Letter Cite This: Nano Lett. XXXX, XXX, XXX-XXX
pubs.acs.org/NanoLett
Enhanced Triggering of Local Anesthetic Particles by Photosensitization and Photothermal Effect Using a Common Wavelength Alina Y. Rwei,†,‡ Bruce Y. Wang,†,‡ Tianjiao Ji,† Changyou Zhan,† and Daniel S. Kohane*,† †
Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States S Supporting Information *
ABSTRACT: On-demand pain relief systems would be very helpful additions to the armamentarium of pain management. Near-infrared triggered drug delivery systems have demonstrated the potential to provide such care. However, challenges remain in making such systems as stimulus-sensitive as possible, to enhance depth of tissue penetration, repeatability of triggering, and safety. Here we developed liposomes containing the local anesthetic tetrodotoxin and also containing a photosensitizer and gold nanorods that were excitable at the same near-infrared wavelength. The combination of triggering mechanisms enhanced the photosensitivity and repeatability of the system in vitro when compared with liposomes with a single photoresponsive component. In vivo, on-demand local anesthesia could be induced with a low irradiance and short irradiation duration, and liposomes containing both photosensitizer and gold nanorods were more effective than those containing just one photoresponsive component. Tissue reaction was benign. KEYWORDS: Sodium channel blocker, on-demand, phototriggerable, photosensitive, plasmonic nanoparticles, surface plasmon resonance, photochemical
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liposomes comprising unsaturated lipids, which contained a photosensitizer in addition to a local anesthetic.5,8 Upon irradiation with 730 nm light, the photosensitizer produced singlet oxygen, peroxidating the unsaturated lipids in the liposome bilayer, making it more permeable, and releasing the encapsulated drug. Both approaches were triggered by near-infrared light. Light is an attractive energy source for externally triggered systems because of the ease of controlling its irradiation parameters (such as irradiance, wavelength, and duration), and the established guidelines for human use. Near infrared light allows deeper tissue penetration than UV and visible wavelengths of light. Here we hypothesized that combining the two approaches (Scheme 1c) would produce an on-demand nerve block system with high photosensitivity and repeatability. We developed a liposomal formulation comprising unsaturated lipids and loaded with photosensitizers and gold nanorods that could both be excited by a single wavelength. To achieve this, gold nanorods were synthesized to have strong absorbance at 730 nm, the wavelength at which the photosensitizer also had peak absorbance. As the local anesthetic, we used the ultrapotent agent tetrodotoxin (TTX). Tetrodotoxin’s potency allows a large
pioids are currently the most widely used drugs in pain treatment,1 but have many side effects.2 In many cases, pain relief could be provided by local anesthesia, but it tends to be of relatively brief duration.3 Controlled release systems have been developed for prolonged duration local anesthesia4 but once they have been administered the resulting level of pain relief cannot be adjusted in accordance with the patients’ changing needs. To address this unmet need, we have developed systems where the release of local anesthetics changes in response to a noninvasive external trigger such as light.5,6 Such systems would allow the patient to control the timing, degree, and duration of local analgesia. The sensitivity of such systems to stimuli is of great importance, as it can affect many key performance characteristics, particularly the depth in tissue at which they can be triggered and/or the degree of tissue injury incurred by intervening tissues in triggering at a given depth. Here we have investigated the feasibility of enhancing triggerability of drug release by combining two different triggering mechanisms in one device. We have followed two principal approaches for triggering release of local anesthetics from liposomes. In one (Scheme 1a), we tethered gold nanorods to the surface of thermosensitive liposomes.6,7 Upon irradiation with 808 nm light, the gold nanorods converted light energy to heat by surface plasmonic resonance, which raised the local temperature above the lipid transition temperature (Tc), triggering anesthetic release from the liposomes. In a second approach (Scheme 1b), we used © XXXX American Chemical Society
Received: September 28, 2017
A
DOI: 10.1021/acs.nanolett.7b04176 Nano Lett. XXXX, XXX, XXX−XXX
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Nano Letters Scheme 1. Schematic of Liposomes for Light-Triggerable Drug Deliverya
a
(a) Liposomes with gold nanorods tethered onto the surface. (b) Liposomes with unsaturated lipids have photosensitizers encapsulated within the bilayer. (c) Liposomes tethered with gold nanorods and also loaded with photosensitizers.
Figure 1. Absorption spectra of (a) PdPC in ethanol, (b) GNR in PBS, (c) liposomal formulations of PdPC and GNR in ethanol.
Figure 2. Photothermal and photochemical effects. (a) Photothermal conversion efficiency of liposomal formulations upon irradiation of different irradiances. (b) Singlet oxygen quantum yield upon irradiation of liposomes (see Materials and Methods for details). (c) Absorption spectra of liposomal formulations in PBS before and after irradiation. * P < 0.01.
effective payload to be encapsulated, and its hydrophilicity makes it readily encapsulated in liposomes. The photosensitivity and
repeatability of the system was demonstrated in vitro, and its efficacy in vivo in a rat model of sciatic nerve blockade. B
DOI: 10.1021/acs.nanolett.7b04176 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 3. Phototriggered release from dye-loaded liposomes. Dye release of liposomal formulations with respect to (a) irradiation duration (at a constant 55 mW/cm2), (b) irradiance (all for 5 min), (c) number of triggered events (blue line, with irradiation; red line, no irradiation; arrows indicate irradiation at 730 nm for 3 min at 55 mW/cm2) * P < 0.01.
Results. Characterization of PdPC and/or GNR Loaded Liposomes. The photosensitizer 1,4,8,11,15,18,22,25-octabutoxyphthalocyaninato-palladium(II) (PdPC) was synthesized by a metal-insertion reaction, as reported.5 PdPC showed an absorption peak at 729 nm in ethanol5,8,9 (Figure 1a). To allow photosensitization and plasmonic photothermal heating to be triggered by irradiation at a single wavelength, the gold nanorods (GNR) were synthesized to match the absorption profile of PdPC. The synthesized GNR absorbed light between 700 and 800 nm wavelengths with an absorption peak at 757 nm (Figure 1b). The synthesized gold nanorods were imaged by TEM, showing a rod morphology with a mean length of 46.6 ± 3.1 nm and mean diameter of 14.6 ± 1.26 nm, yielding an aspect ratio of 3.2 ± 0.3 (Figure S1). PdPC and GNR were then loaded into liposomes (LipoPdPC-GNR, see Materials and Methods for details of liposome synthesis and loading), which had an average diameter of 2.8 ± 1.7 μm. Lipo-PdPC-GNR were imaged by cryo-TEM, showing a vesicular morphology typical of liposomes (Figure S1). The loading of gold and PdPC were 463.2 ± 10.6 μg/mL (loading efficiency 87.1 ± 2.0%) and 99.5 ± 8.2 μg/mL (loading efficiency 99.5 ± 8.2%), respectively. There was no significant difference in GNR loading between Lipo-PdPC-GNR and Lipo-GNR (P = 0.5), nor in PdPC loading between Lipo-PdPC and Lipo-PdPCGNR (P = 0.09). Liposomes loaded with PdPC only (LipoPdPC) showed an absorption peak at approximately 730 nm (Figure 1c), and Lipo-GNR had a broad absorption peak between 700 and 900 nm. Lipo-PdPC-GNR had the PdPC absorption peak at 730 nm and the GNR absorption peak between 700 and 900 nm. These results show that the nearinfrared absorption spectra of PdPC and GNR were preserved upon encapsulation in liposomes and that Lipo-PdPC-GNR should be triggerable by near-infrared light. Phototriggered Thermal and Chemical Effects in Vitro. The phototriggering of liposomal formulations was characterized in vitro. Photothermal heating was quantified by a near-infrared camera from which the photothermal conversion efficiency was determined (see Materials and Methods for details). The
photothermal conversion efficiency of Lipo-PdPC-GNR (27.0 ± 2.8% at 55 mW/cm2) was not significantly different from that of Lipo-PdPC (27.9 ± 1.3% at 55 mW/cm2, p = 0.6) or LipoGNR (29.0 ± 2.0% at 55 mW/cm2, p = 0.3) at irradiances below 55 mW/cm2 (Figure 2a); however, Lipo-PdPC-GNR showed significantly higher photothermal conversion efficiencies at irradiances of 140 and 200 mW/cm2 (Lipo-PdPC and LipoGNR p < 0.001 when compared with Lipo-PdPC-GNR for both irradiances). At 200 mW/cm2, Lipo-PdPC-GNR showed a photothermal conversion efficiency of 50.6 ± 1.1%, whereas Lipo-PdPC and Lipo-GNR had photothermal conversion efficiencies of 22.9 ± 0.9% (p < 0.001) and 25.8 ± 0.6% (p < 0.001), respectively. Irradiation of blank (PBS) liposomes did not result in an increase in temperature, nor did irradiation of PBS at 200 mW/cm2 (Figure S2). To evaluate the photothermal stability of PdPC upon repeated irradiations, the temperature of PdPC-loaded liposome solutions (Lipo-PdPC and Lipo-PdPC-GNR) was measured during three consecutive irradiation events in vitro (Figure S3). Lipo-PdPC reached a peak temperature of 60.9 ± 2.3 °C after irradiation at 200 mW/cm2 for 3 min. After the formulation temperature returned to room temperature, the irradiation was repeated and Lipo-PdPC reached a peak temperature of 64.8 ± 1.8 °C during the second irradiation and 60.4 ± 2.3 °C during the third irradiation. Similarly, Lipo-PdPC-GNR showed a peak temperature of 81.0 ± 4.6 °C, 85.7 ± 1.1 °C, and 86.4 ± 1.1 °C respectively during the first, second, and third irradiation events. The temperature profiles showed no significant difference between irradiation events, demonstrating photostability of the photoactive component. The photochemical production of singlet oxygen by liposomes under irradiation was determined by the singlet oxygen quantum yield, measured with the singlet oxygen sensor green (SOSG) fluorescent indicator10 (see Materials and Methods). The quantum yields of Lipo-PdPC-GNR and Lipo-PdPC were comparable (82.6 ± 8.9% and 91.4 ± 8.8%, respectively) upon irradiation at 730 nm, 55 mW/cm2 (P = 0.2), whereas Lipo-GNR produced negligible amounts of singlet oxygen. No significant C
DOI: 10.1021/acs.nanolett.7b04176 Nano Lett. XXXX, XXX, XXX−XXX
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related to tissue reaction to local anesthetics, (1) the C2C12 myotube cell line used to assess myotoxicity, and (2) the PC12 pheochromocytoma cell line used to assess neurotoxicity. Cells were incubated in 800 μL of medium with 100 μL of test material in a Transwell. The liposomes were irradiated with 730 nm light for 5 min (50 mW/cm2). Cell viability was determined after 96 h incubation. There was no significant cytotoxicity in Lipo-PdPCGNR irradiated or nonirradiated groups compared with the PBStreated, nonirradiated group (Figure S4, P > 0.05 for all comparisons with PBS group). Phototriggered TTX Release in Vitro and Phototriggered Analgesia in Vivo. Tetrodotoxin (TTX) was loaded into LipoPdPC-GNR (Lipo-PdPC-GNR-TTX; mean diameter 4.4 ± 1.4 μm) with a loading efficiency of 14 ± 1% (average TTX concentration of 52.5 ± 3.8 μg/mL). In vitro TTX release was determined by dialyzing TTX-loaded liposomes against PBS in the dark and irradiating at the 5 h time point (Figure S5). LipoPdPC-GNR released 6.5 ± 0.6% in the first 5 h. After irradiation (730 nm, 55 mW/cm2, 5 min) the release increased to 33.1 ± 2.5% at the 9 h time point, whereas without irradiation 11.7 ± 0.3% was released in the same period. Over the same 9 h, cumulative TTX release from Lipo-GNR was 13.6 ± 0.2% with irradiation and 6.6 ± 0.7% without irradiation; Lipo-PdPC yielded a release of 25.5 ± 1.1% with irradiation and 10.8 ± 0.4% without irradiation. These results demonstrated that Lipo-PdPCGNR released a greater amount of drug compared with LipoGNR (P < 0.01, 9 h cumulative release) or Lipo-PdPC (P = 0.04, 9 h cumulative release) after irradiation at 730 nm, 55 mW/cm2, 5 min. In vivo studies were performed where 50 μL of liposomes were injected subcutaneously in the plantar aspect of the rat footpad and neurobehavioral testing was conducted with a Torch Test sensory evaluator as reported.6,12 Effective analgesia was defined as analgesia above 50% maximum possible effect (MPE; see Materials and Methods for details). There was no significant analgesia after injection. The penetration of near-infrared light (730 nm) through rat skin at the footpad was evaluated ex vivo, where rat skin was placed between the light source and a power detector. A total of 27 ±1% of light penetrated through rat skin. Ninety minutes after injection animals were irradiated at 200 mW/cm2 for 3 min at the injection site. In animals injected with Lipo-PdPC-GNR, irradiation triggered effective analgesia lasting 1.5 ± 0.3 h (Figure 4). A second irradiation immediately after the first phototriggered analgesia wore off caused local anesthesia with a duration of 0.8 ± 0.6 h. There were no observable neurobehavioral changes after a third irradiation. Analgesia after
difference in singlet oxygen quantum yield was found upon enhancing the irradiance to 140 mW/cm2 (Figure 2b, P = 0.15 for Lipo-PdPC-GNR and P = 0.86 for Lipo-PdPC). The singlet oxygen was hypothesized to react with the unsaturated lipid components in the lipid bilayer to generate phototriggered lipid peroxidation. This effect was characterized by monitoring the production of conjugated dienes, a product of lipid peroxidation. Conjugated dienes have a distinct absorption peak at approximately 235 nm.11 Upon irradiation at 730 nm (55 mW/cm2, 3 min), Lipo-PdPC-GNR and Lipo-PdPC generated the conjugated diene peak, whereas irradiation of Lipo-GNR did not generate the conjugated diene peak (Figure 2c), indicating that PdPC is necessary for the photochemical effect. Adjustability and Repeatability of Light-Triggered Dye Release in Vitro. The fluorescent dye sulforhodamine B (SRho) was loaded into the liposomes at a concentration high enough to induce self-quenching when encapsulated within the liposome core.5 When SRho was released into the solution, the resulting concentration was much lower, resulting in strong fluorescence.5 Dye release was quantified by monitoring the fluorescence of the liposomal solution. In liposomes irradiated at 55 mW/cm2 (low irradiance), dye release increased with irradiation duration (Figure 3a). The release of SRho from Lipo-PdPC-GNR and Lipo-PdPC was significantly higher than the release from Lipo-GNR after 5 min of irradiation (P < 0.01 for comparisons of both with Lipo-GNR), suggesting that photochemical effects of PdPC were dominant relative to the photothermal effects using these irradiation conditions. Dye release was also dependent on the intensity of irradiation. At irradiances lower than 100 mW/cm2, Lipo-PdPC released up to three times more dye than Lipo-GNR using the same irradiation conditions (3-fold more at 35 mW/cm2; P = 0.01); at irradiances higher than 100 mW/cm2, Lipo-GNR released 50% more than did Lipo-PdPC (P = 0.03 at 200 mW/cm2, Figure 3b). These results suggest that the photochemical effects of PdPC were predominant at lower irradiances while the photothermal effect of GNR were predominant at higher irradiances. Release from Lipo-PdPC-GNR resembled that from Lipo-PdPC at low irradiances and released more dye than either Lipo-PdPC or Lipo-GNR at higher irradiances (P < 0.01 for comparisons of both with Lipo-PdPC-GNR at 200 mW/cm2). The repeatability of phototriggered dye release was investigated by monitoring the fluorescence of the liposomal samples after consecutive irradiations (730 nm, 3 min, and 55 mW/cm2). Irradiation of Lipo-PdPC-GNR induced release of approximately 8% of dye in the following 3 min, followed by slower release. Phototriggered release could be repeated 5 times (Figure 3c); each irradiation event was for 3 min followed by a 3 min interval in darkness. The same irradiation duration and irradiance did not induce significant dye release from Lipo-GNR (P = 0.8 between with and without irradiation). Lipo-PdPC was responsive to the first three irradiation events, but after the third irradiation event the dye release did not slow down to baseline and further release was not triggered by the fourth and fifth irradiations. These results show that Lipo-PdPC-GNR was triggerable in these irradiation conditions while Lipo-GNR was not and that LipoPdPC-GNR could be triggered more times than Lipo-PdPC, demonstrating that the combination of two mechanisms in a single liposomal vehicle provided more effective control of cargo release upon light-triggering. Cytotoxicity. The cytotoxicity of TTX-loaded liposomes with PdPC and GNR was evaluated using two different cell lines
Figure 4. Phototriggered infiltration analgesia of the rat footpad 90 min after subcutaneous injection of 50 μL of liposomes. Time 0 indicates 90 min after injection. Arrows indicate irradiation for 3 min, 730 nm at 200 mW/cm2. D
DOI: 10.1021/acs.nanolett.7b04176 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 5. Temperature profile upon irradiation in vivo. (a) Representative photograph of temperature measurements by a near-infrared camera focusing on the rat footpad. (b) Temperature time courses of rat footpads upon irradiation. Yellow bands indicate irradiations for 3 min at 730 nm and 200 mW/ cm2.
the first irradiation of Lipo-PdPC lasted 0.7 ± 0.1 h but there was no analgesia after the second and third irradiations. Irradiation of Lipo-GNR did not result in analgesia after any of the irradiation events. The potential effects of systemic distribution of TTX or of residual isoflurane were determined by measuring the neurobehavioral response in the contralateral (uninjected) footpad. No systemic effect was observed in any animal at any time point. To demonstrate that the photothermal effect occurred in vivo, the footpad temperature was monitored in vivo at predetermined time points with a near-infrared camera (Figure 5a). In animals injected with Lipo-PdPC-GNR, the footpad temperature increased from 32.8 ± 0.5 °C to 47.5 ± 1.7 °C within the first minute of irradiation (Figure 5b), followed by a plateau up to the third minute of irradiation. Lipo-PdPC and Lipo-GNR followed similar trends, with an average temperature between 43 and 45 °C after 3 min of irradiation. The decreases in average peak temperature with successive irradiation were not statistically significant except in the case of Lipo-PdPC (p ≤ 0.02 at second and third irradiations compared to first). To evaluate the potential effect of photothermal heating of GNR on TTX-induced anesthesia, we coinjected 50 μL of LipoGNR mixed with free TTX (250 μM final concentration) into the rat footpad, followed by irradiation (200 mW/cm2, 3 min). The mean duration of anesthesia was 48.8 ± 19.8 min; without irradiation it was 41.3 ± 31.5 min (P = 0.7). Therefore, the photothermal effect of GNR did not affect the anesthesia induced by TTX. Irradiation of animals injected with liposomes without PdPC or GNR (Lipo) for 3 min increased footpad temperatures to an average of 35 °C; this was not statistically significantly different from uninjected animals after irradiation (P = 0.3). These results show that the liposomes themselves did not generate heat upon laser irradiation. Tissue Reaction. Animals were sacrificed 4 days after injection, and the site of injection in the footpad was harvested and processed by standard hematoxylin-eosin staining procedures. Footpads injected with Lipo-PdPC-GNR showed mild to moderate inflammation (Figure 6, Table 1), as did tissues injected with Lipo (Figure S6), consistent with reaction to injection of particulates.13−16 No significant myotoxicity was observed. Discussion. The combination of triggering by GNRmediated photothermal effects and PdPC-mediated photochemical effects greatly enhanced nerve blockade in vivo. When irradiated with 200 mW/cm2 for 3 min, Lipo-PdPC-
Figure 6. Representative light micrographs of hematoxylin and eosin stained sections of tissue harvested 4 days after injection and irradiation. Scale bars = 100 μm.
Table 1. Inflammation and Myotoxicity Scores from Histology Analysisa,b,c control (nontreated) Lipo-PdPC-GNR + light
inflammation
myotoxicity
0 (0,0) 1.5* (0.75, 2)
0 (0,0) 1 (0,2)
a
Data are medians with 25th and 75th percentiles, n = 4 Inflammation was scored between 0 (normal) and 4 (severe inflammation). Myotoxicity was scored between 0 (normal) and 6 (highest degree of myotoxicity); see Materials and Methods. c* P < 0.05 (n = 4) compared to the control group. b
GNR triggered local anesthesia that lasted for 1.5 ± 0.3 h, whereas Lipo-GNR did not produce any nerve block (Figure 4). Similarly, addition of GNRs increased the magnitude and repeatability of nerve block from Lipo-PdPC (Figure 4).The enhancement of TTX release and of the resulting nerve block may be due to having two separate mechanisms affecting liposome integrity, one photothermal and the other photochemical. The fact that the use of Lipo-PdPC-GNR had a greater anesthetic effect than did Lipo-GNR or Lipo-PdPC implies that the former could achieve a given anesthetic (or other drug delivery) effect at a lower irradiance than the latter two, that is, it is more photoresponsive. This is potentially important in that the use of a lower irradiance is less likely to result in thermal injury. Moreover, the greater responsiveness to a given irradiance could allow triggering at greater tissue depths. Dye release due to photosensitization was activated by a lower irradiance than was that from the photothermal effect (Figure 3b). This phenomenon has also been observed previously when photosensitizers were covalently bound to gold nanostars and activated using a single wavelength;17 photosensitization required less energy to activate compared with photothermal E
DOI: 10.1021/acs.nanolett.7b04176 Nano Lett. XXXX, XXX, XXX−XXX
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Liposome Characterization. Liposome size was determined by the Electrical Sensing Zone method (also termed the Coulter Principle) as previously reported5 using the Multisizer 3 Coulter Counter (Beckman Coulter, Indianapolis, IN). TTX loading was quantified by ELISA (Reagen, Moorestown, NJ). Sulforhodamine B content was assessed by UV-Spectroscopy (λmax = 560 nm) after disrupting the liposomes with octyl β-D-glucopyranoside (OGP). PdPC content was similarly determined using UV− vis (λmax = 730 nm) after dispersing the formulation in ethanol. Gold content was analyzed by ICP-MS. Loading efficiency was calculated by the following equation:
effects (local surface plasmon resonance). Lipo-PdPC-GNR showed a cargo release profile similar to Lipo-PdPC at low irradiances. At higher irradiances, when both photochemical and photothermal effects were activated, Lipo-PdPC-GNR showed more release than Lipo-PdPC or Lipo-GNR. Compared with Lipo-GNR, Lipo-PdPC-GNR had enhanced photosensitivity and was activatable at a lower irradiance (Figure 3a,b, Figure 4). Tissue reaction upon injection and irradiation showed mild inflammation without significant myotoxicity (Figure 6, Table 1). Mild inflammation is often seen with injection of micron-sized particulates.15,16 The absence of myotoxicity is consistent with previous reports5,6 and reflects the facts that (a) singlet oxygen has a short lifetime that limits their distance of travel to approximately 268 nm nanometers,18 (b) photothermal effects of gold nanorods contribute to localized heating but not bulk heating,19 and (c) tetrodotoxin tends to have minimal tissue toxicity.20,21 Here, Lipo-PdPC-GNR showed enhanced performance compared to Lipo-PdPC and Lipo-GNR. This enhancement could also allow a given release event/degree of nerve blockade to be achieved at a lower irradiance, thus improving safety, or at a greater tissue depth at the same irradiance. This work provides proof of concept for the potential effectiveness of dual remote triggering mechanisms for triggering drug release. Materials and Methods. Liposome Synthesis. The photosensitizer 1,4,8,11,15,18,22,25-octabutoxyphthalocyaninatopalladium(II) (PdPC) was synthesized via a metal-insertion reaction5 and gold nanorods (GNR) were synthesized based on seed-mediated growth as reported.22 GNR was synthesized using the following procedure: 11.2 g of cetrimonium bromide (CTAB) was dissolved in 150 mL of deionized water after which 1 mL of gold chloride trihydrate solution (60.59 mg/mL) was added. Borohydride solution (0.58 mg/mL) of 190−230 μL was added to 5 mL of the gold-CTAB solution and stirred at 35 °C for 1 h to grow the seeds. Ascorbic acid (30 mg) and silver nitrate (3.5 mg) were added to the rest of the gold-CTAB solution and the seed solution was added, followed by stirring for 1 h. Excess CTAB was removed by centrifuging the gold nanorod solution and removing the supernatant. PEG-SH solution was added and incubated overnight. The GNR solution was concentrated by centrifugation. Liposomes were synthesized using the thin film hydration method as described.5,14 In brief, 61.3 mg of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Corden Pharma, Plankstadt, Germany), 29.5 mg of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG, Corden Pharma, Plankstadt, Germany), 15 mg of 1,2-dilinoleoylsn-glycero-3-phosphocholine (DLPC, Avanti Polar Lipids, Alabaster, AL), 20 mg of cholesterol (Sigma, St. Louis, MO), and 6.7 mg of SH-mPEG(2000)-DSPE (Nanocs Inc., New York, NY) were dissolved in 15 mL of chloroform/methanol (v/v, 9:1) with or without 0.2 mg of PdPC. The solvent was then set to dissolve on a rotary evaporator (Buchi, New Castle, DE). The resulting lipid film was then hydrated with 15 mL of t-butanol (Sigma, St. Louis, MO) and lyophilized overnight to form a powdery lipid cake. The cake was then hydrated with 2 mL of phosphate buffered saline (PBS), TTX (0.375 mg/mL, Abcam, Cambridge, MA), sulforhodamine dye (10 mg/mL, Sigma, St. Louis, MO), or GNR solution (0.5 mg/mL). The hydrated lipid solution was then subject to 10 freeze−thaw cycles and then dialyzed against PBS for 48 h (molecular weight cut off (MWCO) 1000 kDa, Float-A-Lyzer G2, Spectrum Laboratories, Inc., Rancho Dominguez, CA).
Loading efficiency Mass of compound in particles = Mass of compound added during production of particles
The denominator was 0.1 mg/mL for PdPC and 0.5 mg/mL for GNR. In Vitro Phototriggered Release. Light-triggered release was tested in vitro using sulforhodamine B encapsulated liposomes. The quantification of dye release was determined by fluorescence (excitation/emission = 530 nm/560 nm). Thirty microliters of the liposome solution was transferred into a 96-well plate with round-bottom wells. A 730 nm laser was then positioned 3 cm above the surface on which the plate was placed. After irradiation, 10 μL of liposome solution was diluted in 1 mL PBS and the fluorescence intensity was measured with a plate reader (BioTek, Winooski, VT). Dye release percentage was determined as previously reported, using the following equation: Normalized dye release =
F − F0 Fbreak − F0
where F = fluorescence of liposome sample after irradiation; F0 = fluorescence of liposome sample prior to irradiation; Fbreak = fluorescence of liposome sample after disruption by surfactant octyl β-D-glucopyranoside (OGP) (Sigma-Aldrich, Milwaukee, WI). To evaluate the effect of irradiation duration on cargo release, 100 μL of SRho-loaded liposomes was transferred to the 96-well round-bottom plate. The solution was then irradiated at 55 mW/ cm2 for 5 min and sampled at the predetermined intervals. To study the effect of irradiance on release, the liposome sample was irradiated once for 5 min at a varying iradiances. In all studies, irradiations were performed on four independent samples at each setting (i.e., n = 4). To measure the light-triggered TTX release in vitro, TTXloaded liposomes (100 μL) were placed in a dialysis tube and dialyzed against 14 mL of PBS. The dialyzed solution was replaced with fresh PBS at predetermined time points. The liposomes were incubated in the dark at 37 °C and irradiated (730 nm, 55 mW/cm2, 5 min) at the 5 h time point. The TTX concentration of the dialyzed solutions was measured by ELISA. Repeatability and Post-Irradiation Stability. To assess triggering repeatability and stability of liposomes after irradiation, liposome solution was transferred to a 96-well plate (150 μL/well). The samples were irradiated at 55 mW/cm2 for 3 min and then put in darkness for 3 min. This process was then repeated to achieve a total of five irradiation-rest cycles for each liposome sample (n = 4). Quantification of Photothermal Effects. The photothermal effect of the liposomes was determined by measuring the solution temperature upon irradiation using an FLIR E50 infrared imaging camera (FLIR Systems, Wilsonville, OR) at predeterF
DOI: 10.1021/acs.nanolett.7b04176 Nano Lett. XXXX, XXX, XXX−XXX
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(Costar 3495, pore size 0.4 μm). Irradiation was performed with 730 nm light at 50 mW/cm2 for 5 min. Cell viability was measured by the MTS assay after 96 h incubation. Animal Care. Adult male Sprague−Dawley (Charles River Laboratories, Wilmington, MA) rats weighing 300−450 g were handled and cared for in accordance with protocols approved by the Animal Care and Use Committee at Boston Children’s Hospital. The rats were housed in groups in a 7 am to 7 pm lightdark cycle. In Vivo Phototriggered Anesthesia. Animals were anesthetized with isoflurane/oxygen after which 50 μL of TTX-loaded liposomes (Lipo-PdPC, Lipo-GNR or Lipo-PdPC-GNR) were injected subcutaneously into the rat’s left hind paw with a 23 gauge needle. The nociceptive behavioral test was conducted by documenting an audible vocal response or the withdrawal of the foot upon applying a defined force using a Touch Test sensory evaluator (North Coast Medical, Inc., Gilory, CA).6 A force of 180 g was applied by pressing the evaluator filament onto the rat paw until filament flexion. Anesthetic effect at each time point was quantified by counting how many times the rat did not respond to the Touch Test out of a total of five measurements. This number was then converted to a percentage of the five response to which there was no response. The right hindpaw was used as a control for systemic distribution of drug (there should be no nerve block in the right hindpaw). Animals were anesthetized with isoflurane/oxygen and irradiation was performed at 200 mW/cm2 for 3 min, starting 90 min after injection. The rats then underwent the nociceptive behavioral test as described above at predetermined time points. During irradiation, the temperature of the rat’s hindpaw was recorded using the FLIR infrared camera. Histology. Animals were euthanized by carbon dioxide 4 days after injection. The site of injection in the footpad was collected and standard procedures were followed to produce hematoxylin and eosin-stained slides. The samples were scored for inflammation and myotoxicity according to the scoring system previously reported.27 The inflammation score quantified the degree of severity, where 0 was normal and 4 was severe inflammation. Myotoxicity was scored based on the nuclear internalization and regeneration of myocytes, which are known characteristics of local anesthesia myotoxicity. The scoring scale was as follows: 0 = normal; 1 = perifascicular internalization; 2 = deep internalization (>5 cell layers); 3 = perifascicular regeneration; 4 = deep tissue regeneration (>5 cell layers); 5 = hemifascicular regeneration; 6 = holofascicular regeneration. Statistical Analysis. Statistical comparison of the inflammation and myotoxicity scores were performed with the Mann− Whitney U test due to their ordinal character. All other comparisons were performed by the two-tailed Student’s t test. P < 0.05 was considered significant. To avoid Type 1 error during multiple comparisons, Bonferroni correction was applied: the significance criterion was set as 0.05/M to maintain alpha of 0.05 for each outcome, where M is the number of tests within each hypothesis.
mined time points. Fifty microliters of liposome was irradiated up to a total of 5 min. The temperature of the liposome solution was recorded each minute. The photothermal conversion efficiency was computed as previously reported,23,24 using the following equation: Photothermal Conversion Efficiency =
hA(Tmax − Tmin) − Q 0
(
I 1−
It I
)
where h = heat transfer coefficient; A = surface area of container; Q0 = rate of heat input due to solvent; I = laser power irradiated; It = laser power transmitted. Quantification of Photochemical Effects. Lipid peroxidation from the liposomes was characterized by the formation of a 238 nm absorption peak that resulted from the formation of conjugated dienes.5 Singlet oxygen quantum yield was determined using a fluorescent singlet oxygen indicator, SOSG, as previously reported.25 SOSG solid was dissolved in methanol and was then diluted 100× with PBS. It was mixed with the liposome solution to form a final SOSG concentration of 10 μM. The fluorescence (excitation 504 nm, emission 525 nm) of SOSG endoperoxides (SOSG-EN) was measured by a plate reader (BioTek, Winooski, VT) after irradiation. The singlet oxygen quantum yield was determined by the following equation: rsample
singlet oxygen quantum yield =
A sample rPPIX APPIX
φPPIX
where rsample and rPPIX were the reaction rates (see below) of SOSG with singlet oxygen, produced upon irradiation of liposomes or protoporphyrin IX (PPIX). Asample and APPIX were the absorbance of the liposomes at 730 nm and PPIX at 635 nm respectively at the photosensitizer concentration used in those experiments (0.67 μM). φPPIX was the quantum yield of the reference photosensitizer (PPIX), using a previously reported value of 0.56.26 The reaction rates were determined by the following equation:
r=
d[SOSG − EN] dt
where [SOSG-EN] was the concentration of SOSG-EN, determined by its fluorescence (excitation 504 nm, emission 525 nm). The reaction rate was the slope of the curve relating the fluorescence of the liposomes and the irradiation duration at the reported intensity. Cytotoxicity. C2C12 mouse myoblast cells (ATCC, Manassas, VA) were cultured in DMEM (20% FBS and 1% Penicillin Streptomycin) (Invitrogen, Carlsbad, CA). Cells were seeded in a 24-well plate at 5 × 104 cells/mL in 800 mL media. The cells were then cultured in DMEM (2% horse serum and 1% Penicillin Streptomycin) for 10−14 days for myotubule differentiation. PC12 rat adrenal gland pheochromocytoma cells (ATCC, Manassas, VA, USA) were cultured in DMEM (12.5% horse serum, 2.5% FBS and 1% Penicillin Streptomycin) and seeded in a 24 well-plate at 2 × 104 cells/mL in 800 mL media. The cells were then cultured in DMEM (1% horse serum, 1% Penicillin Streptomycin, and 50 ng/mL nerve growth factor) for 7 days. Cytotoxicity measurements were performed by exposing 100 μL of liposomes to C2C12 or PC12 cells (800 μL of their respective cell culture media) by a 24-well Transwell membrane
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.7b04176. TEM micrographs of gold nanorods and Lipo-PdPCGNR, temperature profile of PBS and Lipo upon G
DOI: 10.1021/acs.nanolett.7b04176 Nano Lett. XXXX, XXX, XXX−XXX
Letter
Nano Letters
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irradiation, photothermal characterization of liposomes, cell viability, phototriggered TTX release in vitro, and light micrographs of hematoxylin and eosin stained sections of tissue (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Daniel S. Kohane: 0000-0001-5369-5932 Author Contributions ‡
A.Y.R. and B.Y.W. contributed equally
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by NIH Grant GM 116920 (to D.S.K.). B.Y.W. is grateful to Professor Daniel Kirschner at Boston College for serving as B.Y.W.’s on-campus advisor and for always pushing him to understand the fundamentals behind the experiments. We would also like to thank Ms. Aishwarya Rajapur for her technical contribution in liposome production and photothermal characterization studies. TEM images were obtained with the support of the Center for Nanoscale Systems (CNS) in Harvard University, a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported by the National Science Foundation under NSF award no. 1541959 CNS. We gratefully thank Dr. Stephen Slocum and the MIT Center for Environmental Health Sciences for their assistance in the ICP-MS measurements. The authors declare no competing financial interests.
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DOI: 10.1021/acs.nanolett.7b04176 Nano Lett. XXXX, XXX, XXX−XXX
