Far‐Red/Near‐Infrared Conjugated Polymer Nanoparticles for Long‐Term In Situ Monitoring of Liver Tumor Growth

The design and synthesis is reported for a fluorescent conjugated polymer (CP), poly{[4,4,9,9‐tetrakis(4‐(octyloxy)phenyl‐4,9‐dihydro‐s‐indaceno[1,2‐b:5,6‐b′]dithiophene)]‐alt‐co‐[4,7‐di(thiophen‐2‐yl)‐2,1,3‐benzothiadiazole]} (PIDT‐DBT), with absorption and emission profiles fallen within far‐red/near infrared (FR/NIR) region and further demonstrate its application in long‐term in vitro cell tracing and in vivo imaging of liver tumor growth. PIDT‐DBT‐Tat nanoparticles (NPs) have an absorption maximum at ≈600 nm with an emission maximum at ≈720 nm in water. In vitro cell tracing studies reveal that PIDT‐DBT‐Tat NPs can trace HepG2 liver cancer cells over 8 d. In vivo imaging results indicate that PIDT‐DBT‐Tat NPs can monitor liver tumor growth for more than 27 d in a real‐time manner. Both in vitro and in vivo studies demonstrate that PIDT‐DBT‐Tat NPs are superior to commercial Qtracker 705 as fluorescent probes. This study demonstrates for the first time the feasibility for long‐term in vivo imaging of tumor growth by utilizing CP‐based fluorescent probes, which will encourage the development of NIR fluorescent CPs for in vivo bioimaging.


DOI: 10.1002/advs.201500008
as indispensable tools in cancer research, clinical trials and medical practice, due to their abilities to visualize the location of tumors and assess their biological processes. [ 5 ] Among in vivo studies, long-term in situ imaging of tumors is of vital signifi cance in precise diagnosis of cancer, image-guided surgery, and monitoring of the treatment process. [ 6 ] Despite the various available bioimaging techniques, such as magnetic resonance imaging, [ 7 ] positron emission tomo graphy, [ 8 ] single photon emission computing tomo graphy, [ 9 ] radiography, [ 10 ] and ultrasound, [ 11 ] fl uorescence imaging with easier maneuverability is an unique nonionizing technique in providing high sensitivity and spatiotemporal resolution. [ 12,13 ] To effi ciently realize long-term in situ in vivo fl uorescence imaging of tumors, bright and stable near infrared (NIR) (650-900 nm) fl uorescent probes are preferentially employed to accumulate within the site of interest through passive (such as enhanced permeability and retention effect) or active (binding to a receptor on the cell surface) targeting strategies. [ 14,15 ] Among them, organic fl uorophores have been historically applied in in vivo applications due to their commercial availability and easy functionalization. [ 16 ] However, their use for long-term or real-time in vivo imaging is hampered by their intrinsic drawbacks such as small Stokes shifts and poor photostability. [ 17 ] As compared to discrete organic fl uorophores, fl uorescent nanoparticles (NPs) have advantages in prolonged intracellular retention, [ 18 ] which is benefi cial for long-term studies. To date, several types of fl uorescent NPs (noble metal NPs, [ 19 ] upconversion NPs, [ 20 ] and hybrid NPs [ 21 ] ) have been employed for highly sensitive optical imaging of cancer at both cellular and animal levels. However, inorganic nanoparticles face the diffi culties in biodegradability and some also show obvious toxicity. [22][23][24] Fluorescent NPs with great biocompatibility and stable fl uorescence in biological environments are preferential for long-term in vivo imaging applications.
Fluorescent conjugated polymers (CPs) based NPs have recently received great attention in bioimaging applications. [25][26][27] They have been used for targeted in vitro/in vivo cellular imaging, intracellular biomolecule imaging, and in vivo small molecule imaging due to their high brightness, good photostability, low cytotoxicity as well as readily tailored optical properties. Despite of their success in bioimaging, CP NPs have not been used for real-time monitoring of tumor growth, largely due to the lack of highly emissive CPs with suitable absorption and emission profi les. The currently available CP The design and synthesis is reported for a fl uorescent conjugated polymer (CP), poly{ [4,4,9,9-

Introduction
Cancer is one of the major causes of mortality in the world and the incidence of cancer continues to increase. Effective real-time monitoring of tumor growth is critical to the success of cancer therapy and improvement of patient outcomes. [1][2][3] However, evaluating tumor growth typically relies on the gross examination of tumor size through physical examination. [ 4 ] This approach severely limits the use of clinically relevant orthotopic tumor models and gives little insight in the tumor processes being affected by the treatment. Therefore, development of techniques with ability to noninvasively image tumor growth processes would be benefi cial in the evaluation of potential antitumor strategies. In vivo imaging technologies have emerged based far-red (FR)/NIR fl uorescent NPs are mainly synthesized through chemical incorporation of narrow-band-gap moieties into CP backbones or physical blending of CP and FR/NIR fl uorescent acceptors (e.g., organic dyes, quantum dots (QDs), and CPs). [ 27c,d , 28 ] In these cases, the polymer absorption is strongly dependent on the donor component and the short wavelength is not suitable for in vivo bioimaging. Very recently, we have successfully shifted the absorption maxima of highly emissive FR/NIR fl uorescent CP NPs to ≈488 and ≈530 nm, by simultaneous incorporation of two narrow-band-gap moieties into CP backbone to realize effi cient intra-and intermolecular energy transfer. [ 29 ] As part of our continuous efforts in developing bright FR/ NIR CP NPs, in this contribution, we report the design and synthesis of poly{ [4,4,9,9- The polymer design takes into consideration that alternating strong electron-rich (IDT) and electron-defi cient (DBT) units could lead to a strong intramolecular charge transfer band at long wavelength. In addition, the four 4-(octyloxy)phenyl substitutions could minimize π stacking between conjugated backbones to favor high fl uorescence in NPs. We further fabricated the CP NPs with cell penetrating peptide, human immunodefi ciency virus type 1 (HIV-1) trans-activating transcriptional activator (Tat), on the surface (PIDT-DBT-Tat) and demonstrated their application in long-term in vivo monitoring of liver tumor growth. In vitro cell tracing studies revealed that PIDT-DBT-Tat NPs can trace HepG2 liver cancer cells over 8 d. The NP-labeled cells were further transplanted into the nude mice liver to monitor the liver tumor growth, which showed that the fl uorescent signals can be clearly detected for 27 d. Both in vitro and in vivo studies reveal that PIDT-DBT-Tat NPs are superior to commercial Qtracker 705 as fl uorescent probes. This study demonstrates for the fi rst time the feasibility of using CP NPs for real-time long-term in vivo monitoring of tumor growth.

Fabrication and Characterization of NPs
Scheme 1 b shows the schematic illustration of the fabrication of PIDT-DBT-Tat NPs. The NPs were fabricated by a modifi ed nanoprecipitation method. [ 31 ] More specifi cally, 1,2-Distearoylsn -glycero-3-phosphoethanolamine-N -[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000 ) and its maleimide modifi ed derivative DSPE-PEG 2000 -Mal were chosen as matrix. Upon addition of a well dissolved DSPE-PEG 2000 / DSPE-PEG 2000 -Mal/PIDT-DBT (1/1/1) mixture in THF solution into tenfold Milli-Q water under continuous ultrasonication, the hydrophobic PIDT-DBT and DSPE components are likely to be embedded into the particle core while the hydrophilic PEG and PEG-Mal segments are extended outside into the aqueous environments to render the NPs with abundant maleimide groups on the surface. The PIDT-DBT-Mal NPs were then conjugated with cell penetrating peptide HIV-1 Tat (RKKRRQRRRC) to afford PIDT-DBT-Tat NPs via click coupling reaction between the maleimide groups on NP surface and thiol groups of peptides at the C-turminus. The obtained nanoparticles were kept in refrigerator at 4 °C, and no obvious suspensions were observed for 2 months.
The volume average hydrodynamic diameters of PIDT-DBT-Tat NPs were studied by laser light scattering (LLS), revealing that PIDT-DBT-Tat NPs have a volume average hydrodynamic diameter of ≈56 nm with a narrow size distribution (Scheme 1 c). The size and morphology of PIDT-DBT-Tat NPs were further studied by transmission electron microscopy (TEM). As shown in Scheme 1 d, the NPs are clearly distinguished with a spherical shape, which have a mean diameter of ≈49 nm. The slightly smaller size relative to that revealed by LLS is due to shrinkage of polymeric NPs in dry state. [ 32 ] In addition, the hydrodynamic diameter of PIDT-DBT-Tat NPs does not show any obvious change even after incubation at 37 °C in phosphate-buffered saline (PBS, pH = 7.4) solution for 14 d, suggesting that PIDT-DBT-Tat NPs have excellent colloidal stability ( Figure S1, Supporting Information). Scheme 1 e shows the UV-vis absorption and photoluminescence (PL) spectra of PIDT-DBT-Tat NPs in water. The NPs have an absorption peak at ≈600 nm with an emission maximum at ≈720 nm, which gives rise to a remarkably large Stoke shift of ≈120 nm, endowing the NPs with great potentials to minimize background interference for in vivo bioimaging application. The absorption spectrum also has a long tail extending over 750 nm, allowing excitation in the FR/NIR region, which is a great advantage for in vivo imaging.

In Vitro Cell Tracing
To evaluate the performance of PIDT-DBT-Tat NPs as a fl uorescent probe, we fi rst studied their in vitro cell tracing ability. HepG2 liver cancer cells were chosen as a model cell line and Qtracker 705 Labeling Kit was used as the benchmark due to its matched emission maximum with PIDT-DBT-Tat NPs. For in vitro cell tracing studies, the HepG2 cancer cells were fi rst incubated with 2 × 10 −9 M PIDT-DBT-Tat NPs and Qtracker 705 at 37 °C for 24 h. The labeled cells were then subcultured in the absence of the probe for designated days and the fl uorescence profi les were studied using fl ow cytometry by counting 10 000 events ( λ ex = 561 nm, 710/50 nm bandpass fi lter). As shown in Figure 1 a, the effi cient labeling rate of HepG2 cells upon incubation with PIDT-DBT-Tat NPs remains ≈100% till day 2 as compared to the untreated cells. The labeling rate drops to 97.3% and 78.1% after continuous culture for 3 and 4 d, respectively. After 5 d, 53.1% of the cells are still effi ciently labeled, while the labeling rate is 23.5% at day 7. On the contrary, only 75.9% and 43.4% of Qtracker 705-treated cells are effectively labeled at day 2 and day 3, while the labeling rate further drops to 18.1% and 4.6% at day 4 and day 6 (Figure 1 b), respectively. Compared to Qtracker 705, these results suggest that PIDT-DBT-Tat NPs are more suitable for long-term cell tracing studies with higher fl uorescence intensity and longer tracing period.
The effi cient labeling of HepG2 cells after incubation with PIDT-DBT-Tat NPs was further confi rmed by confocal laser scanning microscopy (CLSM). As shown in  color-coded projection of PIDT-DBT-Tat NP-labeled cells was also obtained, revealing that the NPs are mainly distributed in cytoplasm ( Figure S2, Supporting Information). In addition, we also studied the confocal imaging of cells incubated with 2 × 10 −9 M of PIDT-DBT NPs (without Tat) under the same experimental conditions. As shown in Figure S3, Supporting Information, only very weak fl uorescence intensity can be detected from the PIDT-DBT NP-treated cells ( Figure S3b, Supporting Information) while much stronger fl uorescence signal from the PIDT-DBT-Tat NP-treated cells was observed, suggesting the much lower internalization effi ciency of the nonfunctionalized NPs. The results indicate that the conjugation of Tat peptide on NP surface is essential to enhance the internalization of NPs into living cells. [ 33 ] Moreover, the cytotoxicity of PIDT-DBT-Tat NPs was evaluated using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. As shown in Figure  S4, Supporting Information, the metabolic viabilities of HepG2 and NIH/3T3 cells show negligible difference and remain above 90% after incubation with 2, 4, and 8 × 10 −9 M PIDT-DBT-Tat NPs for 48 h. The viability of NIH/3T3 cells after incubation with 72 h still remains above 85% ( Figure S5, Supporting Information), suggesting that the NPs are of low cytotoxicity during the test period, which is benefi cial to long-term in vivo studies.

In Vivo Monitoring of Liver Tumor Growth
The ability of PIDT-DBT-Tat NPs for real-time in vivo monitoring of liver tumor growth was subsequently evaluated. Surgical operation was conducted at the abdomen of Balb/c nude mice and 4 × 10 6 of HepG2 cells labeled with 2 × 10 −9 M PIDT-DBT-Tat NPs or Qtracker 705 were directly injected into the parenchymal cells of the left lobe of liver (two groups, n = 3 for each group). Meanwhile, a nude mouse that underwent the same surgical operation without injection of labeled HepG2 cells was used as a control. After injection and suture, the in vivo fl uorescence images of these mice were recorded using an IVIS Spectrum Imaging System. As shown in Figure 3 , the suture at surgical site shows intense fl uorescence upon excitation at 640 nm. After 6 d, the suture was totally absorbed and the cut closed up with no background fl uorescence detectable from the surgical site. As shown in Figure 3 , the mice injected with PIDT-DBT-Tat NPlabeled HepG2 cells shows intense fl uorescence at the surgical site at day 6, due to the high labeling effi ciency and effi cient NIR emission of PIDT-DBT-Tat NPs. The fl uorescence signal remains detectable even after 27 d. On the contrary, no fl uorescence can be detected from the mice injected with Qtracker 705-labeled cells at day 6 under the same experimental setup. These results suggest that PIDT-DBT-Tat NPs can serve as a long-term in vivo tumor tracing probe with advantages over commercial Qtracker 705 in real-time fl uorescence imaging.
Upon 42 d postinjection of PIDT-DBT-Tat NP-labeled HepG2 cells, one mouse was sacrifi ced to collect the liver tissue. It can be clearly seen that the transplanted cancer cells have grown into solid tumors ( Figure S6, Supporting Information) and spread all over the whole liver to emit intense fl uorescent signal ( Figure 4 a). On the contrary, the liver tissues from mouse without HepG2 cell transplantation show no fl uorescent signal under the same experimental conditions. These results indicate the ability of PIDT-DBT-Tat NPs for tracing tumor growth during cancer cell proliferation and invasion without detectable interference in the process. The whole tumor was then mounted and imaged upon excitation at 590 nm using onephoton excited fl uorescence microscope. The effi cient penetration depth of fl uorescence from PIDT-DBT-Tat NPs in tumor tissues was studied by taking images layer-by-layer at a 3 µm interval. The 3D color-coded projection of deep tissue image reveals that the fl uorescent signal from the NP-labeled cells can be detected at 230 µm depth in the liver tumor upon excitation at 590 nm, proving the ability of the NIR emissive PIDT-DBT-Tat NPs in deep tissue imaging.   The ex vivo fl uorescence imaging of organs (liver, heart, intestine, spleen, kidney, and lung) at day 1 and day 7 after cancer cell transplantation was also performed as shown in Figure S7, Supporting Information. It shows that no fl uorescence could be detected from other organs except liver, suggesting the majority of PIDT-DBT-Tat NPs labeled cancer cells remain in the liver and negligible amount of them migrate to other organs during the test period. As the cells are directly injected to a particular site on liver (parenchymal cells) during the experiments, they prefer homing in the organ but not migrating to other organs.

Conclusion
To conclude, we report the design and synthesis of a fl uorescent conjugated polymer PIDT-DBT with absorption and emission profi les fallen within FR/NIR region. We further demonstrated the preparation of their NIR emissive NPs for long-term in vitro cell tracing and in vivo imaging of liver tumor growth. PIDT-DBT-Tat NPs show an absorption maximum at ≈600 nm and an emission maximum at ≈720 nm in water, which is the fi rst CP used for in vivo cancer cell tracking. Our studies have shown that PIDT-DBT-Tat NPs are superior to Qtracker 705 as fl uorescent probes. In vivo imaging results indicate that PIDT-DBT-Tat NPs can monitor liver tumor growth for more than 27 d in a real-time manner. This study demonstrates for the fi rst time the feasibility of utilizing CP-based fl uorescent probes for longterm in vivo imaging of tumor growth, which will encourage the development of NIR fl uorescent CPs for in vivo bioimaging.
Cytotoxicity of PIDT-DBT-Tat NPs : The metabolic activities of HepG2 cells were evaluated using methylthiazolyldiphenyltetrazolium bromide (MTT) assays. Cells were seeded in 96-well plates (Costar, IL, USA) at an intensity of 4 × 10 4 cells mL −1 , respectively. After 24 h incubation, the old medium was replaced by PIDT-DBT-Tat NPs suspension at concentrations of 2, 4, and 8 × 10 −9 M , and the cells were then incubated for 48 h, respectively. To eliminate the UV-vis absorption interference of the PIDT-DBT-Tat NPs at 570 nm, the cells incubated with the PIDT-DBT-Tat NPs without posttreatment by MTT were used as the control. After designated time intervals, the wells were washed twice with 1× PBS buffer and 100 µL of freshly prepared MTT (0.5 mg mL −1 ) solution in culture medium was added into each well. The MTT medium solution was carefully removed after 3 h incubation in the incubator. Dimethyl sulfoxide (DMSO) (100 µL) was then added into each well and the plate was gently shaken for 10 min at 24°C to dissolve all the precipitates formed. The absorbance of MTT at 570 nm was monitored by the microplate reader (Genios Tecan). Cell viability was expressed by the ratio of the absorbance of the cells incubated with PIDT-DBT-Tat NP suspension to that of the cells incubated with culture medium only. The viability of NIH/3T3 cells after incubation with PIDT-DBT-Tat NPs for 48 and 72 h were also carried out following the same procedures.
In Vitro Cell Tracing : HepG2 human liver cancer cells were cultured in 6-well plates (Costar, IL, USA) to achieve 80% confl uence. After medium removal and washing with 1× PBS buffer, 2 × 10 −9 M PIDT-DBT-Tat NPs or Qtracker 705 in DMEM medium were then added to the wells. After incubation at 37 °C for 24 h, the cells were washed with 1× PBS buffer and detached by 1× tripsin and resuspended in culture medium. Upon dilution, the cells were subcultured in 6-well plates containing cell culture coverslips for 2, 4, and 6 d, respectively. After designated time intervals, the cells were trypsinalized to be suspend and fi xed with 4% paraformaldehyde for 15 min. The fl uorescence intensities of cells were analyzed by fl ow cytometry measurements using Cyan-LX (DakoCytomation) and the histogram of each sample was obtained by counting 10 000 events ( λ ex = 561 nm, 710/50 nm bandpass fi lter). A batch of blank cells without any treatment was used as the control group. The suspended cells from the day 0 sample for fl ow cytometry test were also imaged using Leica TCS SP 5X upon excitation at 590 nm with a 600-800 nm bandpass fi lter.
Animals : Male Balb/c nude mice were obtained from the Biological Resource Centre (Biopolis, Singapore). Mice were housed in groups (5 per cage) and provided with standard mouse chow and water at libitum. The cages were maintained in a room with controlled temperature (25 ± 1 °C) and a 12 h light/dark cycle (light on at 7:00 am). All animal experiments were performed in compliance with guidelines set by the Institutional Animal Care and Use Committee (IACUC), SingHealth.
In Vivo Cell Tracing : HepG2 cells were incubated with 2 × 10 −9 M PIDT-DBT-Tat NPs or Qtracker 705 overnight at 37 °C. The cells were then trypsinalized and resuspended in DMEM medium at 2 × 10 7 cells mL −1 . Mice were anaesthetized with intraperitoneal injection of 50/5 mg kg −1 of ketamine/diazepam solution followed by intramuscular injection of 5 mg kg −1 of baytril preoperatively. The surgical site at abdomen was cleaned and a midline incision was made in the abdomen to expose the left lobe of liver. The cell suspension was directly injected into the parenchymal cells of the liver. Abdomen was then sutured with 5/0 maxon (monofi lament polyglyconate synthetic absorbable suture) and skin closed with 5/0 prolene (polypropylene suture). Mice were kept warm after the surgery and returned to their cages when fully awake. After designated time intervals postsurgical operation, the mice were imaged using an IVIS Spectrum Imaging System (Xenogen Co., Alameda, CA, USA) while under anesthesia using 1%-2% of isofl urane in oxygen. The fl uorescence images were recorded with 1 s exposure using a fi lter 720/20 nm upon excitation at 640 nm. The autofl uorescence was removed using the software of IVIS Spectrum Imaging System. www.MaterialsViews.com www.advancedscience.com Adv. Sci. 2015, 2, 1500008