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Multi-Color Liposome Mixtures for Selective and Selectable Cargo Release Upendra Chitgupi, Shuai Shao, Kevin Carter, Wei-Chiao Huang, and Jonathan F Lovell Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b05025 • Publication Date (Web): 31 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018
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Nano Letters
Multi-Color Liposome Mixtures for Selective and Selectable Cargo Release Upendra Chitgupi, Shuai Shao, Kevin A. Carter, Wei-Chiao Huang, Jonathan F. Lovell* Department of Biomedical Engineering, University at Buffalo, State University of New York, Buffalo, NY 14260, USA
ABSTRACT: Many approaches exist for stimuli-triggered cargo release from nanocarriers, but few can provide for on-demand release of multiple payloads, selectively. Here, we report the synthesis of purpurin-phospholipid (Pur-P), a lipid chromophore that has near infrared absorbance red-shifted by 30 nm compared to a structurally similar pyropheophorbide-phospholipid (Pyr-P). Liposomes containing small amounts of either Pur-P or Pyr-P exhibited similar physical properties and fluorescence self-quenching. Loaded with distinct cargos, Pur-P and Pyr-P liposomes were mixed into a single colloidal suspension and selectively released cargo depending on irradiation wavelength. Spatiotemporal control of distinct cargo release was achieved by controlling multicolor laser placement. Using basic orange and doxorubicin anthraquinones, multidimensional cytotoxicity gradients were established to gauge efficacy against cancer cells using light-released drug. Wavelength selectivity of cargo release was maintained following intramuscular administration to mice. KEYWORDS: Liposomes, nanocarriers, stimuli-responsive, controlled release, photoactivatable, porphyrin INTRODUCTION Triggered release of cargo from carriers is an area of research interest.1-5 Liposomes are a prototypical nanoscale carrier.6, 7 Intrinsic mechanisms of triggering cargo release include enzyme activation8, and pH9. Thermal triggered cargo release from liposomes has been developed for decades and advanced to clinical studies.10-12 Light activation has also been explored for releasing cargos.13-24 We have shown that liposomes incorporating small amounts of porphyrin-phospholipid (PoP) release cargo in response to near infrared light.25-27 Most PoP-related studies have made use of pyropheophorbide-a phosphatidylcholine, although other PoPs have been explored, including metal-chelated versions of this28-30 and related metallochlorins31, 32, bacteriochlorophylls33, 34 and texaphyrin35 conjugates. These have modulated light absorption, enabling wavelength-selective PoP excitation. In this work, multi-color PoP liposome mixtures are developed and used for selective and selectable light-activated cargo release. RESULTS AND DISCUSSION Multi-color liposomes Purpurin-phospholipid (Pur-P) was synthesized with the same route as pyropheophorbide-phospholipid (Pyr-P), but using purpurin-18 as a starting material (Figure S1). Figure 1A shows the structure of both PoPs; Pyr-P and Pur-P. Unless indicated otherwise, liposomes were formed with cholesterol (CHOL), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylethanolamine-polyethylene glycol (2 kDa) (PEG-lipid) and PoP with a molar ratio [CHOL:DSPC:DOPC:PEG-lipid:PoP] of [50:33:10:5:2]. DOPC was included since prior studies have found that unsaturated lipids increase the rate of PoP-mediated, light-triggered cargo release.36
Figure 1. Multicolor PoP liposomes. A) Structure of PoPs used in this study. B) Absorbance of liposomes containing 2 mol. % PoP in PBS. Laser irradiation wavelengths used in this study are shown with arrows C) Hydrodynamic size of Pyr-P and Pur-P liposomes D) Polydispersity index of Pyr-P and Pur-P liposomes E) Brightness of liposomes containing varying amounts of PoPs. The aqueous absorption of liposomes containing 2 mol. % PoP is shown in Figure 1B. Pyr-P liposomes have an absorption peak at 665 nm, whereas Pur-P liposomes have a red-shifted peak near 700 nm, likely owing to delocalization of π electrons in the additional carbonyl group in the Pur-P exocyclic ring. This difference in absorption enables selective Pyr-P liposome excitation with a 665 nm laser and selective Pur-P liposomes excitation with a 690 nm laser. Pyr-P liposomes were 120 nm in diameter while Pur-P liposomes were slightly larger with a diameter of 160 nm (Figure 1C). The polydispersity index of Pyr-P liposomes was slightly higher than Pur-P liposomes, with a value of 0.16, compared to 0.06 (Figure 1D). To characterize photophysical properties, liposomes were formed with increasing amounts of Pyr-P or Pur-P. The emission of the PoPs is shown in Figure S2. At higher PoP concentrations, both Pyr-P and Pur-P liposomes exhibited fluorescence self-quenching (Figure S3). Both types of liposomes containing 2 mol. % PoP had the highest fluorescence and were used for further study. Previously, 2% Pyr-P was found to be optimal for triggered release.37 Fluorescence self-quenching correlates with singlet oxygen self-quenching38 and the release mechanism is related to photooxidation.36 When PoPs were loaded at selfquenching concentrations (10 mol. %), fluorescence selfquenching was maintained in serum, unlike the free porphyrins, indicating stability and lack of exchange with serum factors (Figure S4).
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liposomes were mixed, and then added to every single well in 384-well plate (Figure 3A). Next, only selected parts of the plate was irradiated, first with a 665 nm laser to trigger release from Pyr-P liposomes (Figure 3B). SRB release from Pyr-P liposomes was readily observed only in the laser-exposed portions of the plate. In those same wells, minimal release of BO from Pur-P liposomes occurred. Next, a 690 nm laser was used to release BO from Pur-P liposomes in an adjacent portion of the well. The laser irradiation area was selected in such a way that there was an overlap in the center of the well plate. Figure 3C shows the overlayed image, demonstrating that the areas in which both 690 and 665 laser was irradiated released both SRB and BO (shown in yellow). To ensure specificity of cargo release was related to the multicolor activation of the liposomes rather than the cargo properties, the liposomes were loaded with the opposite cargos and irradiation was repeated (Figure 3D). Release occurred selectively based on the wavelength of irradiation and the liposome type with good spatial control.
Figure 2. Selective, selectable light triggered cargo release of Doxorubicin (Dox) or Basic Orange (BO) from PoP liposomes. Indicated liposomes mixtures were subjected to laser Real-time release of liposomal cargo triggered with a A) 665 nm laser or B) 690 nm laser. C,D) Light-triggered release of indicated cargos from liposomes with indicated irradiation. Selective cargo release To assess selective release from multi-color liposomes, Pyr-P and Pur-P liposomes were loaded with doxorubicin (Dox) and basic orange (BO) respectively, via ammonium sulfate gradient. These two types of liposomes were then combined and irradiated with a 665 nm laser at 125 mW/cm2, to selectively excite Pyr-P liposomes. As shown in Figure 2A, in these conditions, only Dox was released from Pyr-P liposomes, with irradiation with minimal release of BO from Pur-P liposomes. In contrast, when a 690 nm laser was used to selectively excite Pur-P liposomes, only BO release was induced (Figure 2B). It was observed that the Dox release rates were slower than the BO release, a phenomenon which has been reported previously,39 suggesting the difference in release rates have to do with the cargo leakage rates, rather than differences in PoP-mediated lightinduced membrane permeabilization. Figure 2C and Table S1 summarizes those results, demonstrating that by selecting the appropriate wavelength, cargo can be selectively released. In a control experiment, the liposomes were used to load the opposite cargos of the preceding experiments. As shown in Figure 2D, selectivity was still maintained when cargo loading was reversed. In general, less selectivity was observed with 665 nm excitation, likely owing to the partial excitation of the Pur-P liposomes at that same wavelength. Light-triggered release responses that are nonlinear with fluence have been observed with actively-loaded cargo in PoP liposomes.36 Selective, spatial control of release To demonstrate selective, selectable release with spatial control, sulforhodamine B (SRB), a hydrophilic fluorophore was passively loaded into Pyr-P liposomes. SRB was chosen since it is a bright fluorophore and does not have spectral overlap with other cargos used. BO was also used due to its brightness. Pyr-P and Pur-P
Figure 3. Selective and selectable spatial control of release. A) Fluorophore-loaded Pyr-P and Pur-P liposome were mixed and disturbed to each well of a 384 well microplate. B) Sequential irradiation of the microplate with the indicated pattern of two different laser irradiation wavelengths. C) Fluorescence image of a 384 well microplate with mixed liposomes in every well, selectively irradiated as indicated. D) Similar experiment as in C, but cargo was loaded in the opposite types of PoP liposomes. Released SRB is shown in red, BO in blue and the overlapping signal in yellow. Multidimensional gradients of released cargos We next sought to use light to establish gradients of released cargo. Time-based release studies were carried out with SRB and BO loaded Pyr-P and Pur-P liposomes, respectively. Samples were subjected to 665 nm and 690 nm laser irradiation both individual-
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Nano Letters
ly and sequentially as shown in Figure 4A. With increasing 665 nm irradiation, SRB could be increasingly released from the Pur-P liposomes while with increasing 690 nm irradiation triggered increasing release of BO from Pur-P liposomes. When the liposomes were increasingly irradiated with both 665 nm and 690 nm, both SRB and BO were released in increasing amounts. Thus, these results show that selectable gradients of cargo can be released selectively with light. Figure S5 shows that light gradients can be used to create continuous released cargo gradients. Next, multi-dimensional cargo release gradients were used to examine the chemotherapeutic drug toxicity. The cytotoxic anthraquinones Dox and BO were loaded into Pyr-P and Pur-P liposomes, respectively. Without laser irradiation, minimal drug release was observed during a 3 hour period (Figure S6). Following incubation in serum, light-irradiation induced cargo release, indicating both liposomes and cargo were intact and that the PoP was still embedded in the bilayer (Figure S7). Liposomes were incubated with human glioblastoma cells, irradiated immediately. When empty PoP liposomes were used, laser irradiation did not decrease cell viability (Figure 4B). However, the drug-loaded liposomes inhibited cell growth in an increasing fashion when exposed to light (Figure 4C). This is due to increasing amounts of drug being released from the liposomes and being made more available for cell uptake. In this manner, gradients could be useful for assessing synergies between multiple drugs using free drug concentrations established by light-triggered release.
Figure 4. Multidimensional gradients of released cargos. A) Selective and increasing release of SRB and/or BO based on irradiation time. B) Cell viability of U87 cells incubated with empty PyrP or Pur-P liposomes irradiated as indicated. C) Cell viability of U87 cells treated with BO-loaded Pyr-P and Dox-loaded Pur-P liposomes treated with indicated laser. One asterisks denotes statistically significant with P
