Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Rational Design of Photosynthesis-Inspired Nanomedicines Published as part of the Accounts of Chemical Research special issue “Nanomedicine and Beyond”. Kara M. Harmatys,§ Marta Overchuk,†,§ and Gang Zheng*,†,‡,§ †
Acc. Chem. Res. Downloaded from pubs.acs.org by UNIV PARIS-SUD on 04/26/19. For personal use only.
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada ‡ Department of Medical Biophysics, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada § Princess Margaret Cancer Centre, University Health Network, 101 College Street, Toronto, Ontario M5G 1L7, Canada CONSPECTUS: The sun is the most abundant source of energy on earth. Phototrophs have discovered clever strategies to harvest this light energy and convert it to chemical energy for biomass production. This is achieved in light-harvesting complexes, or antennas, that funnel the exciton energy into the reaction centers. Antennas contain an array of chlorophylls, linear tetrapyrroles, and carotenoid pigments spatially controlled by neighboring proteins. This fine-tuned regulation of protein-pigment arrangements is crucial for survival in the conditions of both excess and extreme light deficit. Photomedicine and photodiagnosis have long been utilizing naturally derived and synthetic monomer dyes for imaging, photodynamic and photothermal therapy; however, the precise regulation of damage inflicted by these therapies requires more complex architectures. In this Account, we discuss how two mechanisms found in photosynthetic systems, photoprotection and light harvesting, have inspired scientists to create nanomedicines for more effective and precise phototherapies. Researchers have been recapitulating natural photoprotection mechanisms by utilizing carotenoids and other quencher molecules toward the design of photodynamic molecular beacons (PDT beacons) for disease-specific photoactivation. We highlight the seminal studies describing peptidelinked porphyrin-carotenoid PDT beacons, which are locally activated by a disease-specific enzyme. Examples of more advanced constructs include tumor-specific mRNA-activatable and polyionic cell-penetrating PDT beacons. An alternative approach toward harnessing photosynthetic processes for biomedical applications includes the design of various nanostructures. This Account will primarily focus on organic lipid-based micro- and nanoparticles. The phenomenon of nonphotochemical quenching, or excess energy release in the form of heat, has been widely explored in the context of porphyrin-containing nanomedicines. These quenched nanostructures can be implemented toward photoacoustic imaging and photothermal therapy. Upon nanostructure disruption, as a result of tissue accumulation and subsequent cell uptake, activatable fluorescence imaging and photodynamic therapy can be achieved. Alternatively, processes found in nature for light harvesting under dim conditions, such as in the deep sea, can be harnessed to maximize light absorption within the tissue. Specifically, high-ordered dye aggregation that results in a bathochromic shift and increased absorption has been exploited for the collection of more light with longer wavelengths, characterized by maximum tissue penetration. Overall, the profound understanding of photosynthetic systems combined with rapid development of nanotechnology has yielded a unique field of nature-inspired photomedicine, which holds promise toward more precise and effective phototherapies.
1. INTRODUCTION The sun is the most abundant source of energy on earth and is crucial for bioenergy production. Phototrophs have discovered ways to harvest this light energy and convert it to chemical energy via a process of photoinduced charge separation. Complex arrays of chlorophyll dyes, carotenoid pigments, and matrix proteins are involved in the transport of exciton energy that funnels down to a reaction center (RC) on a 10−100 ps time scale.1 The reaction centers are surrounded by lightharvesting complexes (LHCs) that contain a high concentration of chromophores. The function of LHCs, or antenna complexes, is to maximize the absorption cross section (∼100 fold) of the © XXXX American Chemical Society
RC for efficient capture of photons. The chromophores involved in photosynthetic light-harvesting include chlorophylls, linear tetrapyrroles, and carotenoids, which all have high molar extinction coefficients ∼100 000 M−1 cm−1.2 Surprisingly, nature has a limited synthetic versatility of available chromophores. It has designed clever strategies for “choosing” specific pigments and tailoring the pigment−protein spatial arrangements depending on the need for a broad spectral cross section to collect more light or the need to avoid dye quenching Received: February 28, 2019
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DOI: 10.1021/acs.accounts.9b00104 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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extremely low. They can achieve this by engaging pigments with different wavelengths to cover a wider range of light in the absorbance spectrum, broadening the absorption bands and/or tuning the wavelength by modulating the pigment−protein or pigment−pigment supramolecular interactions. For example, LHCII in purple bacteria tunes its protein scaffold to control the organization of bacteriochlorophyll a dyes, resulting in an absorption spectral range from 800 to 850 nm.2 Another interesting light-harvesting mechanism developed by green sulfur bacteria relies on pigment−pigment intermolecular interactions for efficient exciton energy transfer. Green sulfur bacteria contain highly efficient light-harvesting organelles called chlorosomes that allow them to thrive in extremely lowlight conditions at a depth up to 150 m underwater (Figure 2A).6
during light harvesting. The mechanisms of light harvesting under low-light conditions and photoprotection against oxidative damage under excess light conditions in photosynthetic systems have gained attraction for biomimetic reconstruction for the use in solar cells and biosupramolecular electronics.3 We will discuss these regulation mechanisms further in the context of phototheranostic agent design for biomedical applications (Figure 1).
Figure 1. Photosynthesis-inspired design of phototheranostic agents. (A) Nature provides mechanisms of photoprotection in excess light conditions and light harvesting in dim light conditions. (B) Application of photosynthesis-inspired photophysical processes for theranostic agent design. Left panel: Activatable photodynamic therapy beacons and nanoparticles. Right panel: J-aggregation-induced wavelength modulation for effective light harvesting.
Figure 2. Natural ordered dye self-assemblies as an inspiration to overcome light-tissue penetration. (A) Schematic representation of chlorosome light-harvesting structures found in green sulfur bacteria that inhabit deep sea waters. Ordered aggregation of bacteriochlorophyll c results in exciton coupling and a bathochromic absorbance shift. (B) Propagation of light with different wavelengths through tissue. Major endogenous absorbers prevent shorter wavelengths of light from penetrating into deeper tissue layers, leaving a 700−900 nm window where the intrinsic absorbance is minimal for maximum light penetration.
Protection Mechanisms under Excess Sunlight Conditions
Safety mechanisms are implemented in antenna systems to reduce photodamage and the associated oxidative stress under excess sunlight conditions. LHCs contribute to the regulation of energy flow to the RCs, which is essential for photosynthetic organisms that are exposed to varying light intensities. Carotenoids (CARs) are photosynthetic pigments involved in light harvesting and protection against photo-oxidative damage. They absorb energy in the blue spectral region and can transfer that energy to (bacterio)chlorophylls for efficient light harvesting.4 In addition, they participate in the protection against oxidative damage by quenching the excited states of chlorophylls. Another way light-harvesting organisms protect themselves against damaging reactive oxygen species (ROS) is by dissipating the excess excitation energy in the antenna into heat. This process of nonphotochemical quenching (NPQ) releases heat before it can initiate harmful chemical species from the photosynthetic RCs.5 Generally, this quenching relies on rapid changes in pigment−pigment interactions, which are achieved by tightly regulated protein conformational changes. NPQ can be triggered by low pH conditions causing a charge gradient across the photosynthetic membrane that diverts the excitation energy into heat before it can be transferred to the RCs. CARs are also involved in NPQ; if a triplet state is generated, it is promptly quenched by CARs that safely dissipate the thermal energy to the surrounding environment.
Chlorosomes are ellipsoid structures around 100 nm in length containing >250 000 bacteriochlorophyll (BChl) c/d/e molecules that self-organize via noncovalent interactions without protein assistance in a hydrophobic environment encased by a lipid monolayer. Dye organization is constructed by metal coordination between a magnesium atom and 31-O, hydrogen bonding between the 31-OH with 13-CO, and π−π stacking. This results in unique supramolecular structures with large exciton interactions indicative of a broadening and red-shifting of the Qy absorption into the near-infrared (NIR) spectral range. The extended coverage of the light spectrum in combination with the fast excitation energy transfer leads to efficient charge separation. The observed absorption band broadening is attributed to a mixture of BChl c/d/e homologues, which is ideal for light harvesting in comparison to narrowed absorption bands observed in synthetic J-aggregates. These natural aggregates then absorb the light and transfer the excitation energy down into a protein-BChl a membrane baseplate complex, which then funnels down to the Fenna−Mathews− Olson protein complex and ends in the photosynthetic RCs. Overall, the exceptional light-harvesting ability and the lack of a complex protein scaffold are major reasons why chlorosomes are a great design inspiration for artificial light-harvesting systems. Kundu and Patra recently outlined the strategies of light-
Harvesting Mechanisms under Diminished Sunlight Conditions
Light-harvesting antenna systems found in phototrophs have adapted clever strategies to collect more available light necessary to survive in environments where the photon counts are B
DOI: 10.1021/acs.accounts.9b00104 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 3. Activatable PDT beacons. (A) Protease beacons are photoactivated upon cleavage of a disease-specific linker that separates the photosensitizer-quencher (PS-Q) pair. (B) An intact porphyrin-carotenoid PS-Q construct shows diminished 1O2 luminescence and restoration of photoactivity upon caspase-3 linker cleavage. Adapted with permission from ref 10. Copyright 2004 American Chemical Society. (C) Nucleic acid PDT beacons are activated upon the linker binding to the target mRNA sequence. (D) A linear superquencher PDT beacon shows higher activation with each additional quencher upon target nucleic acid hybridization. Adapted with permission from ref 19. Copyright 2010 American Chemical Society. (E) Cleavage of the zipper PDT beacon results in the release of a cell-penetrating peptide, which facilitates intracellular delivery of a PS. (F) Confocal images indicate greater cell uptake and cleavage of the zipper PDT beacon (right image) in comparison to the control beacon (left image). Adapted with permission from ref 18. Copyright 2009 American Chemical Society. (G) Adsorption of a chlorin e6 PS-nucleic acid conjugate to the single-walled carbon nanostructure results in fluorescence quenching and (H) thrombin protein target binding to the nucleic acid aptamer results in significant fluorescence enhancement. Adapted with permission from ref 21. Copyright 2008 American Chemical Society.
harvesting systems based on inorganic and organic nano-
these unique photophysical properties and tailoring them to
particles, but this Account will focus primarily on exploiting
specific nanomedicine and biomedical applications.7 C
DOI: 10.1021/acs.accounts.9b00104 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Our group applied nature’s strategy of controlled 1O2 scavenging toward the design of activatable PDT probes. Initial studies from our lab describe covalently linked porphyrin− carotenoid constructs as photodynamic therapy molecular beacons, termed PDT beacons.10 PDT beacons consist of a photosensitizer (PS), a disease-specific linker, and a 1O2 quencher/scavenger (Q). The close proximity between the PS and Q prohibits 1O2 generation or fluorescence until the linker is cleaved by a disease-specific enzyme. This general construct is illustrated in Figure 3A. As a first-generation design, pyropheophorbide a (pyro) was chosen as the PS, a carotenoid was selected as the Q moiety, and a short caspase-3-cleavable peptide sequence was the designated linker. When the pyropeptide-CAR beacon is intact, the CAR is in close proximity to the PS to efficiently decrease 1O2 generation and lifetime (Figure 3B). Caspase-induced cleavage separates the Q and PS, which allows photoactivation of the PS. In exploration of an alternative activation strategy, we generated a new class of nucleic acid-triggered PDT beacons containing a single-stranded oligonucleotide linker that forms a hairpin structure in which the sequence is complementary to a target mRNA (Figure 3C).11 The hairpin conformation brings the PS and Q close together to maximize fluorescence and 1O2 quenching. A similar strategy was implemented by Cló and coworkers for the design of a PS with DNA sequence-controlled on-and-off switching of 1O2 generation.12 Natural CARs are suitable quenchers in PDT beacon constructs because of their efficient light-harvesting and photoprotective role in directly scavenging 1O2. There is a very high level of photoprotection in nontargeted cells, however, the beacon loses some PDT potency toward targeted cells since the cleaved CAR moiety retains its 1 O2 scavenging activity. Further consideration into quenchers beyond the photosynthetic CAR is necessary for future design of effective PDT beacons.
Photoprotection and Light Harvesting in Nanomedicine
Naturally derived chromophores have been extensively applied toward photomedicine and photodiagnosis. Specifically, porphyrin dyes in combination with light are widely used for cancer treatment with photodynamic therapy (PDT).8 Upon photon absorption, a photosensitizer is promoted into a short-lived excited singlet state, which leads to fluorescence emission of a photon or internal conversion as heat generation. However, if a photosensitizer molecule undergoes a transition to its excited triplet state through intersystem crossing, it can either transfer the energy directly to an organic molecule within the cell (Type I process) or interact with molecular oxygen (Type II photodynamic process), generating singlet oxygen. Both mechanisms result in localized cell death and tissue destruction. Unlike during photosynthesis, generation of ROS at the site of disease is a desired outcome of PDT. Early photosensitizers, while effective for PDT, lack tissue specificity, which often results in severe and prolonged light toxicity. To address this problem and design disease-specific phototheranostic agents, scientists mimicked nature’s way of controlling ROS generation by introduction of a nearby quencher moiety or aggregation-induced self-quenching of the photosensitizer. In the first strategy, a photosensitizer molecule is placed in close proximity to a quencher molecule via a linker, which can then be cleaved or unfolded upon interaction with a disease-specific stimulus. The second strategy involves photosensitizer incorporation into a nanostructure to achieve structure-dependent photoactivation. These two strategies, which are inspired by nature’s mechanisms of photoprotection in light-harvesting organisms, are discussed in a greater detail in sections 2 and 3 of this Account, respectively. Photodiagnosis uses contrast agents in combination with light to visualize various structures and pathologies within the body. Effective light collection in conditions of limited light penetration and the design of systems that can sense external environmental stimuli are two major challenges in photodiagnostic agent design. Nanostructures that recapitulate dye assemblies found in natural light-harvesting systems, such as chlorosomes, can achieve light absorbance into the desired NIR spectral region (Figure 2A). The “optical window” between 700 and 900 nm is where light absorption and scattering from endogenous absorbers is minimized in biological tissues (Figure 2B). Furthermore, these nanostructure-specific optical properties, such as fluorescence quenching or J-aggregation, can be useful for activatable fluorescence or ratiometric imaging. The biomedical utility of nanostructures that mimic high-ordered dye assemblies in natural light-harvesting systems is described in section 4.
Beyond Carotenoid Quenchers
In our search toward PDT beacon designs with maximum quenching efficiency, a small beacon library was created in which pyro was conjugated to a variety of Förster resonance energy transfer (FRET) quenchers.13 Quenchers with spectral overlap greater or equal to J = 5.1 × 1013 M−1 cm−1 nm4 were the most efficient, reducing up to 90% of the pyro fluorescence and 1O2 production. Due to the high FRET efficiency and 1O2 quenching between pyro and black hole quencher 3 (BHQ3), pyro-BHQ3 PDT beacon constructs were further investigated in cancerassociated linkers, such as matrix metalloproteinases (MMPs). MMP7-triggered pyro-beacon photoactivation was validated in solution, cancer cells and multiple tumor mouse models overexpressing MMP7.14−16 Although promising, some challenges concerning conventional protease-cleavable PDT beacons have arisen: (1) quenching is dependent upon natural linker peptide folding that places Q in close proximity to the PS, (2) activation of most protease-probes occurs extracellularly, which could induce offtarget toxicities, and (3) the probes rely on passive and nonspecific delivery to target cells. A novel strategy from the Tsien lab can overcome these delivery challenges using activatable cell-penetrating peptides (CPPs).17 A polyargininebased domain is fused to a negatively charged inhibitory domain between a protease-cleavable linker. Once separated, the polycationic CPP and its attached cargo can penetrate the cell membrane. We applied this concept toward PDT beacons that contain a polycation/polyanion electrostatic “zipper” hairpin-
2. PHOTOSYNTHESIS-INSPIRED MOLECULAR BEACONS Porphyrin−Carotenoid Conjugates
Carotenoids have unique photophysical properties that protect photosynthetic organisms from light-induced damage. They quench the chlorophyll triplet excited state at a rate much faster than singlet oxygen (1O2) sensitization and they can also directly scavenge the 1O2.4 In order to investigate the structural requirements needed for carotenoid photoprotection, covalently linked carotenoid−porphyrin dyads were synthesized.9 This photoprotective mechanism has inspired scientists to utilize the carotenoid−porphyrin interaction for activatable fluorescence and photodynamic agent design. D
DOI: 10.1021/acs.accounts.9b00104 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Figure 4. Porphyrin-containing photoactivatable nanostructures, such as (A) liposomes, (B) poly(vinyl alcohol) polymer nanoparticles, and (C) lipoproteins. These structures reveal aggregation-induced self-quenching, resulting in the loss of fluorescence signal and heat dissipation that can be utilized for PA imaging, activatable fluorescence imaging, and phototherapies. (D) Application of porphyrin-lipid-containing liposomal structure for in vivo PA and activatable fluorescence imaging. Adapted with permission from ref 26. Copyright 2011 Springer Nature Publishing. (E) Application of PVA-porphyrin-containing nanostructure for in vivo photothermal therapy, fluorescence imaging, and drug release. Adapted with permission from ref 32. Copyright 2017 Ivyspring International Publisher. (F) Application of porphyrin-lipid-containing lipoprotein mimetic for its structurally driven selfquenching and activatable fluorescence and singlet oxygen generation. Adapted with permission from ref 35. Copyright 2015 American Chemical Society.
serve as a scaffold for dye assembly while simultaneously solubilizing and stabilizing these hydrophobic assemblies in an aqueous solution. These porphyrin nanoassemblies can undergo similar photophysical processes observed in photosynthetic systems, including self-quenching, resonance energy transfer, and exciton coupling.22 Furthermore, organic nanostructures can serve as a biocompatible delivery vehicle for these photosynthesis-inspired dye assemblies for biomedical applications. Depending on the specific dye−dye intermolecular interactions, these nanostructures can be applied toward various phototheranostic modalities, such as structure-dependent and/ or ratiometric fluorescence imaging, photodynamic, and photothermal therapy.23 In this section, we will describe how nanostructures mimic natural supramolecular dye assemblies that have phototheranostic capabilities.
linked PS-Q pair that dissociate upon specific cleavage (Figure 3E).18 This universal next-generation PDT beacon concept enhances delivery by facilitating cell entry of the unquenched polycation-PS (Figure 3F). Additionally, a more advanced nucleic acid PDT beacon was designed that comprises the first linear superquencher architecture.19 Sequential BlackBerry quencher phosphoramidites were added at the 5′ terminus. The triple quencher had nearly 90-fold higher fluorescence activation in comparison to the single and double quencher constructs at 30- and 40-fold activation, respectively, and was comparable to the 100-fold activation observed using gold as a molecular beacon quencher (Figure 3D).20 Finally, the Tan lab explored single-walled carbon nanotubes (SWNTs) as efficient quenchers for PDT beacon designs. They comprise a chlorin e6 (Ce6) photosensitizer, a SWNT quencher, and a ssDNA aptamer linker, as illustrated in Figure 3G.21 SWNTs are a suitable quencher because they can also protect the adhered DNA probes from nuclease digestion. Up to 98% quenching of Ce6 was observed with a 20-fold fluorescence enhancement by specific thrombin protein binding (Figure 3H). This novel approach can regulate 1O2 generation with a variety of targets and does not rely on a hairpin structure of peptide selffolding. In summary, PDT beacons can recapitulate the photoprotective properties observed within photosynthetic membranes. Moving forward, self-quenching between chlorophyll photosensitizers within larger constructs, such as nanoparticles, is a promising approach toward photoprotection and activation. This will be further discussed in the next section.
Nanostructure-Induced Activation
Aggregation-induced quenching is one way to achieve structuredependent photophysical properties. Encapsulation of relatively high amounts of dye into a nanoparticle often leads to proximityinduced interactions (π−π stacking or hydrophobic interactions), which result in ground-state coupling and the formation of nonfluorescent complexes.24 This process termed nonphotochemical quenching is utilized by natural photosynthetic systems for photoprotection through dissipation of excess energy as heat. Variations of highly quenched porphyrin-based agents have been designed for photothermal therapy and/or photoacoustic (PA) imaging that effectively convert highintensity light energy into heat for localized tissue ablation or acoustic wave generation.25 Furthermore, since fluorescence can be easily restored upon nanostructure dissociation, selfquenching has been widely used for the design of the intensometric activatable agents.23 A prime example of a structurally driven self-quenching nanoparticle is the porphysome, a liposome-like nanovesicle that consists of an amphiphilic porphyrin-lipid conjugate self-
3. PHOTOSYNTHESIS-INSPIRED PORPHYRIN NANOSTRUCTURE ASSEMBLY While molecular beacons provide an opportunity to explore dye−dye interactions with high precision, they do not recapitulate the complex molecular assemblies present in natural photosynthetic systems. On the contrary, a nanostructure can E
DOI: 10.1021/acs.accounts.9b00104 Acc. Chem. Res. XXXX, XXX, XXX−XXX
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Accounts of Chemical Research assembled in the bilayer structure (Figure 4A).26 Exceptionally high porphyrin content (>80 000 molecules per particle) and strong π−π stacking of the porphyrin rings results in >99.9% quenching of fluorescence and singlet oxygen generation (SOG), which is restored upon nanostructure disruption. Such high fluorescence and SOG quenching results in effective heat generation upon laser irradiation, which combined with high porphysome stability and favorable pharmacokinetics, enables photothermal tumor ablation (Figure 4D).26,27 Furthermore, the inherent metal chelating properties of pyropheophorbide a enables porphysome transformation into a multimodal positron emission tomography (PET) agent for simultaneous lesion detection and fluorescence-guided surgical resection.28 Our group has extensively explored porphysome applications for PET, fluorescence, and photothermal therapy in a variety of murine29 and rabbit30 models. Additionally, Zou and coauthors explored porphyrin−peptide conjugates that were assembled into highly stable nanodots that exhibit fluorescence quenching and highly effective light-to-heat energy conversion for in vivo PA imaging and photothermal generation.31 Luo and co-workers developed (poly(vinyl alcohol)-porphyrin polymeric nanoparticles capable of optical/PET imaging, photodynamic/photothermal therapy, and drug delivery (Figure 4B).32 The PVA-porphyrin conjugate building block allows for self-assembly in the presence of drugs or imaging agents in a single nanoplatform through a cost-effective “onepot” fabrication process. Similar to porphysomes, they exhibit unique architecture-dependent fluorescence self-quenching and photoproperties. Specifically, they demonstrated effective photothermal tumor ablation that was further enhanced by doxorubicin codelivery. Upon nanostructure disruption, they showed activatable fluorescence imaging and photodynamic therapy (Figure 4E). Finally, more complex multilevel porphyrin nanoarchitectures have been developed for tumor microenvironment-responsive combination chemo-phototherapy.33 Specifically, pyro was conjugated to doxorubicin via a pH-sensitive hydrazine bond. These monomers assembled into small micelle-like structures, which in the presence of PEG2000, cross-linked to form 80 nm aggregates. These larger aggregates remained highly stable after systemic injection, however upon the exposure to a lower pH within the tumor microenvironment, these nanoassemblies dissociated and released free doxorubicin and pyro, resulting in fluorescence activation.
uptake is more suited for activatable photodynamic rather than photothermal therapy (Figure 4F). The presence of a stabilizing synthetic mimetic peptide enabled long plasma circulation time (∼10 h) and smaller size (
