Article pubs.acs.org/molecularpharmaceutics
Amphiphilic Biodegradable PEG-PCL-PEI Triblock Copolymers for FRET-Capable in Vitro and in Vivo Delivery of siRNA and Quantum Dots Thomas Endres,†,⊥ Mengyao Zheng,†,⊥ Ayşe Kılıç,‡ Agnieszka Turowska,§ Moritz Beck-Broichsitter,† Harald Renz,‡ Olivia M. Merkel,*,∥ and Thomas Kissel† †
Department of Pharmaceutics and Biopharmacy, Philipps-Universität Marburg, Ketzerbach 63, 35037 Marburg, Germany Institute of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps-Universität Marburg, Baldingerstraße 1, 35043 Marburg, Germany § Sterna Biologicals GmbH & Co/KG, Biomedizinisches Forschungszentrum (BMFZ), Hans-Meerwein-Straße 2, 35043 Marburg, Germany ∥ Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan 48202-3489, United States ‡
ABSTRACT: Amphiphilic triblock copolymers represent a versatile delivery platform capable of co-delivery of nucleic acids, drugs, and/or dyes. Multifunctional cationic triblock copolymers based on poly(ethylene glycol), poly-ε-caprolactone, and polyethylene imine, designed for the delivery of siRNA, were evaluated in vitro and in vivo. Moreover, a nucleic acid-unpacking-sensitive imaging technique based on quantum dot-mediated fluorescence resonance energy transfer (QDFRET) was established. Cell uptake in vitro was measured by flow cytometry, whereas transfection efficiencies of nanocarriers with different hydrophilic block lengths were determined in vitro and in vivo by quantitative real-time PCR. Furthermore, after the proof of concept was demonstrated by fluorescence spectroscopy/microscopy, a prototype FRET pair was established by co-loading QDs and fluorescently labeled siRNA. The hydrophobic copolymer mediated a 5-fold higher cellular uptake and good knockdown efficiency (61 ± 5% in vitro, 55 ± 18% in vivo) compared to its hydrophilic counterpart (13 ± 6% in vitro, 30 ± 17% in vivo), which exhibited poor performance. FRET was demonstrated by UV-induced emission of the acceptor dye. Upon complex dissociation, which was simulated by the addition of heparin, a dose-dependent decrease in FRET efficiency was observed. We believe that in vitro/in vivo correlation of the structure and function of polymeric nanocarriers as well as sensitive imaging functionality for mechanistic investigations are prerequisites for a more rational design of amphiphilic gene carriers. KEYWORDS: triblock copolymer, siRNA, nanocarrier, QD-FRET, theranostic nanomedicine
■
especially for pulmonary co-delivery of drugs and nucleic acids.6 Because of their modular design and potential to be optimally engineered, amphiphilic block copolymers capable of selfassembly to various types of colloids (e.g., micelles, nanoparticles)3,7,8 are especially suitable for multifunctional delivery of hydrophobic drugs (e.g., paclitaxel3), nucleic acids,4 and/or dyes.9 Recently, amphiphilic block copolymers comprising poly(ethylene glycol) (PEG), poly-ε-caprolactone (PCL), and PEI segments were shown to be effective, stable, biodegradable, and biocompatible nucleic acid delivery vectors, particularly for siRNA.10−12 Self-assembly in an aqueous milieu leads to nanocarriers with a core−corona structure, where the stabilizing
INTRODUCTION The development of safe and effective nonviral gene or small interfering RNA (siRNA) delivery systems represents a strong scientific barrier to clinical translation.1 Notably, the rational development of new nucleic acid delivery platforms requires knowledge of the delivery process, particularly the cellular uptake mechanism, endolysosomal escape, and payload unpackaging on a systemic and cellular level need to be understood. To gain mechanistic insights into these areas, efficient tracking and sensing formats are required that can be carried out with in vitro/in vivo experiments. Ever since the early attempts at using cationic homopolymers such as polyethylene imine (PEI) for complexation of nucleic acids, a constant evolution of vector technology led to smart multifunctional delivery vehicles.2 In this context, tailored multifunctional polymeric vectors for simultaneous transport of different therapeutic agents has gained increasing interest,3−5 © 2014 American Chemical Society
Received: Revised: Accepted: Published: 1273
December 12, 2013 January 21, 2014 March 4, 2014 March 4, 2014 dx.doi.org/10.1021/mp400744a | Mol. Pharmaceutics 2014, 11, 1273−1281
Molecular Pharmaceutics
Article
In the literature, QDs most often serve as a central nanoscaffold around which diverse functionalities are chemically tethered,18,27 or they serve for labeling via covalent linkage.19,28,29 Herein, we report a straightforward and versatile approach of tagging siRNA carriers via physical entrapment. The encapsulation of hydrophobic QDs into the PCL core of amphiphilic PEG-PCL-PEI nanocarriers combines nucleic acid delivery and imaging capabilities in a single platform. To fabricate a FRET-capable delivery vehicle designed for the elucidation of the siRNA delivery process, QD-loaded carriers were complexed with Alexa Fluor 647 (AF647)-labeled siRNA. However, depending on the application, the QDs employed in this prototype theranostic delivery system can be easily exchanged for arbitrary nanoscaled hydrophobic substances. The aim of the current study was the evaluation of a multifunctional and QD-FRET-capable siRNA delivery platform in vitro and in vivo. We believe that theranostic imaging and delivery formats have the potential to provide deeper insights into the siRNA delivery process, leading to the design of more rationally tailored colloidal nucleic acid carriers.
and protecting hydrophilic PEG shell surrounds the hydrophobic PCL core. The PCL domain can increase the affinity of the delivery system for the cell membrane, and it serves as a reservoir for hydrophobic drugs or dyes. The ratio of hydrophilic and hydrophobic segments has been shown to have a strong impact on the physicochemical and biological characteristics of these vectors.10 Although increasing PEG shell thickness is known to reduce toxicity and to increase colloidal stability,10 this often leads to poor transfection activity.13 However, pronounced stealth properties may prove advantageous for prospective receptor-mediated targeting approaches.14 PEI, located at the interface between hydrophilic and hydrophobic moieties, enables electrostatic interaction with nucleic acids.12 Polyplexes that are assembled of PEI or PEGPEI and nucleic acids exclusively by electrostatic interactions are often prone to disassembly, especially in a medium of high ionic strength (e.g., under serum conditions).15 In contrast to those systems, amphiphilic PEG-PCL-PEI carriers are formed by a self-assembly process even in the absence of counterions. In addition to its cargo functionality, the hydrophobic core region further increases the carrier stability.13,16 Moreover, carriers may be reproducibly stored in the absence of nucleic acids under dry conditions. Afterward, rehydration and nucleic acid loading can take place directly before administration.12,17 Because of their unique properties such as wideband excitation, narrow emission spectra, high quantum yields, and photo/chemical stability, luminescent semiconductor quantum dots (QDs) have emerged as powerful tools to help better understand the behavior and intracellular fate of nanoparticulate delivery systems.18 The QD emission wavelength can be tuned in the full range from the UV to near IR (NIR), allowing excitation and visualization in biological environments of cells and tissues for use as in vitro and in vivo imaging probes.19,20 The presence of a core−shell structure (e.g., cadmium selenide/zinc sulphide, CdSe/ZnS)18 and/or encapsulation into polymer matrices21 can reduce heavy-metal cytotoxicity and prevent leaching. Moreover, hydrophilic polymer coatings enable application of hydrophobic QDs in aqueous environments.22 QDs have been shown to replace conventional fluorophores as more powerful donors for fluorescence resonance energy transfer (FRET).18 FRET involves the transfer of excitation energy from a donor molecule in an excited state to a proximal acceptor owing to a suitable excitation spectrum. With two fluorophores independently attached onto a pair of distinct biomolecules, FRET has been shown to be a versatile tool for probing a variety of biological processes.23,24 The incorporation of QDFRET-based sensing formats into nucleic acid delivery carriers may be useful to provide a deeper understanding of fundamental aspects of gene therapy by functioning as a highly sensitive on/off switch for dissociation in the course of the unpacking procedure,18,25 which is a crucial step in nucleic acid delivery. On one hand, to be effective, the nucleic acid carrier has to be designed to avoid premature release and to protect its payload from degradation in the bloodstream or the endosomal compartment. On the other hand, the delivery system also needs to release the genetic materials at the target site.26 FRETbased monitoring capabilities facilitate the design of a carrier exhibiting the necessary complexation and decomplexation properties. Subsequent to carrier−nucleic acid dissociation, both fluorophores may be individually tracked (e.g., via fluorescence microscopy).
■
MATERIALS AND METHODS Reagents and Chemicals. Noncoding control dicer substrate interfering RNA (DsiRNA), DsiRNA against the human and murine housekeeping gene glyceraldehyde 3phosphate dehydrogenase (hGAPDH_DsiRNA, mGAPDHDsiRNA), and AF 647-labeled DsiRNA were supplied by Integrated DNA Technologies (IDT, Leuven, Belgium). Fetal calf serum (FCS) was obtained from Cytogen (Sinn, Germany). Heparin sodium (150 000 IE/g) was procured from SERVA Electrophoresis (Heidelberg, Germany). Firststrand cDNA synthesis kit and RNase I (no. EN0531) were obtained from Fermentas (St. Leon-Rot, Germany). QuantiFast SYBR green PCR kit, Hs_GAPDH_primer, Mm_GAPDHprimer, Hs_β-actin-primer, Mm_β-actin-primer, and DNase I were purchased from Qiagen (Hilden, Germany). Hydrophobic CdSe/ZnS core−shell QDs with an emission wavelength of 605 nm (organic QDot 605 ITK), SYBR Gold reagent, Lipofectamine 2000 (LF), and PureLink RNA mini kit were purchased from Invitrogen (Karlsruhe, Germany). Balb/c mice were purchased from Harlan Laboratories (Horst, The Netherlands). SKOV3 cells were obtained from ATCC, LG Promochem (Wesel, Germany). Additional chemicals and solvents, which are not mentioned in detail, were supplied by Sigma-Aldrich (Steinheim, Germany) at the highest grade commercially available. Polymer Synthesis and Characterization. PEG-PCLPEI triblock copolymers were manufactured by a three-step synthesis-route and characterized as described in detail elsewhere.10 Briefly, in the first reaction step, hydroxyterminated monomethyl-PEG (of different molecular weights) was employed as a macroinitiator for the tin(II) 2-ethylhexanoate (Sn(Oct)2)-catalyzed ring-opening polymerization of ε-caprolactone (amount calculated to result the designated PCL block length). Subsequently, the end group of the resulting PEG-PCL diblock copolymer was modified by reaction with acryloyl chloride in the second reaction step. In the third reaction step, linear PEI of 2500 Da was coupled onto the modified PEG-PCL copolymer by Michael-type microaddition. Block lengths for PCL and PEI were 10 000 and 2500 Da, respectively. To monitor the impact of the PEG chain length on transfection efficiency, carriers with two different PEG segment lengths (500 and 5000 Da) were chosen. 1274
dx.doi.org/10.1021/mp400744a | Mol. Pharmaceutics 2014, 11, 1273−1281
Molecular Pharmaceutics
Article
solution (BD Biosciences, San Jose, CA). Cell suspensions were measured on a FACS CantoTM II (BD Biosciences, San Jose, CA) with excitation at 633 nm and an emission filter set to 660 nm bandpass. Ten-thousand viable cells were evaluated in each experiment, and the geometric mean fluorescence intensity (MFI) was calculated as the mean value of three independent measurements. Data acquisition and analysis was performed using FACSDiva software (BD Biosciences, San Jose, CA). Results are presented as mean values ± standard deviation of three independent experiments. Transfection Efficiency in Vitro. In vitro transfection efficiency was determined by quantitative real-time PCR (qRTPCR). SKOV3 cells were seeded in 6-well-plates at a density of 1.5 × 106 cells/well 24 h before transfection. Subsequently, fresh medium (containing 10% FCS) and NS complexed with hGAPDH_DsiRNA or negative control DsiRNA at N/P = 5 were added to each well to reach a final siRNA concentration of 100 pmol/well. LF 2000 (0.5 μL/10 pmol of siRNA), a commercially available transfection reagent, served as a positive transfection control and was used with both the GAPDHtargeting siRNA and the scrambled negative control sequence. After 4 h of incubation at 37 °C, the medium was exchanged, and cells were incubated for another 20 h before they were washed with cold PBS and lysed with lysis buffer (PureLink RNA mini kit). Afterward, mRNA was isolated from culture cells using the PureLink RNA mini kit (with additional DNase I digestion, 18 U per isolation) and reverse transcribed to cDNA employing the first-strand cDNA synthesis kit on a TGradient thermocycler (Biometra GmbH, Goettingen, Germany). qRTPCR was performed by employing the SYBR green PCR kit, QuantiFast primers (Qiagen, Hilden, Germany), and a RotorGene3000RT-PCR thermal cycler (Corbett Research, Sydney, Australia). Hs_GAPDH-primers were used to quantify hGAPDH gene expression, and Hs_β-actin-primers were utilized as an internal standard to determine the relative expression levels for each gene. Calibration curves for GAPDH and β-actin mRNA were prepared by serial dilutions of cDNA of the blank sample (untreated cells). Measurements were carried out in triplicate. Results are presented as mean values ± standard deviation. Confocal Laser Scanning Microscopy (CLSM). To visualize the in vitro fate of FRET complexes in SKOV3 cells, QD-loaded carriers were prepared and complexed with AF647siRNA at N/P = 5 as described earlier. Cells were seeded at a density of 1.5 × 104 cells per well in 8-well chamber slides. Twenty-four hours after seeding, cells were treated with freshly prepared carrier complexes to reach a concentration of 50 pmol siRNA per well and incubated in medium containing 10% FCS for 4 h at 37 °C. Subsequently, cells were washed with PBS (pH 7.4), quenched with 0.4% trypan blue solution, washed again with PBS, and fixed using 4% paraformaldehyde in PBS. Fixed cells were nucleus-stained with DAPI (Molecular Probes, Eugene, OR, USA), washed with PBS twice, and embedded utilizing FluorSave reagent (Calbiochem, Merck Biosciences, Darmstadt, Germany). CLSM was subsequently performed on a Zeiss Axiovert 200 M (Jena, Germany). A diode laser (405 nm) and an argon laser (488 nm) were chosen for excitation; emission was detected in the blue (420−480 nm, DAPI), green (545−635 nm, QD), and red (>650 nm, AF647) channels. QD fluorescence was pseudocolored as green to distinguish it from red AF647 fluorescence.
Accordingly, the block copolymers PEG500-PCL10 000PEI2500 and PEG5000-PCL10 000-PEI2500 were used in the course of the following investigations. Assembly of Nanocarriers. Nanocarriers were prepared by a solvent-displacement technique as described in detail elsewhere.10 Briefly, carriers were assembled by dissolving 1 mg of polymer in 200 μL of acetone. The resulting solution was subsequently injected into 1 mL of magnetically stirred (300 rpm) double-distilled and filtered water. After injection of the organic phase, the resulting colloidal suspension was stirred for 3 h under reduced pressure at room temperature to remove organic solvent. Nanosuspensions (NSs) were characterized and used directly after preparation. QD Loading of Nanocarriers. Hydrophobic QDs were stored as a 1 μM stock dispersion in decane. Prior to use, QDs were transferred from decane to acetone by injection of 125 μL of a QD dispersion into a magnetically stirred (300 rpm) 2.5 mL phase of acetone. Subsequently, the liquid was evaporated for 3 h with stirring under reduced pressure, and acetone was refilled in 10 min intervals. The resulting reddish dispersion was used directly after preparation. To load nanocarriers, QDs in acetone were co-dissolved with polymers at various polymer/ QD ratios. To this end, 25, 50, 75, 100, and 200 μL of QDs dispersed in acetone were mixed with pure acetone to reach a final volume of 200 μL, and the respective polymers were added to reach a final concentration of 5 mg/mL. The assembly of nanocarriers (containing 1.25, 2.5, 5.0, 7.5, and 10.0 pmol of QDs per milligram of polymer) was carried out in an analogous manner to the formulation of nanosuspensions without QDs described earlier. Complexation of Nanocarriers with siRNA. Freshly prepared nanocarriers (blank or QD-loaded) were complexed with siRNA (or AF647-siRNA) at certain ratios of positively charged nitrogens per negatively charged phosphates (N/P ratio) by addition of the appropriate amount of aqueous siRNA solution (c = 100 μM) to an aliquot of NS (c = 1 mg/mL) followed by vigorous mixing. siRNA-loaded nanocarriers were characterized and used directly after preparation. Size Measurements. The average particle sizes and size distributions were determined by dynamic light scattering (DLS) using a Zetasizer NanoZS/ZEN3600 (Malvern Instruments, Herrenberg, Germany). Analyses were performed at a temperature of 25 °C using samples appropriately diluted with filtered and double-distilled water. The particle mean diameter (z ave.) and the width of the fitted Gaussian distribution, which is displayed as the polydispersity index (PDI), were calculated from data of at least 10 runs and are displayed as the mean value of three independent measurements ± standard deviation. Cellular Uptake in Vitro. Cellular uptake of nanocarriers was determined by flow cytometry measurements, as described in detail elsewhere.30 Nanocarriers were assembled in triplicate and complexed with AF647-siRNA at N/P = 5 as described earlier. SKOV3 (ATCC, LG Promochem, Wesel, Germany) cells were seeded at a density of 8 × 104 cells per well in 24-well plates 24 h prior to the experiment. Subsequently, freshly prepared carrier complexes were added to reach an siRNA concentration of 50 pmol per well. After different incubation times (15−240 min), cells were washed with phosphatebuffered saline (PBS) and incubated for 5 min with 0.4% trypan blue to quench extracellular fluorescence. Afterward, cells were detached using 100 μL of trypsin and treated with 900 μL of PBS solution containing 10% FCS. Cells were then collected by centrifugation and resuspended in 300 μL of FACS Cellfix 1275
dx.doi.org/10.1021/mp400744a | Mol. Pharmaceutics 2014, 11, 1273−1281
Molecular Pharmaceutics
Article
fluorescence values as a function of the emission wavelength were calculated from data of at least 10 reads. SYBR Gold Assay. To mimic FRET-switching functionality, fluorescent siRNA was displaced from the carrier by addition of heparin, whereas in parallel experiments with unlabeled siRNA, SYBR Gold intercalation assays were applied to quantify the degree of displacement. The QD-bearing nanoparticle batches, complexed with AF647-siRNA at N/P = 10, were standardized to the same polymer concentration to account for the absorption of the fluorescent intercalation signal by the presence of water-insoluble polymer and were added to opaque FluoroNunc 96-well plates (Nunc, Thermo Fisher Scientific, Langenselbold, Germany) for analysis. Nanocarriers without siRNA served as blank control. Samples were incubated for 10 min in the dark after adding SYBR Gold reagent and analyzed with the TECAN Safire 2 by applying 485 nm excitation and 520 nm emission wavelengths. Each data point was recorded as the mean value of 10 runs. Samples were prepared in quadruplicate, and values are presented as mean ± standard deviation. Subsequent to SYBR Gold measurements, fluorescence spectra of FRET complexes with and without heparin were recorded as described earlier.
In Vivo Experiments. Six-week-old BALB/c mice (∼20 g) were used for in vivo experiments. All animal experiments were carried out according to the German Law of Protection of Animal Life and were approved by an external review committee for laboratory animal care. On the day of the experiments, mice were anaesthetized with xylazine (13 mg/kg) and ketamine (65 mg/kg). Mice (n = 5 for each group) were intubated through the mouth and trachea with a flexible tube from a 24-gauge catheter (BD Insyte, Becton Dickinson GmbH, Heidelberg, Germany). Freshly prepared nanocarriers were complexed with mGAPDH_DsiRNA or negative control siRNA, respectively, at N/P = 5. After 15 min of incubation, NS containing 35 μg of siRNA (within a volume of 50 μL) per mouse were intratracheally instilled into the lungs. Control mice were treated with the same volume of a 5% glucose solution. After 5 days, mice were sacrificed, and lungs were inflated and fixed in situ with 4% paraformaldehyde after bronchoalveolar lavage (BAL). For the BAL, tracheae were cannulated, and lungs were rinsed twice with 1 mL of fresh icecold PBS. Samples for microscopic analysis were embedded in paraffin. The deparaffinized slices (3 μm) were embedded with FluorSave to protect the fluorophores. For measuring in vivo transfection efficiency, tissue samples were placed into a mortar together with a small amount of liquid nitrogen and ground into a powder using the pestle. After addition of 1 mL of peqGOLD TriFast (PEQLAB Biotechnologie GmbH, Erlangen, Germany) into the frozen tissue powder, the tissue suspension was incubated at room temperature for 5 min. Subsequently, the suspensions were each added to 200 μL of chloroform and mixed by shaking for 15 s. After incubation for 15 min at room temperature, the suspensions were centrifuged for 5 min at 12 000g to form a pellet, and the supernatant was transferred into a tube for mRNA isolation. After addition of 500 μL of isopropanol followed by vigorous mixing, the mRNA containing solution was stored at −80 °C for 30 min. Then, the sample was thawed and centrifuged at 4 °C and 12 000g for 10 min. The supernatant was discarded, and the pellet was vigorously mixed with 1 mL of an aqueous ethanol solution (75%). The sample was subsequently centrifuged (4 °C, 12 000g, 5 min) one more time, and the resulting pellet was stored in ethanol at −80 °C. The specimen was thawed, the supernatant was discarded, and the pellet was dried under reduced pressure for 30 min. Lastly, the pellet was dissolved in 60 μL of RNase-free water and incubated for 10−15 min at 55 °C. Samples were stored at −80 °C, and isolated mRNA was reverse-transcribed and subjected to qRT-PCR as described earlier. Fluorescence Spectra. Fluorescence spectra demonstrated and quantified FRET-induced AF647 emission upon QD excitation in the UV range. Carriers were loaded with 7.5 pmol of QDs per milligram of polymer as described earlier and complexed with siRNA at N/P = 10. To evaluate the appropriate amount of AF647 for optimal FRET efficiency, AF647-siRNA was mixed with unlabeled siRNA prior to complexation to reach various AF647/QD ratios (0, 0.25, 0.5, 1, 2, 4 mmol AF647-siRNA per milligram of polymer) at a constant N/P ratio. Uncomplexed, free AF647-siRNA was measured for comparison. Fluorescence was analyzed 30 min after complexation in opaque FluoroNunc 96-well plates (Nunc, Thermo Fisher Scientific, Langenselbold, Germany) with a TECAN Safire 2 plate reader (Männedorf, Switzerland). Emission wavelength step size was set to 1 nm. Relative
■
RESULTS To delineate a relationship between polymer structure and nanocarrier performance, the influence of the PEG shell thickness was evaluated. Two PEG chain lengths (PEG500 and PEG5000) with different abilities to shield the cationic PEI charges were investigated to demonstrate the impact on cell interaction and ultimately transfection efficiency. Intracellular uptake was quantified via flow cytometry (Figure 1). Whereas
Figure 1. In vitro cell uptake of nanocomplexes with different PEG shell thicknesses determined by flow cytometry (λex, 633 nm, λem, 660 nm); measurements were carried out in triplicate, and values are presented as the mean ± standard deviation.
PEG500-PCL10 000-PEI2500 vectors showed a rapid cell uptake within 2 h, the fluorescence of cells incubated with PEG5000 carriers remained at the baseline level. Fluorescence of nanocarriers that merely bind to the cell surface was quenched by trypan blue so that only intracellular fluorescence was measured. Both siRNA- and QD-treated samples contained 95% viable cells, as measured by flow cytometry. Therefore, no QD-mediated toxicity was observed after 2 h of incubation. Flow cytometry results were in good agreement with transfection efficiencies that were not only determined in vitro but also in vivo (Figure 2A). Nanocarriers constructed from polymers with short PEG segments (PEG500) showed superior transfection efficiency (61 ± 5% knockdown in vitro, 55 ± 18% knockdown in vivo) in comparison with the nanocarriers composed of PEG5000-PCL10 000-PEI2500 (13 1276
dx.doi.org/10.1021/mp400744a | Mol. Pharmaceutics 2014, 11, 1273−1281
Molecular Pharmaceutics
Article
Figure 2. (A) In vitro and in vivo knockdown efficiency of nanocomplexes with altered PEG segment length. In vitro transfection efficiency (SKOV3 cells) was determined by qRT-PCR. Hs_GAPDH-primers were used to quantify hGAPDH gene expression, and Hs_β-actin-primers were utilized as internal standards to determine relative expression levels for each gene. LF was used as a positive transfection control with both the GAPDHtargeting siRNA and the scrambled negative control sequence. Values are presented as the mean ± standard deviation (n = 3). (B) Fluorescence microscopy image of lung tissue instilled with carrier complexes from PEG500-PCL10 000-PEI2500 carriers and AF647-siRNA after lavage. Uptake of siRNA into the alveolar epithelium is represented by the fluorescently labeled siRNA shown in red.
± 6% knockdown in vitro, 30 ± 17% knockdown in vivo). Scrambled siRNA was used as negative control and showed the toxicity of the delivery system indirectly. Decreased normalized GAPDH expression after transfection with the negative control siRNA samples prepared with LF and PEG500 polymer (Figure 2A) indirectly suggested off-target effects or toxicity of these systems. To determine the spatial distribution of AF647-siRNA containing carriers in the lung after intratracheal administration, histology specimens of fixed lung samples were investigated by CLSM (Figure 2B), and emission was predominantly observed in the alveolar region of the lung tissue. Because all lungs underwent BAL, adsorbed nanoparticles were removed during lavage, and only intracellularly internalized particles are shown in the tissue sections. To mimic the concept of loading the hydrophobic PCL moieties for theranostic purposes, QDs were encapsulated during the solvent-displacement process. Subsequently, to obtain a co-loaded complex with FRET functionality, AF647siRNA was used for complexation (Figure 3). This was investigated solely for the well-performing PEG500-PCL10 000-PEI2500 nanocarrier. QD605 (λem, 605 nm) and AF647 (λex, 647 nm; λem, 679 nm) were chosen as a FRET donor− acceptor pair. The QD emission and AF647 excitation spectra overlap well between 600 and 650 nm (a prerequisite for successful FRET). Furthermore, the acceptor and donor are required to be in close proximity (
