-π stacking in polar, biological media.2,

3

This reduces their effective solution concentrations, thus

diminishing the efficacy of the PS. These drawbacks severely limit the use of PS in traditional systemic drug delivery or topical applications. Zinc (II) phthalocyanine (ZnPc) generates reactive singlet oxygen upon photoexcitation4 and is a promising PS due to its low toxicity in its non-excited state, yet it suffers from the aforementioned limitations of other PS materials. Further, in the absence of a targeting moiety, ZnPc lacks cellular/subcellular organelle specificity when administered to the body.5 Previous approaches to improve the clinical implementation of ZnPc have included its structural modification to improve water solubility and to decrease its propensity for self-aggregation.3,6,7 Although chemical modifications (including the addition of charged, water-soluble substituents) render the ZnPc more stable in aqueous media, delivery to cells still necessitates the use of 3 ACS Paragon Plus Environment


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surfactants or organic solvents, which can be toxic.3,8,9 Further, the introduction of charged substituents can reduce the ability of ZnPc to interface with cellular structures such as the plasma membrane.7,9 Hence, there remains a critical need for new methods to improve the solubility and specific cellular delivery of ZnPc. Nanoparticle (NP)-mediated drug delivery seeks to harness the unique attributes of NPs to enhance or augment the efficacy of on-board drug cargos. Multiple NP formulations (including liposomes, polymers, silica matrices, and inorganic NPs) have been investigated for the targeted delivery of PS. Indeed, multiple studies have shown that targeting of PS to the plasma membrane, mitochondrion, or nucleus increases PS therapeutic efficacy while minimizing toxicity to healthy cells.10-12 This is largely attributed to localized singlet oxygen generation in the immediate vicinity of biological membranes coupled with the peroxidation of unsaturated phospholipids.12,13 Studies have been aimed at improving ZnPc loading, solubility, enhanced permeability and retention in tissue, targeted delivery and controlled release.2,

14-16

However,

reports of the use of NP formulations of ZnPc that can be specifically tethered to the plasma membrane are limited.10 For example, Kim et al. reported improved PDT efficacy of ZnPcloaded fusogenic liposomes that delivered ZnPc to the plasma membrane, yet these materials can alter cellular homeostasis upon fusion with the plasma membrane.10 Ideally, NP-ZnPc formulations could be designed to facilitate excitation of the ZnPc moiety via Förster resonance energy transfer (FRET) which offers the advantages of efficient excitation, minimal photobeaching, and better depth of penetration when longer wavelength light can be used. NP carriers have been designed to mediate FRET excitation of ZnPc using lanthanide upconverting nanoparticles (UCNPs) coupled with near-infrared (NIR) stimulation for better depth of tissue penetration.2,17-20 These approaches have encountered limitations, however, arising from


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complicated synthesis/purification schemes, steric stability of inorganic UCNPs, leakage of the ZnPc, and high toxicity associated with the UCNPs themselves.2 To overcome the technical challenges facing the design and implementation of NP-based ZnPc systems, herein we have prepared a liquid crystal NP (LCNP)-based nanocarrier bioconjugate to augment the PS properties of ZnPc. The salient features of the system include the co-encapsulation of ZnPC with multiple copies of the FRET donor, perylene (PY), within the LCNP core, and the targeted tethering of the ensemble NP to the plasma membrane. In our previous work, we demonstrated the utility of the LCNP platform for the controlled modulation of on-board drug and dye cargos with bright fluorescence tracking. For example, PY-loaded LCNPs were easily trackable in cells due to the high fluorescence quantum yield of PY (~1.0). When decorated with transferrin and loaded with the chemotherapeutic doxorubicin, the LCNP system showed a ~40-fold improvement in the IC50 over free DOX in human embryonic kidney cells.21 More recently we showed the specific membrane-targeted delivery of the potentiometric dye 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO).21,22 Along with the enhanced membrane persistence and residence time, the delivery of DiO as a LCNP-cholesterol bioconjugate resulted in a ~40% attenuation of the cytotoxicity of free DiO delivered from bulk solution. Using these latter studies as motivation, here we have prepared a LCNP system whose inner hydrophobic core is loaded with multiple cargoes during synthesis. Specifically, the core of the LCNP is co-loaded with PY and ZnPc (PY-ZnPc-LCNP). The surface of the LCNP is further functionalized with a PEGylated cholesterol moiety that facilitates the targeted tethering of PYZnPc-LCNP to the plasma membrane bilayer of living cells. The purpose of co-loading the PY and ZnPc into the LCNP core is multifold: 1) to prevent the self-aggregation of ZnPc inside the matrix of LCNP core, 2) to track/image the particles in cells using fluorescence-based imaging,



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3) to optically excite the ZnPc via FRET by using the photoexcited PY as the energy donor, and 4) to ultimately facilitate two-photon (TP) excitation of the ZnPc which takes advantage of the large two-photon absorbance of PY.23,24 We show that the plasma membrane targeted tethering of PY-ZnPc-LCNP and efficient generation of singlet oxygen via FRET excitation significantly improves cell killing ability. In FRET-induced PDT, PY-ZnPc-LCNP facilitates 70% greater killing of HeLa cells compared to when LCNPs loaded with ZnPc alone were used in direct excitation mode. Cumulatively, this report demonstrates the highly multifunctional nature of the PY-ZnPc-LCNP composite wherein multiple hydrophobic cargos are encapsulated in the LCNP carrier and the ensemble facilitates the targeted tethering of PY-ZnPc-LCNP to the plasma membrane for augmented PDT applications.

RESULTS AND DISCUSSION Rationale for the design of hybrid LCNPs for PDT. Our goal was to design and implement a NP-based system that could simultaneously improve ZnPc colloidal stability, localize the ZnPc to the desired site of action, and augment ZnPc-driven 1O2 generation through photophysical processes (e.g., FRET). One critical design criterion was to keep the ZnPc (a Type II PS)2 localized inside the NP during PDT, where the generated 1O2 could diffuse to the immediate vicinity of the targeted cells and tissues. This simultaneously minimizes the inherent nonphotoexcited (‘dark’) toxicity while enabling excitation of the ZnPc non-radiatively via FRET when paired with an appropriate dye donor. This configuration can result in ZnPc-mediated 1O2 generation with minimal photobeaching.25,26 Further, since photogenerated 1O2 has a very short life-time ( 1×10-7 s) and a very limited diffusion distance (~30 nm) in biological environments,27


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the localization of the ZnPc-NP construct within a few nanometers of targeted organelles (e.g., plasma membrane) is desired. To pursue our goal, we synthesized a hybrid LCNP-based delivery system for the encapsulation and targeted tethering of ZnPc to the plasma membrane bilayer of living cells. The formulation comprises LCNPs that are co-loaded with PY and ZnPc in their hydrophobic core; the NP core is suitably nonpolar and serves as a host for both species. Scheme 1 shows the proposed working principle for PDT generation from the PY-ZnPc-loaded LCNP system. The LC agent used herein forms a covalently crosslinked polymeric matrix that structures the PY and ZnPc (present at a molar ratio of ~6:1 PY:ZnPc) in close proximity to each other to facilitate light harvesting via enhanced FRET. The energy transfer from PY enables ZnPc to be excited efficiently at a higher electronic level and generate more 1O2 compared to when ZnPc is excited directly (vide infra). Additionally, the PY dye allows for visualization/tracking of the LCNPs. The LCNP surface is further functionalized with PEGylated cholesterol (PEG2000-Chol) for appending the LCNP to the exofacial leaflet of the plasma membrane to place the ZnPc in close proximity to phospholipids of the membrane bilayer. This close membrane tethering of the LCNPs enables efficient diffusion of generated 1O2 to improve cell killing via irreversible peroxidation of membrane lipids.28,29



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Scheme 1. Schematic of the perylene (PY)- and zinc phthalocyanine (ZnPc)-loaded hybrid liquid crystal nanoparticle (LCNP) tethered to the plasma membrane by PEGylated cholesterol (PEG2000-Chol). Excitation of ZnPc via FRET from the PY donor generates reactive singlet oxygen (1O2) resulting in lipid peroxidation, the undermining of plasma membrane integrity, and cell death.

Synthesis and characterization of hybrid LCNPs. LCNPs loaded with PY, ZnPc or both were synthesized following the procedure described in refs.21, 22 The carboxylate moiety on the LCNP surface provides stability in aqueous media and presents a functional handle for the attachment of cell-targeting ligands. Figure 1 shows schematically the components and the as-synthesized LCNPs used in this study. Upon synthesis of the LCNPs, free carboxylate groups on their surface 8 ACS Paragon Plus Environment

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were functionalized with NH2-PEG2000-Chol via EDC coupling (Figure 1G) as described elsewhere.22 Full physicochemical characterization of the LCNPs are shown in SI Figures S1 and S2. As anticipated, negatively charged, unconjugated LCNPs showed strong mobility toward the cathode.

However, after functionalization with NH2-PEG2000-Chol the LCNP-PEG-Chol

particles exhibited minimal migration toward the positive electrode, confirming the reduction in negative surface charge due to amide bond formation (SI Figure S1A). Dynamic light scattering (DLS) analysis further confirmed a significant increase in the hydrodynamic size of the LCNPs (SI Figure S1B) and zeta potential measurements showed a reduction in overall negative surface charge as a result of PEG2000-Chol conjugation (SI Figure S1C). Cumulatively, these data provided strong evidence of the successful conjugation of PEG2000-Chol to the NP surface. This conjugation procedure resulted in LCNPs that maintained sufficient negative surface charge (-20 to -40 mV) to ensure colloidal stability in solution. Nanoparticle conjugates showed excellent stability for up to six weeks when stored at 4 °C (data not shown). Finally, transmission electron microscopy (TEM) of the PEG2000-Chol conjugated LCNPs confirmed the spherical shape of the LCNPs and an average diameter of less than 100 nm (SI Figure S2).



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Steady state

and

time-resolved

Figure 1. Schematic of the perylene (PY), zinc phthalocyanine (ZnPc), or hybrid (PYZnPc) loaded liquid crystal nanoparticles (LCNPs). (A) Chemical structures of the compounds utilized to prepare the LCNPs: an acrylate liquid crystal cross-linking agent (DACTP11, top row) and a carboxyl-terminated polymerizable surfactant (AC10COONa, bottom row). Chemical structures of the (B) PY chromophore and (C) ZnPc PS drug. (D-F) Schematic representation and photographs of various LCNP suspensions: (D) PY-LCNP, (E) ZnPc-LCNP, and (F) PY-ZnPc-LCNP. (G) EDC coupling of NH2-PEG2000-Chol to the carboxylate groups on the surface of the various LCNPs mediates targeting to the plasma membrane. spectroscopic properties of the LCNPs. UV-vis spectra of the LCNPs showed absorption peak maxima (λabs) at ~530 nm for PY (PY-LCNPs), ~600 nm for ZnPc (ZnPc-LCNPs), and ~615 nm for ZnPc in and PY-ZnPc-LCNPs (Figure 2 A,B). The λabs for PY in LCNP is close to that observed for PY dissolved in organic solvents (chloroform:methanol, 3:1 v:v). However, we noted a significant change in the absorption spectra of ZnPc when it was incorporated into the LCNPs compared to its absorption in chloroform:methanol (Figure 2B and SI Figure S3A). These spectral properties strongly suggested the encapsulation of PY and ZnPc in the hydrophobic core of the LCNP. We confirmed this by UV-vis spectroscopy after digestion of the LCNPs with mixed organic solvents (chloroform:methanol (3:1 v:v)) where the digested LCNPs showed characteristic absorbance maxima of ~528 nm and ~672 nm for PY and ZnPc,


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respectively (SI Figure S3B). Clearly, PY and ZnPc maintained their chemical integrity and optical properties during synthesis of the LCNPs. From the UV-vis spectra, the PY:ZnPc ratio in the core of the LCNPs was calculated to be ~6:1 (SI Figure S4). Steady state fluorescence measurements were then performed on the LCNPs to determine if PY and ZnPc functioned as a FRET donor/acceptor pair (Figure 2C). The fluorescence spectra were measured by exciting the PY-ZnPc-LCNPs using two different wavelengths: 600 nm (for direct excitation of ZnPc) and 532 nm (for direct excitation of PY). As observed in the spectra, the excitation of ZnPc in the FRET configuration provided more than 45 and 60 times greater fluorescence emission intensity compared to the direct excitation of ZnPc in the PY-ZnPc-LCNP and ZnPc-LCNP formats, respectively. Concurrently, the emission intensity of PY in PY-ZnPc-LCNPs appeared to be much lower in the context of PY-LCNPs. Steady state emission spectra of PY-LCNP and PYZnPC-LCNP were further analyzed by spectral deconvolution to calculate the FRET (FRETE) using the equation FRETE =1-FLDA/ FLD, where FLDA and FLD are the fluorescence emission of the donor (PY) in the presence of acceptor (ZnPc) and the donor alone, respectively. The steady state FRET efficiency was calculated to be 68%. In addition, the relative quantum yield (φ) of PY measured relative to a fluorescein standard (φ, 0.92 at pH ~11) decreased from 0.98 to 0.15 when co-loaded with ZnPc in LCNP. These results provided strong evidence that PY and ZnPc engaged in FRET in the context of LCNP. We additionally calculated FRETE using timecorrelated single photon counting (TCSPC) fluorescence lifetime measurements (Figure 2E) using the equation FRETE =1-τDA/ τD, where DA and D are the lifetimes of the donor (PY) in the presence of acceptor (ZnPc) and the donor alone, respectively.30,31 The reduced fluorescence lifetime of the PY donor in the presence of the ZnPc acceptor confirmed that the PY-ZnPc pair engaged in FRET (Table S1). In the presence of ZnPc, the average fluorescence lifetime of PY



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decreased form 4.1 ns to 1.3 ns, corresponding to a FRETE of ~68%, which matched that determined from the steady state data. Given the R0 value for the PY-ZnPc pair of 81.6 Å, the center-to-center PY-ZnPc distance (r) in the LCNP was calculated to be 53.4 Å. These data provide strong evidence that the incorporation of PY in the ZnPc-LCNP not only facilitated FRET excitation of ZnPc but also significantly amplified ZnPc emission. Similar observations of amplification of acceptor emission have been reported in other FRET pair systems where a molar excess of donor over acceptor coupled with homo-FRET processes contribute to the acceptor emission.32-35 In the PY-ZnPc-LCNP system, it is likely that both of these phenomena are occurring as there is significant spectral overlap between PY absorption and emission (JPYPY=3.08×10

-14

mol-1cm-3), and PY emission and ZnPc absorption (JPY-ZnPc =1.60×10-12 mol-1cm-3)

(SI Figure S5A and B). The energy transfer process between the PY donor and ZnPc acceptor is further supported by electronic energy level analysis (SI Figure S5C). 36-38


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ROS

generation efficiency of PY-ZnPCLCNPs.

We

first

characterized

the

ROS

generating ability of the LCNP system using a in

cuvette-based assay and the commercial ROS probe, Cell ROX®. As show Figure 3A, upon irradiation for 12 min the fluorescence emission intensity of the ROS probe increased

~1,400% Figure 2. Steady state and time resolved spectral properties of the PEG-Chol conjugated LCNPs. (A) Normalized absorption spectra of LCNPs in 0.1x PBS (pH 7.4). (B) Inset view of select region of panel A showing spectral detail in the region of absorbance of 0 – 0.1 a.u. (B) Fluorescence spectra of the LCNPs excited at close to the absorbance maxima of PY and ZnPc (~532 nm and ~600 nm, respectively). Samples were prepared by diluting stock solutions of LCNPs into 0.1x PBS to a final concentration of PY and ZnPc of ~24 uM and ~4.0 µM, respectively, across the different types of LCNP. (D) Inset view of select region of panel C showing spectral detail in the region of fluorescence intensity of 0 – 20000 a.u. (E) Fluorescence decay profile of PY in absence and presence of ZnPc in PY-LCNP and PY-ZnPc-LCNP, respectively.

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when PY-ZnPc-LCNPs were excited at 532 nm to drive FRET excitation of the ZnPc from the PY donor. Comparatively, the fluorescence intensity of the probe increased only ~500%, ~650% and ~800% when ZnPc was excited directly at 638 nm in the context of ZnPc-LCNPs, free ZnPc (in DMSO), and PY-ZnPc-LCNPs, respectively (SI Figure S6 for spectral data). This corresponds to a ~1.8 to 2.8-fold increase in the efficiency of ROS generation when ZnPc is excited in the FRET configuration. Control experiments revealed negligible increase of emission from the ROS probe upon irradiation of LCNPs lacking PY or ZnPc (Figure 3A and SI Figure S6). Further, kinetic analysis revealed that the rate of ROS generation in the FRET excited PYZnPc-LCNP construct was 12 times faster than when ZnPc was excited directly (Figure 3B). This result is likely attributable to the efficient excitation of ZnPc via enhanced FRET from multiple surrounding PY moieties in the LCNP core. Similar results of enhanced ROS generation via FRET-mediated excitation (e.g., in light harvesting π-conjugated polymer systems and chromophore-to-PS FRET systems) have been reported elsewhere.39,40 However, considering the size of the LCNPs (radius ~50 nm) and diffusion distance (~30 nm) of singlet oxygen, it is obvious that not all the singlet oxygen generated within the particle may diffuse out and contribute to the PDT. Nonetheless, singlet oxygen generated in the periphery of the LCNP core should efficiently diffuse from the LCNP and contribute to PDT. Such is the case in other NPencapsulated PDT systems where, for example, UCNPs of diameter 50-100 nm were able to mediate efficient diffusion of ROS from the NP core.2


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Importantly,

we

observed

minimal leakage of the PY or ZnPc

from the

LCNP core. Figure 3C shows the

results

of

samples to

dialysis

at 37°C.

fluorescence

analysis

after

of PY-ZnPc-LCNP were subjected (against 20% BSA in 1x PBS) for 24 h The emission intensity for both PY and ZnPc showed

negligible change

(

Hybrid Liquid Crystal Nanocarriers for Enhanced Zinc Phthalocyanine

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