MAY 2008 Volume 19, Number 5 Copyright 2008 by the American Chemical Society
COMMUNICATIONS A Novel Near-Infrared Indocyanine Dye-Polyethylenimine Conjugate Allows DNA Delivery Imaging in ViWo Andrea Masotti,*,†,‡ Paola Vicennati,†,‡ Federico Boschi,§ Laura Calderan,§ Andrea Sbarbati,§ and Giancarlo Ortaggi‡ Chemistry Department, SAPIENZA Universita` di Roma, P.le Aldo Moro 5, 00185 Rome, Italy, and Department of Morphological-Biomedical Sciences, Section of Anatomy and Histology, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy. Received September 14, 2007; Revised Manuscript Received February 27, 2008
Near-infrared (NIR) fluorescence light has been applied to monitor several biological events in ViVo since it penetrates tissues more efficiently than visible light. Dyes exhibiting NIR fluorescence and having large Stokes shift are key elements for this promising optical imaging technology. Here, we report the synthesis of a novel conjugate between a near-infrared indocyanine dye and an organic polyamine polymer (polyethylenimine, PEI) (IR820-PEI) with high chemical stability and good optical properties. IR820-PEI absorbs at 665 nm, emits at 780 nm, and displays a large Stokes shift (115 nm). Moreover, the reported conjugate is able to bind DNA, and the delivery process can be monitored in ViVo with noninvasive optical imaging techniques. These characteristics make IR820-PEI one of the most effective and versatile indocyanine dye polymeric-conjugate reported so far.
Near-infrared (NIR)-absorbing dyes represent an intriguing avenue for extracting biological information from living subjects since they can be monitored with safe, noninvasive optical imaging/contrasting techniques (1–7). Optical imaging represents a rapidly expanding field, with direct applications in pharmacology, molecular and cellular biology, and diagnostics. More sensitive optical sensors and new, powerful probes such as semiconductor nanocrystals (8–11), fluorescent proteins (12, 13), or NIR fluorescent molecules (14–16) have been synthesized and studied in the past few years. * Andrea Masotti, Chemistry Department - SAPIENZA Universita` di Roma, 00185 Rome, Italy. Tel: (+39) 06 4991 3341; Fax: (+39) 06 490631; E-mail:
[email protected]. † These two authors equally contributed to the work. ‡ SAPIENZA Universita` di Roma. § University of Verona.
While light in the visible range is routinely used for intravital microscopy, imaging of deeper tissues (>500 µm to cm) requires the use of near-infrared light, as hemoglobin and water, the major absorbers of visible and infrared light, respectively, have their lowest absorption coefficient in the NIR region (650-900 nm). The advantages of imaging in the NIR region (700-1100 nm) are numerous: the absence or significant reduction of background absorption, fluorescence, and light scattering along with high sensitivity, the availability of low-cost sources of irradiation (but no radiation is necessary for acquisition), and the versatility of different reporter probes. Numerous long-wavelength fluorophores belonging to the BODIPY (17), rhodamine (18, 19), and oxazine (20) have been used, but only cyanine dyes (21–23) have been reported to display large molar extinction coefficients, moderate-to-high fluorescence quantum yields, and a broad wavelength tunability (24–26).
10.1021/bc700356f CCC: $40.75 2008 American Chemical Society Published on Web 04/23/2008
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Scheme 1a
a
Synthesis of 1: DMF, TEA, H2N(CH2)5COOH, 85 °C 3 h. Synthesis of IR820-PEI: DMF, EDC · HCl, 1, rt 18 h.
Heptamethine dyes with a rigid chloro-cyclohexenyl ring in their moiety have increased photostability and a better fluorescence quantum yield, and also provide a useful reactive site for further chemical substitution (27, 28). However, reactions replacing the central chlorine atom, with phenol (26) or thiophenol (29) have led to chemically unstable probes (30). Only heptamethine cyanine dyes containing C-N bonds were reported to display large Stokes shifts (around 100-140 nm) and considerably stronger fluorescence that might be attributed to an excited-state intramolecular charge transfer (ICT) between the donor and acceptor in the same dyes (31). Recently, we reported an in Vitro study focusing on polyethylenimine (PEI) and PEI derivatives able to form complexes (polyplexes) with DNA and to deliver (or transfect) it to cells (32). This polyamine polymer having a high cationic chargedensity potential is able to establish electrostatic interactions with DNA and compact it efficiently and has been employed as a versatile vector for several biomedical applications (i.e., gene therapy) (33). Once penetrated into cells, PEI destabilizes the endosomal membrane due to the protonation of its numerous amino groups (“proton sponge” effect). The influx of protons and counterions into endosomes leads to membrane lysis and to the release of their internal contents (DNA). The conjugation of PEI with an NIR dye is aimed at obtaining a multifunctional delivery vector whose localization can be monitored in ViVo with noninvasive techniques and that may serve to identify potential transfection sites and assess its efficiency to deliver DNA. To obtain an NIR-PEI conjugate, we chose the recently reported indocyanine IR-820 (See Scheme 1) among the heptametine NIR dyes, since its heterocyclic nitrogen atoms bear two alkyl-sulfonate groups that improve photostability and provide a sphere of solvation in water preventing this dye from aggregation. Attempts to directly replace the meso-chlorine atom of IR820 with an amino group of PEI were unsuccessful. We hypothesized that the close proximity between the dye and the hindered polymeric structure could prevent this substitution. Thus, we
Figure 1. Visible/NIR absorption spectra of IR-820 (solid line), 1 (dotted line), and IR820-PEI dyes (dashed line) in MeOH (0.1 mM, 25 °C).
replaced the meso-chlorine atom with a less-hindered carboxyterminal amino linker (6-aminohexanoic acid), obtaining an intermediate (1) suitable for further conjugations (Scheme 1). The successful conjugation of 1 with PEI allowed us to obtain a near-infrared dye-polymer conjugate (IR820-PEI) highly soluble in water. IR820-PEI absorbs at 665 nm (Figure 1) and emits at 780 nm displaying a large Stokes shift (115 nm) (Figure 2). These characteristics make this system more suitable for use as fluorescence probes than do common cyanine dyes. To investigate the feasibility of employing IR820-PEI as a vector able to bind and deliver DNA, agarose gel retardation assays (Figure 3) were performed. IR820-PEI is able to bind DNA similarly to PEI itself, while intermediate 1, being negatively charged, has no effect, as expected. These results confirm that, when PEI is derivatized at a small percentage of substitution, it substantially retains all the physicochemical and acid-base properties of the starting polymer (32).
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Figure 2. Emission spectra of 1 (solid line) and IR820-PEI (dotted line) dyes in MeOH (0.1 mM, 25 °C). The absorption spectrum (arbitrary unit) of IR820-PEI is also reported for comparison.
Figure 3. Gel retardation assay (agarose 1%) of IR820-PEI/DNA (lane 5) compared to PEI/DNA (lane 3) and 1/DNA (lane 4). DNA ladder (lane 1) and reference β-Gal pDNA (lane 2) are also reported.
Figure 4. Optical images of a nude mouse with 2 mg/mL tail vein injection of IR820-PEI: preinjection (a), and after 1 min (b), 1 h (c), 2 h (d), and 3 h (e) from the injection. 3D reconstruction of mouse body and the organs of interest (f): 1, brain; 2, lungs; and 3, liver. Color bar on the right side indicates the signal efficiency of the fluorescence emission coming out from the animal.
The properties of the novel NIR system IR820-PEI and its ability to deliver DNA in ViVo (IR820-PEI/DNA) were assessed using a VivoVision Systems, IVIS 200 Series Imaging System (Xenogen Corporation, Alameda, CA, USA). Figure 4 shows the optical images of a nude mouse before (Figure 4a) and after 1 min (Figure 4b), 1 h (Figure 4c), 2 h (Figure 4d), and 3 h from the injection of IR820-PEI (Figure 4e). Figure 4f shows
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Figure 5. Optical images of a nude mouse with 2 mg/mL tail vein injection of IR820-PEI-DNA: preinjection (a), and after 1 min (b), 1 h (c), 2 h (d), and 3 h (e) from the injection. 3D reconstruction of mouse body and the organs of interest (f): 1, brain; 2, lungs; and 3, liver. Color bar on the right side indicates the signal efficiency of the fluorescence emission coming out from the animal.
the 3D reconstruction of the mouse body and the organs of interest (1, brain; 2, lungs; 3, liver). After i.v. injection of IR820PEI, a signal enhancement localized in the regions corresponding to the brain and liver was observed. CCD camera collects light coming out from the skin of the animal without any a priori information regarding the deepness of the sources. However, excitation and emission photons employed in our experimental sessions have a mean path before absorption of 1-2 cm, and this property depends on the optical characteristic of tissues themselves. Thus, since the light can pass up to 2 cm through the animal body, sources located up to 2 cm below the skin can be visualized. However, in order to unambiguously localize the fluorescent dye accumulated in specific anatomical districts, a much more detailed study should be taken into account. Figure 5 shows the optical images of a nude mouse before (Figure 5a) and after 1 min (Figure 5b), 1 h (Figure 5c), 2 h (Figure 5d), and 3 h from the injection of IR820-PEI/DNA (Figure 5e). Figure 5f shows the 3D reconstruction of the mouse body and the organs of interest (1, brain; 2, lungs; 3, liver). Also, in this case, after the i.v. injection of the IR820-PEI/DNA, optical images shown signal enhancement, in the region corresponding to the brain, lungs, and liver. Optical imaging data suggest that, after intravenous tail vein injection of IR820-PEI or IR820-PEI/DNA, a predominant hepatic accumulation occurs. This effect is most likely due to a nonspecific electrostatic interaction between IR820-PEI (or IR820-PEI/DNA) conjugate and the negatively charged plasmatic membranes of capillary endothelial cells (i.e., sulfated proteoglycans, glycosylaminoglycans, etc.) that may explain the initial NIR dye biodistribution thought brain and lungs (the first highly vascularized organ encountered by NIR dyes after intravenous tail vein injection and in which blood flow is slower due to the capillary circulation). After 1 h, the two NIR systems began to accumulate in the liver, a typical store organ, and after 3 h, the signal intensity increased. The signal is clearly visible even after 24 h from the injection (see Figure 1S in Supporting Information). The reported distribution observed for IR820-PEI and IR820-PEI/DNA is in good agreement with published works on other systems like NIR-nanoparticles (34–36) and NIR-nanoprobes (37). However, the fluorescence signal acquired by the optical imaging device might be due both to IR820-PEI/DNA complex and to unbound IR820-PEI. So, to discriminate between the contributions of these two species, we assessed the ability of IR820-PEI to bind a fluorescent oligonucleotide (randomized sequence) 5′-labeled with a Cy5.5 fluorochrome (Cy5.5-ODN) (Figure 6). This modified oligonucleotide emits in the red region
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In conclusion, IR820-PEI is one of the most attractive nearinfrared indocyanine dye-polymer conjugate molecules that should be further investigated for its potential use as a multifunctional system for targeted gene delivery by conjugation with selected molecules (i.e., folic acid, folate-PEG, etc.) or specific antibodies.
ACKNOWLEDGMENT
Figure 6. Gel retardation assay (agarose 4%) of IR820-PEI complexed with 0.3 (lane 3), 0.6 (lane 4), 1 (lane 5), 1.5 (lane 6), 2.5 (lane 7), and 3 nmol (lane 8) of Cy5.5-ODN. DNA ladder (lane 1) and reference Cy5.5-ODN (1 nmol) are also reported.
Authors thank Prof. M. Barteri and Dr. R. De Carolis for helpful discussions and MIUR (Ministero dell’Universita` e della Ricerca) (PRIN 2005 - prot. 2005039758) for financing. Supporting Information Available: Materials and Methods section. This information is available free of charge via the Internet at http://pubs.acs.org.
LITERATURE CITED
Figure 7. Optical images of a nude mouse with 2 mg/mL tail vein injection of PEI/IR820-DNA: pre injection (a), and after 4 min (b), 1 h (c), 2 h (d), and 3 h (e) from the injection. Color bar on the right side indicates the signal efficiency of the fluorescence emission coming out from the animal.
and not in the near-infrared region (like IR820-PEI). For this reason, a differential localization of the cDNA and the delivery vector is possible through a sequential acquisition of the fluorescence emission first in the red then in the NIR region. Unfortunately, even if a partial accumulation of IR820-PEI/ Cy5.5-ODN was observed in the liver, a spurious fluorescence emission coming out from the food ingested by the animal before the experiment was observed (see Figure 2S in Supporting Information). However, even if this experiment is neither significant nor conclusive, DNA accumulation in the liver is reported, and the region concerned is the same as that involved when using the PEI-IR820/(unlabeled)DNA complex. Therefore, in order to overcome this problem and clearly see the localization of DNA (and not that of the delivery vector), we repeated the acquisition injecting a calf thymus DNA labeled with the same NIR dye used for IR820-PEI (indicated as IR820DNA) (see Supporting Information) complexed with a solution of unlabeled PEI. In this way, it was possible to follow the fluorescence in the NIR region of the PEI/IR820-DNA complex and to obtain indications, at least in the initial stages, on DNA localization. DNA accumulated in the liver analogously to IR820-PEI/DNA (Figure 7). These results indicate, at least in a first approximation, that DNA is transported and/or delivered in ViVo in the same regions, but additional assays are required to investigate the effective transfection in ViVo of this novel synthesized system. Liver uptake or nonselective accumulation into undesired organs is an obvious problem that should be taken into account, in the near future, for the development of efficient tumortargeting therapies.
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