A smart and versatile theranostic nanomedicine platform based on nanoporphyrin

Abstract

Multifunctional nanoparticles with combined diagnostic and therapeutic functions show great promise towards personalized nanomedicine. However, attaining consistently high performance of these functions in vivo in one single nanoconstruct remains extremely challenging. Here we demonstrate the use of one single polymer to develop a smart 'all-in-one' nanoporphyrin platform that conveniently integrates a broad range of clinically relevant functions. Nanoporphyrins can be used as amplifiable multimodality nanoprobes for near-infrared fluorescence imaging (NIRFI), magnetic resonance imaging (MRI), positron emission tomography (PET) and dual modal PET-MRI. Nanoporphyrins greatly increase the imaging sensitivity for tumour detection through background suppression in blood, as well as preferential accumulation and signal amplification in tumours. Nanoporphyrins also function as multiphase nanotransducers that can efficiently convert light to heat inside tumours for photothermal therapy (PTT), and light to singlet oxygen for photodynamic therapy (PDT). Furthermore, nanoporphyrins act as programmable releasing nanocarriers for targeted delivery of drugs or therapeutic radio-metals into tumours.

Figure 1. Design, synthesis and characterizations of nanoporphyrins

(a) Schematic illustration of a multifunctional nanoporphyrin self-assembled by a representative porphyrin- telodendrimer, PEG^5k-Por₄-CA₄, comprised of 4 pyropheophorbide-a molecules and 4 cholic acids attached to the terminal end of a linear PEG chain. (b) Schematic illustration of nanoporphyrins as a smart “all-in-one” nanomedicine platform against cancers. (c) TEM image of nanoporphyrins (stained with phosphotungstic acid, PTA). (d) The absorbance spectra of NPs before and after chelating different metal ions. (e) Fluorescence emission spectra of NPs in the presence of PBS (red) and SDS (blue). Excitation: 405 nm. (f) Near-infrared fluorescence imaging of nanoporphyrin solution (10 μL) in the absence and in the presence of SDS with an excitation bandpass filter at 625/20 nm and an emission filter at 700/35 nm. (g) Single oxygen generation of NPs in PBS and SDS upon light irradiation (690 nm at 0.25 w/cm² for 60 seconds) measured by using SOSG as an indicator^, (n=3). Concentration-dependent photo-thermal transduction of NPs: (h) thermal images and (i) quantitative temperature change curve (n=2). The temperature of nanoporphyrin solution (10 μL) in the absence and in the presence of SDS was monitored by a thermal camera after irradiation with NIR laser (690 nm) at 1.25 w/cm² for 20 seconds.

Figure 2. Design, synthesis and characterizations of disulfide crosslinked nanoporphyrins

Schematic illustration of a representative crosslinkable porphyrin-telodendrimer (PEG^5k-Cys₄-Por₄-CA₄), comprised of 4 cysteines, 4 pyropheophorbide-a molecules and 4 cholic acids attached to the terminal end of a linear PEG chain. Ebes was used as a spacer. (b) TEM imaging of the disulfide crosslinked nanoporphyrins (CNPs) (stained with PTA). (c) The fluorescence emission spectra of the CNPs in the presence of PBS and SDS in the comparison with the non-crosslinked NPs. Glutathione (GSH) was used as a reducing agent to break the disulfide crosslinking. Excitation: 405 nm. These CNPs also showed very weak red fluorescence emission with a peak value at 680 nm when excited at 405 nm (green curve). When the fluorescence emission spectra of the CNPs were recorded in the presence of SDS, there was increase in the peak emission at 680 nm (black curve). However, the peak value was significantly lower than that of the non-crosslinked NPs in the presence of SDS at the same porphyrin concentration. Upon addition of GSH, the CNPs were completely broken down and the peak at 680 nm in the fluorescence emission spectra increased further to a similar value as that of NPs in the presence of SDS (pink curve). (d) The loading efficiency of doxorubicin into CNPs and size change of doxorubicin loaded CNPs (CNP-DOX) versus the level of drug loading. The loading efficiency is defined as the ratio of drug loaded into nanoparticles to the initial drug content. The volume of the final NP solution was kept at 1 mL and the final concentration of the telodendrimer was 20 mg/mL. (e) Schematic illustration of FRET-based approach for study of the real-time release of doxorubicin from nanoporphyrins in human plasma. FRET signal (f) and changes in apparent FRET efficiency (E_app) (g) of CNP-DOX in human plasma with irradiation at 24 hrs and 28 hrs, respectively. Irradiation: 1.25 w/cm² for 5 min. The apparent FRET efficiency reflects the distance between the FRET pair and is calculated as E_app = I_A/(I_A + I_D), where I_A and I_D represent acceptor and donor intensities, respectively. (h) Changes in apparent FRET efficiency (E_app) of CNP-DOX in human plasma treated with GSH (10mM) at 24 hrs. (i) Cumulative DOX release profiles from CNP-DOX measured by dialysis method at 37 °C. Aliquots of CNP-DOX solution were pre-treated with light (1.25W/cm², 5 mins) or GSH (10 mM) or no treatment and then dialyzed against diluted human plasma. Data were reported as the average percentage of DOX accumulative release for each triplicate sample.

Figure 3. Intracellular delivery and photo-cytotoxicity of nanoporphyrins against cancer cells

(a) Uptake of DOX (green) loaded CNPs (red) in SKOV3 ovarian cancer cell line. Cells were cultured in the 8 well chamber cover glass slides overnight. Cells were pre-treated with Hoechst 33342 (blue) for 10 minutes for nucleus staining followed by incubation with CNP-DOX (DOX: 0.1mg/ml) in PBS. Imaging was acquired at 2 hrs using Deltavision deconvolution microscope. (Scale bar = 30 μm). The viability of SKOV3 ovarian cancer cells after (b) 2 hrs exposure to 4.4μM CNPs followed by illumination with various levels of NIR light, and (c) incubation with CNPs or 5-ALA for 24 hrs followed by exposure to NIR light at 0.07 W cm⁻² for 60 seconds. ROS mediated cell death after CNPs and light treatment of SKOV3 ovarian cancer cells: (d) Cells were treated with or without 2.2 μM CNPs for 24 hrs and loaded with DCF-DA for 30 min. After treatment with NIR light at 0.07 W cm⁻² for 60 seconds, images were acquired by fluorescence microscopy to detect ROS production; (e) SKOV3 ovarian cancer cells were incubated with 2.2 μM CNPs for 24 hrs in 96-well black-wall plate, stained with 40 nM of DiOC₆(3) (Green, ΔΨm^high) for 20 min at the end of incubation to evaluate mitochondria membrane potential(ΔΨm), and followed by illumination of a portion of each well to elicit PDT effect. The illumination area was marked with “L”. 24 hrs later, the cells were stained with propridium iodide (PI) for cell death. (f) Cells were treated with different concentrations of CNPs for 24 hrs followed by PDT. 24 hrs later, caspase3/7 activity was measured by SensoLyte® kit (Anaspec, Fremont, CA). (g) Cell morphology after PDT. SKOV3 ovarian cancer cells were cultured on 8-well chamber slides and treated for 24 hrs with PBS, CNPs alone and combination of CNPs and light ( at 0.07 W cm⁻² for 60 seconds ). Cells were then fixed and stained with Hema3® after 2 hrs. Cells treated with CNPs+light exhibited obvious nucleus swelling, cell rounding, membrane damage, and cytoplasm aggregation. (h) Cytotoxicity effect from combination of doxorubicin with CNPs mediated photo-therapy: SKOV3 ovarian cancer cells were treated with CNPs alone, doxorubicin loaded crosslinked nanoporphyrins (CNP-DOX) or doxorubicin loaded standard micelles (NM-DOX) with various concentrations of DOX and/or CNPs for 24 hrs. After washing, cells were exposed with light and cell viability was measured after 24 hrs. * p<0.05, one-way ANOVA.

Figure 4. In vivo blood elimination kinetics and biodistributions

(a) In vivo blood elimination kinetics of NPs and CNPs at a dose of 5mg/kg body weight (n=3 for each group). (b) Gd distribution in tissues of SKOV3 ovarian cancer mice 24 hrs post-injection of Gd-DTPA, Gd-NP and Gd-CNPs, measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent Techologies, 7500ce). Three mice were used in each experiment. Mice received Gd-DTPA, Gd-NP, and Gd-CNP at the dose of Gd 20 mg/kg. After 24 hrs, mice were sacrificed. Tumor and major orangs were collected and weighted. Tissue samples were treated with 200 μl of nitric acid under 60°C for 2 hrs till most tissues were digested. Fat was digested by adding another 200 μl of 50% H₂O₂ for 2-4 hrs and total volume was adjusted to 1 ml. Equal amounts of clear samples were used for ICP-MS analysis to detect Gd concentration. Results were normalized to original tissue weight and present as mg Gd per gram tissue (n=3 for each group).

Figure 5. Nanoporphyrin mediated NIRF imaging in animal models

(a) Representative in vivo NIRF imaging of nude mice bearing SKOV3 ovarian cancer xenograft following intravenous injection of NPs and CNPs (NP dose: 25 mg/kg). The white arrow points to the tumor site. (b) Representative ex vivo NIRF imaging of SKOV3 ovarian cancer xenograft 24 hrs post-injection of NPs (left) and CNPs (right). (c) Quantitative NIRF fluorescence in tumor SKOV3 at 24 hrs post-injection of NPs and CNPs ( (n=4) (NP dose: 25 mg/kg). Images were analyzed as the average signal in the region of interest (ROI) in tumor and normalized to muscle. *** p<0.001, t-test. (d) Projection images of the distribution of CNPs in SKOV3 tumor at 1, 24, 48 hrs post injection observed by Large-Scale-Imaging (LSI) laser scanning confocal microscope. Red: CNPs; Green: Dextran-FITC labelled tumor blood vessel.. (e) The representative in vivo and ex vivo NIRF light imaging of transgenic mice with mammary cancer (FVB/n Tg(MMTV-PyVmT) at 24 hrs post-injection of CNPs (NP dose: 25 mg/kg). The white arrows point to the tumor site. Tumor volume was calculated by (L*W²)/2, where L is the longest, and W is the shortest tumor diameter (mm). (f) The accumulation of CNPs in lung metastasis of breast cancer in transgenic mice at 24 hrs post injection observed by LSI laser scanning confocal microscope. The white arrow points to the metastatic site. Red: nanoporphyrins; Green: Dextran-FITC labelled tumor blood vessel.

Figure 6. Nanoporphyrin mediated MRI and PET imaging in animal models

(a) In vitro MRI signal of Gd-NPs in the absence and in the presence of SDS obtained by T1-weighted MR imaging on a Bruker Biospec 7T MRI scanner using a FLASH sequence. (b) Representative coronal and axial MR images of transgenic mice with mammary cancer (FVB/n Tg(MMTV-PyVmT) using a FLASH sequence pre-injection and after injection of 0.15 mL Gd-NPs (Gd dose: 0.015 mmole/kg). The white arrow points to the tumor site. (c) PET image of nude mice bearing SKOV3 ovarian cancer xenografts at 4, 8, 16, 24, 48 hrs post-injection of ⁶⁴Cu-labeled NPs (150-200 μL, ⁶⁴Cu dose: 0.6-0.8 mCi). The white arrow points to the tumor site. (d) 3D coronal MR images of nude mice bearing A549 lung cancer xenografts using a FLASH sequence at 4 or 24 hrs post-injection with 0.15 mL of ⁶⁴Cu and Gd dual-labeled NPs (150-200 μL, ⁶⁴Cu dose: 0.6-0.8 mCi, Gd dose: 0.015 mmole/kg). The white arrow points to the tumor site. (e) PET-MR images of tumor slices of nude mice bearing A549 lung cancer xenograft at 4 or 24 hrs post injection of dual-labeled NPs. White arrow points to the necrotic area in the center of the tumor.

Figure 7. ROS production and heat generation in blood and at tumor tissue

(a) ROS production of blood drops drawn from nude mice bearing implanted tumor xenografts 1 min post-injection of CNPs and NM-POR (Por dose: 5 mg/kg) after light exposure, and was measured by DCF-DA as ROS indicator. Light dose: 0.1 W for 60 s and 300 s. **p< 0.002, ***p< 0.001, t-test. The temperature of blood drops drawn from nude mice bearing implanted tumor xenografts 1 min post-injection of (b) CNPs and (c) NM-POR (Por dose: 5 mg/kg) after light exposure. Light dose: 0.1 W for 300 seconds. The temperature was monitored by a FLIR thermal camera. (d) The temperature changes (ΔT) at tumors of nude mice bearing implanted SKOV3 tumor xenografts 24 hrs post-injection of PBS, CNPs, and NM-POR (Por dose: 5 mg/kg) after light exposure (n=5). Light dose: 1.25 W/cm² for 120 seconds. The temperature was monitored by a FLIR thermal camera. ***p< 0.001, one-way ANOVA. (e) ROS production at tumors of nude mice bearing implanted tumor xenografts 24 hrs post-injection of PBS, CNPs and NM-POR (Por dose: 5 mg/kg) after light exposure (n=5). Light dose: 1.25 W/cm² for 120 seconds. Measured by using DCF-DA as a ROS indicator. *p< 0.01, ***p< 0.001, one-way ANOVA.

Figure 8. Nanoporphyrin mediated multimodal therapy in animal models and imaging-guided delivery

Representative thermal images of tumors in transgenic mice with mammary cancer (FVB/n Tg(MMTV-PyVmT) after light irradiation at 24 hrs post-injection of (a) CNPs and (b) PBS. (c) Temperature change in tumors in transgenic mice injected with CNPs and PBS before and after irradiation. (d) ROS production at tumor site in transgenic mice treated with CNPs and PBS (control) for 24 hrs followed by laser irradiation by using DCF-DA as an indicator. p<0.025, t-test. (e) Histopathology of tumors from mice injected with PBS or CNPs 24 hrs after irradiation. The light dose was 1.25 W cm⁻² for 2 min while the NPs dose was 25 mg/kg (equivalent to 5 mg/kg of Por) for a-e. (f) Tumor volume change of transgenic mice with mammary cancer (n=8) treated with CNPs, CNP-DOX and NM-DOX (standard micelles without porphyrin)²¹ on day 0, 7, and 14 (black arrow) followed by light exposure on the tumors in the mice in all groups at 24 hrs post-injection (red arrow). p< 0.01, one-way ANOVA. DOX dose: 2.5 mg/kg, NP dose: 25 mg/kg (equivalent to 5 mg/kg of Por), light dose: 1.25 W cm⁻² for 2 min. (g) Pictures of transgenic mice at day 34 of the treatment. (*: mammary tumors) (h) Tumor volume change of mice (n=8) bearing SKOV3 ovarian cancer xenograft treated with CNPs and CNP-DOX at day 0, 4 and 8 (black arrow) followed by irradiation on day 1, and 9 (red arrow). PBS and NM-DOX were injected for comparison. DOX dose: 2.5 mg/kg, NP dose: 25 mg/kg (equivalent to 5 mg/kg of Por), light dose: 0.25 W cm⁻² for 2 min. p< 0.01, one-way ANOVA. (i) MRI guided PTT/PDT: representative MR images of mice injected with Gd-NPs (Gd dose: 0.015 mmole/kg) before and after laser irradiation. White arrows indicate the tumor sites. Images were collected at 0 (pre-injection), 4, 24, 48, 72, 96 and 168 hrs post-injection. MR imaging showed large signal voids (blue arrows) at tumor sites 24 hrs after irradiation (48 hrs post-injection) at a dose of 1.25 W cm⁻² for 3 min and the tumors were completely ablated at 168 hrs (7 days) post-injection.