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A Quantum Dot-Protein Bioconjugate That Provides for Extracellular Control of Intracellular Drug Release Lauren Field, Scott A. Walper, Kimihiro Susumu, Guillermo LasarteAragones, Eunkeu Oh, Igor L. Medintz, and James B. Delehanty Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00357 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018

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Bioconjugate Chemistry

A Quantum Dot-Protein Bioconjugate That Provides for Extracellular Control of Intracellular Drug Release Lauren D. Field1, Scott A. Walper1, Kimihiro Susumu2,3, Guillermo Lasarte-Aragones1,4, Eunkeu Oh2,3, Igor L. Medintz1, James B. Delehanty1*

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Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, USA 2 Optical Sciences Division, Code 5600 U.S. Naval Research Laboratory Washington, DC 20375, USA 3 KeyW Corporation, Hanover, MD 21076, USA 4 George Mason University, College of Sciences, Fairfax, VA 22030 USA *Address correspondence to: [email protected]

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract The ability to control the intracellular release of drug cargos from nanobioconjugate delivery scaffolds is critical for the successful implementation of nanoparticle (NP)-mediated drug delivery (NMDD). This is particularly true for hard NP carriers such as semiconductor quantum dots (QDs) and gold NPs. Here we report the development of a QD-based multicomponent drug release system that, when delivered to the cytosol of mammalian cells, is triggered to release its drug cargo by the simple addition of a competitive ligand to the extracellular medium. The ensemble construct consists of the central QD scaffold that is decorated with a fixed number of maltose binding proteins (MBP). The MBP binding site is loaded with dye- or drug-conjugates of the maltose analog beta-cyclodextrin (βCD) to yield a QD-MBP-βCD ensemble conjugate. The fidelity of conjugate assembly is monitored by Förster resonance energy transfer (FRET) from the QD donor to the dye/drug acceptor. Microplate-based FRET assays demonstrated that the βCD conjugate was released from the MBP binding pocket by maltose addition with an affinity that matched native MBP-maltose binding interactions. In COS-1 cells, the microinjected assembled conjugates remained stably intact in the cytosol until the addition of maltose to the extracellular medium, which underwent facilitated uptake into the cell. Live cell FRET-based confocal microscopy imaging captured the kinetics of realtime release of the βCD ligand as a function of extracellular maltose concentration. Our results demonstrate the utility of the selfassembled QD-MBP-βCD system to facilitate intracellular drug release that is triggered extracellularly through the simple addition of a well-tolerated nutrient and is not dependent on the use of light, magnetic field, ultrasound or other traditional methods of stimulated drug release. We expect this extracellularly-triggered drug release modality to be useful for the in vitro characterization of new drug candidates intended for systemic delivery/actuation and potentially for on-demand drug release in vivo.

Keywords: drug delivery, quantum dot, fluorescence, ligand, FRET, microinjection, maltose, extracellular triggering, bacterial periplasmic binding protein


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The primary goal of nanoparticle (NP)-mediated drug delivery (NMDD) is to overcome many of the limitations associated with traditional systemic drug delivery. The major limitations are inadequate drug solubility and the need for repeated administration of large drug doses, which can be overcome by the improvement of targeted delivery combined with improved or augmented drug efficacy (reviewed in ref.

1-2

). In this context, NP drug carriers have much to

offer including their: (1) small size for enhanced circulation time and deeper tissue penetration, (2) large drug loading capacity, (3) ability to be functionalized with specific cell-targeting moieties (e.g., peptides), and (4) potential ability to facilitate the controlled, spatiotemporal release of the on-board drug cargo. 3-6 Numerous methodologies are currently under development for the controlled, on-demand active release of NP-associated cargos. These include, for example, the use of incident excitation light for the photocleavage of labile linkers7 and the application of magnetic8-9, ultrasonic10, or radiofrequency fields11 to facilitate drug release from appropriately sensitive nanocarriers. However, these drug release modalities are not without their limitations. Incident light has been used to photothermally heat AuNP- and drug-loaded mesoporous silica NPs12-13 or liposomes14-15 for on-demand drug release. While effective, the tissue penetration depth of even near infrared (NIR) light can be limited to just a few hundred microns which can preclude the use of this drug release modality in deeper tissue applications.16 The alternative, UV light, is effective at producing photo-cleavage of drug from the NP surface but the high energy causes significant DNA and tissue damage at the doses required for optimum efficacy.17 Magnetic, ultrasonic and radiofrequency fields are coupled with the generation of bulk heating of surrounding tissues and necessitates the specific targeting of the NP carriers to the site of application to avoid off-target hyperthermic toxicity.18-19 Given these aforementioned limitations, there is a continuing need for the development of effective yet innocuous modalities for on-demand drug release from NP carriers. The primary design criteria for such new modalities include the: (1) ease of NP-drug conjugate assembly, (2) stability of the complex prior to the triggering of drug release, (3) induction of drug release in a noninvasive manner that does not depend on traditional triggering stimuli (e.g., light or the application of external fields), and (4) ability to monitor the release of the drug cargo in realtime. Ideally, the triggering stimuli should be simple in structure, easy to produce, and inherently nontoxic.

Herein we have designed and functionally tested an

externally-triggered NP-based drug release system that incorporates all of these criteria. The


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system builds upon an optically-based nutrient sensor previously developed by our laboratory and comprises a central semiconductor quantum dot (QD) that serves as both a central assembly scaffold and a Förster resonance energy transfer (FRET) donor.20 Multiple copies of maltose binding protein (MBP) are assembled onto the QD surface facilitated by metal affinity coordination between a polyhistidine tract on the MBP terminus and zinc ions present in the QD shell. This means of conjugate assembly allows for fine control over the number of MBP arrayed around the QD. MBP was specifically chosen as it recognizes the α(14) glucosidic linkage present in maltose and other structurally similar sugar analogs such as beta-cyclodextrin (βCD). Accordingly, βCD-acceptor dye/quencher conjugates bound within the MBP binding pocket engage in efficient FRET with the QD donor. In this optical sensing configuration, the addition of maltose competitively displaces the βCD ligand in a concentration-dependent manner and the ensuing change in FRET tracks the binding affinity of the binding interaction between MBP and maltose.20 In the current study, we sought to determine the utility of this optical sensor design to simultaneously facilitate and report on the controlled intracellular release of βCD-dye/drug conjugates. As proof-of-concept, we conjugated βCD to the organic dye TideFluor3 (TF3) or the chemotherapeutic drug, doxorubicin (DOX). Both βCD and TF3 engage in FRET with a 520 nm-emitting QD donor when bound in the MBP binding pocket. Initial microplate-based assays showed that the QD-MBP constructs assembled with βCD-TF3 or βCD-DOX responded quantitatively to the addition of maltose with binding affinities determined for both βCD conjugates that matched well that of the MBP-maltose interaction. Upon microinjection of the preformed QD-MBP-βCD into the cytosol of COS-1 cells, the subsequent addition of maltose to the extracellular medium, coupled with its rapid cellular uptake, resulted in the time- and concentration-dependent release of the βCD-TF3 and -DOX conjugates that was easily tracked by FRET between the QD donor and acceptor. These results were confirmed by time-resolved measurements of the QD donor excited state lifetime before and after maltose-induced conjugate release. Importantly, the formed complexes were stable in the cellular cytosol over the 4 h experimental window used herein and showed no measurable release of the βCD conjugate prior to the addition of extracellular maltose. Further, the rate of maltose-induced intracellular release of the βCD cargo matched well with the known affinity of maltose binding to sugar


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transporters.21-23 These results method new concept for the controlled extracellular actuation and release of intracellular drug cargo complexes. RESULTS AND DISCUSSION Design and rationale of extracellularly-controlled nanobioconjugate drug delivery system The aim of this study was to design and implement a NP-protein bioconjugate delivery system wherein intracellular drug release from the conjugate could be actuated by the simple addition of a triggering molecule to the extracellular environment. To do this, we engineered a multicomponent, self-assembled drug delivery system utilizing a QD as the central NP scaffold. The QD surface was appended with multiple copies of an MBP carrier where coordination of the protein to the QD was driven by a polyhistidine motif present on the MBP terminus (Figure 1A). This QD-MBP configuration was specifically chosen as it takes advantage of the unique individual physicochemical properties of the QD and MBP as well as the advantageous attributes of the QD-MBP conjugate. The relevant QD properties include their: (1) large surface area-tovolume ratio for maximal cargo loading, (2) controlled, ratiometric self-assembly with histidinetagged peptides and proteins, (3) large quantum yields coupled with high photostability for intracellular tracking, and (4) size-tunable photoluminescence (PL).24-25 These latter attributes make QDs ideal FRET donors for distance-dependent measurements and the tracking of bioconjugate stability/dissociation. The histidine-tagged MBP, once bound to the QD surface, presents its binding pocket in an orientation that avails its loading with dyes or drugs that are conjugated to the cycloamylose, βCD (vide infra).26 Critically, once the ensemble construct is delivered to the cellular cytosol, the addition of a βCD analog to the extracellular medium, coupled with its cellular uptake, induces the specific and competitive displacement and release of the βCD conjugate cargo to the cytosol.


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Figure 1. Design and function of QD-MBP-βCD-dye-and -drug conjugate systems. A) Schematic of the QD-MBP-βCD-TF3 conjugate system. The βCD-TF3 conjugate (hexagon) bound within the MBP binding pocket mediates FRET-sensitized emission of the TF3 acceptor from the photoexcited QD donor. MBP is bound to the ZnS shell of the QD surface by a terminal poly-histidine tail. When these constructs are microinjected into cells, the addition of maltose to the extracellular medium (followed by its internalization by membrane transporters) displaces the βCD-TF3 resulting in increased photoluminescence (PL) from the QD donor and reduced emission of the TF3 acceptor. B) Schematic of the QD-MBP-βCDDOX “on demand” drug delivery system. The βCD-DOX drug conjugate bound within the MBP binding pocket quenches QD PL with minimal emission from DOX. Release of the βCD-DOX is achieved as described above. C) Absorbance and emission spectra for the 520 nm QD donor and both the TF3 and DOX acceptors used in this study. Both the QD-TF3 and QD-DOX donor-acceptor pairs show significant spectral overlap between QD donor emission and acceptor absorption. D) 520 nm emitting QDs capped with CL4 ligands were assembled with increasing ratios of MBP and subjected to gel electrophoresis. Addition of increasing MBP/QD results in attenuated migration of the negatively-charged QDs toward the cathode. ACS Paragon Plus Environment

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This self-assembly design reflects several key elements that are critical to the overall functionality of the conjugate system. First, it utilizes the well-known and thoroughly characterized QD-histidine assembly process that has been shown to facilitate ratiometric assembly of numerous cargos onto the QD surface.27-29 Histidine tags interact with the Zn2+ present in the QD shell via metal-affinity coordination and they can be easily engineered into peptides or proteins. The assembly is rapid (complete within minutes) and has been shown to be stable for days in cellular environments, including the cytosol30-32 and the endolysosomal pathway.30, 33-34 Importantly, histidine-based assembly allows for peptide-mediated QD delivery to specific cellular locations for labeling and tracking31, 33, 35-36, sensing37, and drug delivery38 in addition to being minimally toxic.35 Of particular relevance to this study, histidine-tagged MBP has been shown to self-assemble to the surface of CdSe/ZnS (core/shell) QDs with fine control over the final number of MBP conjugated to each QD.20,

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Second, MBP’s binding pocket

specifically recognizes the α(14) glucosidic linkage present in maltose with a binding affinity of ~0.9 µM.40 As βCD also contains this same α(14) glucosidic linkages, it binds to MBP with a similar affinity (1.8 µM).41 Thus, βCD serves as a structural maltose analog and dye or drug photoexcited acceptors conjugated to βCD can engage in efficient FRET with the QD donor. Finally, upon microinjection of the assembled conjugates into the cytosol of cells, followed by the extracellular addition (and subsequent cellular uptake) of maltose, displacement of the βCD ligand results in the release of the dye or drug ligand with spatiotemporal control. Figure 1 shows the two designs used in the current study where βCD conjugates of the organic fluorophore TF3 (Figure 1A) or the chemotherapeutic drug DOX (Figure 1B) were used to occupy the MBP binding pocket. A unique feature of the two systems described here is their use of FRET to monitor the realtime intracellular association and release of the βCD ligand from the MBP binding pocket. Figure 1C shows the absorption and emission spectra for the 520 nmemitting QD donor and the TF3 or DOX acceptors. QDs with emission centered at 520 nm were specifically chosen for this study as their absorbance (which increases into the UV) allows for their excitation at a wavelength that is far removed from the absorbance of the TF3 and DOX acceptors. Additionally, the QD emission has significant spectral overlap with the absorbance of TF3 and DOX while it does not overlap with their emission. To confirm the efficient assembly of MBP to the current QD scaffold, agarose gel electrophoresis was performed on QD-MBP assemblies. 520 nm-emitting QDs capped with


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compact ligands (CL4) were assembled with increasing ratios of histidine-tagged MBP. The CL4 capping ligand, which was purposefully selected for its small size and its ability to mediate efficient intracellular FRET, imparts an overall net negative charge onto the QD surface.42 Figure 1D shows the attenuated migration of QDs through the gel matrix toward the cathode as a function of increasing MBP/QD ratio. This analysis showed that maximal MBP loading of the QD surface was accomplished at a ratio of ~10 MBP/QD, consistent with structural simulations and results from previous studies.39, 43 At ratios below five MBP/QD discrete bands were seen corresponding to QDs assembled with one and two copies of MBP while at higher ratios the QD remained largely excluded from entering the gel. These results are indicative of saturation of the QD surface with the MBP and are consistent with gel analyses performed elsewhere.44 Based on our previous studies on peptide and protein packing onto the QD surface, a ratio of ten MBP/QD was used for all subsequent experiments to ensure maximal QD surface loading.43

Characterization of the FRET efficiency of QD-MBP-βCD conjugates To understand the efficiency of assembly of the QD-MBP-βCD conjugate systems, we first confirmed and quantified the FRET efficiency (FRETE) of the fully assembled conjugates. Figure 2A shows the resulting spectral traces collected from the QD-MBP-βCD-TF3 conjugate system. It was apparent that an increase in the number of MBP-βCD-TF3 assembled onto the QD surface resulted in a concomitant decrease in QD donor luminescence coupled with an increase in the sensitized emission from the TF3 acceptor over the ratio of 0-10 MBP/QD. Control experiments were performed to ensure that the passivation of the QD surface by binding of the MBP polyhistidine motif was constant for each MBP/QD ratio (i.e., the QD surface was occupied with 10 MBP whether or not all the MBP were loaded with βCD conjugates; see Experimental). Using these spectral data, the emission maxima for the donor and acceptor were plotted using Eq. 1 (in Experimental) to determine the FRETE at each TF3/QD ratio. The R0 (the QD-TF3 separation distance at which the FRETE is 50% in an assembly consisting of one QD donor and one TF3 acceptor) for the QD-TF3 donor-acceptor pair was calculated to be 4.7 nm. These data were then analyzed using Eq. 2 (in Experimental), which relates the FRETE to R0 to determine the calculated QD donor-TF3 acceptor center-to-center separation distance (rDA) for each ratio of MBP-βCD-TF3/QD. This equation takes into account the enhanced FRETE due to the contribution of increasing numbers of acceptors, n, assembled onto the QD surface.43, 45 As


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shown in Figure 2B, the FRETE clearly tracked with the number of βCD-TF3 acceptor complexes arrayed around the central QD donor, with maximal FRET efficiency of ~33% obtained at a ratio of 10 MBP-βCD-TF3 per QD. Further, this analysis resulted in a calculated

Figure 2. FRET analysis of the QD-TF3 and QD-DOX donor-acceptor pairs in QD-MBPβCD conjugates. (A) Emission spectra of the CL4-capped 520 nm-emitting QD-MBP-βCDTF3 conjugates loaded with increasing ratios of MBP-βCD-TF3. Each spectra corresponds to the average number of MBP-TF3 (MBP pre-loaded with βCD-TF3) per QD and the balance of the QD surface (up to a ratio of 10 MBP) was made up with unloaded MBP. This was done to correct for any effects of surface passivation on QD PL. Spectra show decreasing QD PL and increasing TF3 PL that correlate with increasing loaded MBP per QD. (B) Plot of the QDMBP-βCD-TF3 FRETE versus the number of loaded MBP per QD. Line is fit to Eq. 2 in Experimental. (C) Emission spectra of the QD-MBP-βCD-DOX system with increasing ratios of loaded MBP per QD showing decreasing QD PL that correlates to increasing loaded MBP. (D) Plot of the QD-MBP-βCD-DOX FRETE as function of increasing loaded MBP per QD. Line is fit to Eq. 2 using the calculated donor-acceptor separation distance values for each ratio.


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center-to-center QD to TF3 distance (βCD-TF3 within the MBP binding pocket) of ~7.8 nm. Taking into account the ~1.4 nm linker between the βCD and TF3, this calculated distance is consistent with the size of the QDs used (~2 nm radius (SI Figure S7)) and MBP (~3.5 nm radius46) which predicts an approximate 5.5 nm separation distance. Similar analysis was performed on the QD-MBP-βCD-DOX conjugate system. As evidenced by Figure 2C, the decrease in QD donor luminescence again tracked with increasing numbers of MBP-βCD-DOX acceptors arrayed around the QD. In contrast to the TF3 system, however, negligible reemission from the DOX acceptor was observed as a function of QD quenching. This is not surprising given the modest molar absorptivity of DOX (ε = 1.0 x 104 M1

cm-1) and the dependence of DOX intercalation between the DNA base pairs for strong DOX

emission.47-48 Nevertheless, DOX clearly quenched the QD donor in a ratiometric, dosedependent fashion. Analysis of the FRET efficiency as a function of the number of acceptors revealed a considerable difference in the calculated center-to-center distance for the QD-DOX system compared to the QD-TF3 donor-acceptor pair. The separation distance for the QD-DOX pair was determined to be ~4.3 nm, which is ~3.5 nm shorter than that determined for the QDTF3 pair. In this instance, it is likely that the flexible carbon spacer (linear length ~1.4 nm) could allow the DOX to fold back to the βCD, and thus closer to the QD surface. This fact, coupled with the amphiphilic nature of DOX49 and the role of the βCD as an efficient host for amphiphilic cargos (including DOX),50 presents the possibility that the DOX associates with the βCD in a host-guest configuration despite the physical link, resulting in the comparatively shorter QD-DOX separation distance we observed here.

Microplate-based maltose-induced actuation efficiency of QD-MBP-βCD conjugates Having successfully demonstrated that the QD-MBP-βCD bioconjugates engaged in efficient FRET, we next determined the sensitivity and competitive responsivity of the QDMBP-βCD-conjugates to the addition of maltose. To do this, microplate-based assays were performed in which MBP was first loaded with either the βCD-TF3 or βCD-DOX conjugate (at a ratio of 1:5 MBP:βCD conjugate) to ensure complete loading of the binding sites in the MBP. After removal of unbound βCD conjugate (see Experimental), the loaded MBP was then assembled onto CL4-capped 520 nm-emitting QDs (ratio of 1:10 QD:MBP) to form the full ensemble conjugate. Replicate wells of the microplate were loaded with the assembled


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complexes and the system was excited at 400 nm to minimize direct acceptor contribution while spectra of all wells were collected from 450 nm to 750 nm (data not shown). This was performed as an internal reference control to ensure that all of the replicate wells yielded uniform peak baseline intensities prior to the addition of maltose to induce the release of the βCD from the MBP binding pocket.

Maltose was then added to the wells such that the final maltose

concentration ranged from 0 nM to 20 mM. Immediately after delivery the spectra were again

Figure 3: Responsiveness of QD-MBP-βCD-TF3/DOX systems to the addition of maltose. A) Emission spectra of the QD-MBP-βCD-TF3 complexes after addition of increasing concentrations of maltose. Displacement of the βCD-TF3 results in increasing QD PL and decreasing TF3 PL that tracks with increasing maltose concentration. B) Plot of the QD-MBP-βCD-TF3 FRET efficiency determined from QD PL loss versus maltose fit to a sigmoidal binding isotherm. C) Emission spectra of the QD-MBP-βCD-DOX complexes after maltose addition that shows increasing QD PL that correlates to increasing maltose concentration. B) Plot of the QD-MBP-βCD-DOX FRET efficiency as a function of maltose concentration. Curve is fit to sigmoidal binding isotherm function. ACS Paragon Plus Environment

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collected over the same range. Figure 3A shows the resulting spectra for the response of the βCD-TF3 system to the addition of increasing concentrations of maltose. There was a clear increase in QD emission and decrease in TF3 luminescence that tracked with the increasing maltose concentration added to the wells. This provided strong evidence of the dose-dependent, competitive displacement of the βCD-TF3 conjugate from within the MBP binding pocket by the exogenous maltose. The calculated FRETE, when plotted as a function of concentration (Figure 3B), showed an apparent binding dissociation constant (Kd) for the MBP-βCD interaction of ~45 µM, which is only slightly lower than the micromolar affinity of MBP for maltose reported in the literature51-53 (likely due to the influence of the attached TF3 dye). Comparable results were obtained with the maltose concentration-dependent studies on the QD-MBP-βCD-DOX conjugate (Figure 3 C,D). In this conjugate system, only an increase in QD PL emission was observed (vide supra). Importantly, the fit of the data to the binding isotherm resulted in a similar Kd value (~65 µM) for that determined for the TF3 system, indicating similar displacement efficiencies in both QD-MBP-βCD conjugate systems in response to the addition of maltose. Control experiments to monitor the dissociation of the βCD-TF3 or βCD-DOX cargo from the MBP binding pocket in the absence of added maltose showed negligible release of the cargo (95%). The structure of the final βCD-DOX conjugate is shown in Supporting Information (SI Figure S1). Cellular proliferation assays comparing the toxicity of βCD-DOX to free DOX showed that the bioactivity of the βCD-DOX conjugate was within 10% of the free DOX (SI Figure S7D).

Preparation of QD-MBP-βCD Conjugates Initial studies were performed to ensure that the MBP was fully loaded with the βCD conjugates before complexing them with the QD surface. First, aliquots of MBP (in 20% glycerol) were subjected to buffer exchange to increase protein concentration and to remove glycerol. MBP solutions (500 µL) were loaded into 30 kDa Amicon® Ultra 0.5 mL filters (Millipore, MA) and centrifuged for 10 min at 14K rpm. This procedure was repeated two subsequent times, washing with HBSS each time. The concentrated MBP solution was spun off the filter at 1000 rpm for 2 min. To generate QD-MBP- βCD-TF3 or -DOX conjugates, MBP (50 µM) was first incubated with 250 µM βCD-TF3 or βCD-DOX (23 µL total volume) at 4oC overnight. Prior to FRET analysis or microinjection, this solution was diluted to 500 µL and subjected to buffer exchange (as above) to remove unincorporated βCD-conjugates. For cellular injections, the MBP- βCD conjugates were subsequently incubated with 5 µM 520 nm CL4capped QDs for 1 h at room temperature (at a 1:10 QD:MBP final ratio).

Analysis of QD-MBP-βCD Conjugate Responsiveness Microplate-based FRET assays were first performed to determine the responsiveness of the QD-MBP-βCD-TF3 or -DOX constructs to varying concentrations of maltose. Unfiltered βCD-loaded MBP was assembled onto 520 nm-emitting QDs (7.5 pmol QD; ratio 1:10 QD:MBP) for 1 h at room temperature. Control solutions containing TF3 alone were included for all ratios to determine the contribution of direct TF3 (acceptor) excitation, this was not


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performed for the DOX constructs as DOX has negligible direct emission/emission in the absence of DNA at the concentrations used here. The constructs were excited at 400 nm using a Tecan Infinite M1000 microplate reader and emission spectra were collected from 450-700 nm for the TF3 constructs and from 450-600 for the DOX constructs. Initial spectra were collected from each well to ensure uniformity across the wells. Then, increasing concentrations of maltose were added to the wells at a 1:1 (v:v) ratio so the

Eq 1

final

concentration of maltose in each well equated to a range from 50 nM to 20 mM. The spectra were then measured and plotted for each maltose concentration. The FRET efficiency (FRETE) at each maltose concentration was determined using the following equation: FRET = 1 −

F

F

where FDA and FD are the fluorescence intensities of the QD donor in the presence and absence of the βCD-TF3 or -DOX acceptor, respectively. The emission profiles of the TF3 spectra were normalized to the auto-fluorescence produced by corresponding TF3 concentrations alone. To further analyze the FRETE, increasing amounts of loaded MBP were complexed with 7.5 pmol of QD at ratios from 1:1 to 1:10 (QD:MBP). To account for any effect of surface passivation by MBP on QD PL, control experiments were performed where various ratios of loaded/unloaded MBP were assembled onto the QD surface. The spectra were measured to determine the FRETE over increasing ratios of βCD-TF3 or -DOX per QD. The QD-TF3 or -DOX separation (r) distance for each ratio was determined according to the following equation: =

1 +

Eq 2

where R0 is the calculated Förster distance for the donor-acceptor pair. R0 were determined for each pair as in

72

where the κ2 value was set to 2/3 for randomly assembled ensembles. Data

analysis was performed in Excel (ver. 14.0).


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Microinjection Adherent COS-1 cells were injected using an Eppendorf FemtoJet® 4i microinjector controlled by an InjectMan® 4 micromanipulator in a manner similar to that described previously.73 Cells were prepared for injection by washing once with HBSS to remove excess media, then allowed to incubate for 5-10 minutes in fresh HBSS. The solution was then replaced with fresh HBSS prior to injection. The microinjection tip (Eppendorf Femtotips) was loaded with 5-10 µL of the 5 µM QD-MBP-βCD-TF3 or -DOX constructs with excess βCD-TF3 or DOX removed by filtration. Injection time (0.48 sec) and pressure (300 hPa) was maintained for uniformity of injection volume and to produce an even distribution of QDs throughout the cytosol. As an internal control for the QD-MBP-βCD-DOX injections, red-emitting 655 nm QDs capped with CL4 at a concentration of 300 nM were added to the complexes prior to filtration and loading into the tip.

Maltose Delivery A minimum of 50 cells were microinjected with either the QD-MBP-βCD-TF3 or -DOX constructs and the dishes were transferred to a Nikon A1RSi confocal microscope and placed within a humidified stage-top incubator maintained at 37 °C. For each time course, five locations were chosen per plate and seven slices (each 1.5 µm) were taken per z-stack and 2X averaging was performed per 512 X 512 pixel image. This timing regimen allowed for an entire set of images to be captured in 1 minute, 43 seconds with time points taken every two minutes for a total of 30 minutes for each maltose delivery. Maltose solutions in HBSS were added between the final image of the first time point and the beginning of the second time point so that the final concentration of maltose in the plate ranged from 1.25 mM to 30 mM.

Image Acquisition and Analysis Images were captured using the spectral imaging modality on the Nikon A1RSi confocal imaging system. Briefly, a 32 PMT array captured the emission intensity over a range of wavelengths as designated by the experimental setup. Three wavelength 'bin' sizes were available for use: 2.5, 6, and 10 which facilitate capture of a maximum wavelength range of 80, 192 and 320 nm, respectively, where the PMT spectral range extends from 400 nm to 750 nm. For our experimental setup, ranges were chosen to include the QD peak and the TF3 (470 nm - 660 nm)


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or the QD and the red QD control (480 nm - 740 nm), and given the scale of these ranges the largest bin size (10 nm) was chosen. The images produced by this setup were compilations of the intensities for each of the PMTs false colored for their delineated wavelength range. The pinhole was set completely open to compensate for the inherent dimness of the spectral image acquisition. As each PMT recorded a maximum intensity associated with its designated wavelength range, a spectral intensity profile was produced for each pixel within the acquired image. To analyze this data, specific regions of interest (ROIs) were chosen to focus on the cytosolic environment and the spectral profiles for the brightest three slices (of the 7 originally captured) were exported into Microsoft Excel (ver. 14.0). These were then summed together to give the overall intensity for that ROI at that time point. Data was compiled for all time points and either the donor/acceptor ratio (TF3 samples) or the normalized donor PL increase of the QD peak (DOX samples) was calculated for each ROI and averaged over all the individual ROIs taken at each concentration. Donor PL increase was calculated using Eq. 1, and the 520 nm QD peak intensity was normalized to the 655 nm QD peak intensity (used as an internal control), which should not be affected by maltose delivery and remain constant throughout experimental acquisition. Analysis was performed in Excel and graphs were plotted using GraphPad Prism (ver. 7).

ASSOCIATED CONTENT Supporting Information. Supporting Information is available free of charge on the ACS Publications website. Additional experimental details include the synthesis of maltose binding protein, gel electrophoresis, cell culture, and fluorescence lifetime imaging.

Acknowledgements The authors acknowledge the NRL NSI and Base Funding Program (Work Units MA041-06-41T008-15 and MA041-06-41-4943). L.D.F. was a Ph.D. candidate in the Fischell Department of Bioengineering, University of Maryland, College Park, MD, USA and is currently supported by a postdoctoral fellowship from the National Research Council. The Maryland NanoCenter and its NispLab supported TEM measurements.


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A Quantum Dot-Protein Bioconjugate That ... - ACS Publications

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