Hybrid Amyloid Quantum Dot Nano-Bio Assemblies to Probe Neuroinflammatory Damage

Various oligomeric species of amyloid-beta have been proposed to play different immunogenic roles in the cellular pathology of Alzheimer’s Disease. The dynamic interconversion between various amyloid oligomers and fibrillar assemblies makes it difficult to elucidate the role each potential aggregation state may play in driving neuroinflammatory and neurodegenerative pathology. The ability to identify the amyloid species that are key and essential drivers of these pathological hallmarks of Alzheimer’s Disease is of fundamental importance for also understanding downstream events including tauopathies that mediate neuroinflammation with neurologic deficits. Here, we report the design and construction of a quantum dot mimetic for larger spherical oligomeric amyloid species as an “endogenously” fluorescent proxy for this cytotoxic assembly of amyloid to investigate its role in inducing inflammatory and stress response states in neuronal and glial cell types. The design parameters and construction protocol developed here may be adapted for developing quantum dot nano-bio assemblies for other biological systems of interest, particularly neurodegenerative diseases involving other protein aggregates.

Precursor Synthesis: Prior to synthesis of CdSe cores and shelling to generate CdSe/CdS core/shell QDs, both ~1 M TOP:Se and ~0.08 M Cd-oleate were synthesized for each respective reaction.For 1 M TOP:Se, 680 mg Se pellets and 8.5 mL TOP were combined in a scintillation vial and mixed at 60 o C overnight in a glovebox to result in a clear solution.A small amount of secondary phosphines, 90 uL DPP, was added to help promote efficient QD nucleation. 1 For 0.08 M Cd-oleate, 250 mg CdO, 2.6 mL OAc, and 20 mL ODA were combined in a side-arm storage flask (ChemGlass; cat: AF-0522) and connected to a Schlenk line.The contents were degassed at room temperature, followed by 90 minutes of heating at 270 o C under an inert gas (N2) environment.The flask was then cooled to room temperature, with 1.3 mL of OAm injected at 150 o C to help prevent solidification of the product, which was stored in a glovebox until needed.
CdSe core synthesis: The CdSe cores were synthesized from an adaptation of procedures previously reported. 2,3 o a 3-neck flask was added 820 mg CdO, 16.2 g TOPO, 37 g HDA, and 3.2 g TDPA.The flask was connected to a Schlenk line and purged with N2 and then kept under this inert gas environment while the contents were heated to 90 o C. The contents were then degassed by 3 cycles of evacuation (<100 mT) and refiling with N2, followed by heating to 320 o C with rapid stirring.Cd-TDPA formation was visually determined via changes in the transparency of the solution from an opaque to translucent solution.The contents were then cooled to 260 o C, and 8.0 mL 1 M TOP:Se was rapidly injected at this temperature while maintaining the same stir rate.The CdSe cores were grown for 2 to 3 hours until the desired size was achieved, as determined by PL measurements of aliquots taken routinely after the 2 hour timepoint.The reaction was then quenched by removing the flask from heat and applying forced air to bring the solution to 200 o C before submerging the flask into a water bath to rapidly cool the solution to 100 o C. The solution was then injected with 40 mL of ButOH and allowed to cool for ~1 hour before performing several rounds of washing followed by two cycles of size-selective precipitation.The final pellet was resuspended in hexane and filtered through a 0.45 μm syringe filter to produce the CdSe stock used for shelling.

CdS shelling:
The CdSe cores were shelled with CdS following protocols previously used by our group and others. 2,3 n a 3-neck flask setup on a Schlenk line, 100nmol of the CdSe stock solution, 3 mL OAm, and 3 mL ODE were added.The flask was degassed for ~1 hr with constant stirring (~800 rpm) at room temperature.The contents were then heated to 115 o C and held at this temperature for ~20 min, followed by returning the flask to atmospheric pressure by refilling with N2 gas.The contents were then heated to 350 o C at a ramp rate of 16 o C/min, during which time the cadmium and sulfur precursors, 0.150 mmol of Cd-oleate and 0.180 mmol of octanethiol, were prepared in a glovebox.The two precursor solutions were each diluted with ODE to a final volume of 3.5 mL and loaded into two separate syringes.The syringes were then loaded onto a dual syringe pump and injected at a rate of 1.5 mL/hr once the solution reached 200 o C. The solution was held at 350 o C for shell growth, with the final shell thickness determined by taking aliquots every couple of minutes and looking at the shift in the peak position of the PL spectra.Once a desired shell thickness was achieved, the reaction was cooled to 200 o C, followed by dropwise addition of 1 mL OlAc.The solution was allowed to further anneal for 1 hr before cooling to 75 o C, before the contents were transferred to falcon tubes for three rounds of washing with hexane and ethanol.The final product is stored in the glovebox until needed.
Peptide Synthesis: The Aβ(1-16)-PEG-CG sequence, DAEFRHDSGYEVHHQK-PEG-CG, was synthesized following a traditional C-to-N solid phase procedure. 4,5 ll mixing steps are done on a rotisserie or tube rotator.First 0.1 mmol of Rink resin was loaded into a fritted syringe and swelled with DCM for 5 min on a rotator, followed by filtration and three rounds of rinsing with DMF via vacuum filtration.Deprotection solution (20% v/v piperidine in DMF) is then added to the resin and mixed for 10-15 min, followed by two rinses with DMF.This deprotection step was repeated.The resin is then loaded with the first amino acid G, by two 90 min couplings with HBTU and HOBt in DMF coupling solution containing a 5-molar excess (0.5 mmol) of all components (Fmoc-Gly-OH, HBTU, HOBt).The loaded resin is then capped with an acetic anhydride solution (20% v/v in DMF).Amino acids are then sequentially coupled with HBTU/HOBt as activators in DMF at a 5-molar excess to the rink resin (0.5 mmol) and mixed for an hour.Between each amino acid, the elongating peptide sequence undergoes two rounds of deprotection, comprised of mixing in deprotect solution for 10 minutes followed by two washes with DMF.For the Fmoc-PEG-OH coupling, HATU and HOAt were used as activators in a ~3% v/v DIPEA:DMF solution.Additionally, the lysine (Fmoc-Lys(Boc)-OH) residue coupling following the PEG amino acid was coupled twice to ensure complete strand elongation at all available sites.The completed peptides undergo a final Fmoc deprotection step, followed by cleavage from the resin using a 95:2.5:2.5 v/v/v TFA:TIPS:water solution.The resulting solution was concentrated and the peptide was precipitated via addition of cold ether.The precipitate was collected via centrifugation and dissolved in a 60:40 v/v MeCN:water mixture prior to purification by preparatory HPLC.
Peptide Purification and Characterization: Peptide purification was performed using a reverse phase Phenomenex Gemini (5 μm, NX-C18, 110 Å, 250 x 50 mm) column on an Interchim PuriFlash 4125 preparatory HPLC with a binary gradient of water and acetonitrile with 0.1% TFA at 100   ⁄ .For fraction collection, UV absorbance of the eluent was monitored at 215 nm and 254 nm.Peptide purity was confirmed using analytical HPLC with a reverse phase Phenomenex Gemini (5 μm, C18, 110 Å, 250 x 4.6 mm) column on a Shimadzu LC-2010A and via MALDI-TOF mass spectroscopy.Peptide concentration for all experiments was determined by analyzing samples via analytical HPLC for comparison to a concentration curve calibrated by amino acid analysis (Figure S2).Appropriate amounts were then aliquoted and lyophilized prior to use.
Protocol: The following details an example reaction scheme involving 1 μmol of the thiol source (Aβ(1-16-PEG-CG) and 1.6 μmol of the maleimide source (DSPE-PEG2k-Mal), however the reaction may, to our experience, be scaled up to at least 10x equivalents.
First, any disulfide bridges formed between monomers of Aβ(1-16)-PEG-CG are reduced via incubation of 1 μmol of Aβ(1-16)-PEG-CG with 10-mole excess (10 μmol) TCEP in degassed PBS pH 7.2.Specifically, 2.8 mg of TCEP is added to a 10 mL round bottom flask and 1 μmol of Aβ(1-16)-PEG-CG is dissolved in 8.7 mL of degassed PBS, then added to the flask to dissolve the TCEP.The flask is capped with a septa and punctured with two needles to allow for purging under constant flow of inert gas (N2 in this case) while stirring at 400 rpm for 18 hours.An aliquot is taken for analysis via thin layer chromatography (7.5% v:v MeOH:CHCl3) to confirm a reaction has occurred; this is indicated by the presence of a species under short and long UV irradiation with a retention factor different than both the thiol and maleimide sources.After confirmation of a product is formed, the reaction is terminated and the reaction volume is purified via dialysis using a 2k MWCO dialysis cassette (ThermoFisher; cat: 87718) using a bath solution of nanopure water and three rounds of purification (bath solution was replaced at hours 2, 5, and 24.The purified product was collected from the dialysis cassette and characterized via analytical HPLC and MALDI-TOF to confirm the observation of a delayed retention peak in HPLC and mass distribution in MALDI-TOF that would be indicative of the product (Figure S2).For MALDI-TOF, the sample was prepared in a matrix composed of α-cyano-4-hydroxycinnamic acid, 0.5% trifluoroacetic acid, and 0.1% NaCl dissolved in EtOH and measured at 60−80% power (Shimadzu Axima Performance).The final DSPE-PEG2k-Aβ solution frozen over dry ice and lyophilized for storage in -20 o C until needed.

ABQD Construction
Protocol: The DSPE-PEG2k-Aβ (0.5 μmol) was dissolved in 0.5 mL of chloroform (CHCl3) and combined with 0.05 nmol of CdSe/CdS QDs in a 1 dram vial.The mixture was sonicated for 5 minutes at power level 6 (VWR; model 250D) to homogenize the two species and disperse any QD aggregates and inverse micelles of DSPE-PEG2k-Aβ.The solution was then dried using a rotary evaporator until a paste like consistency was achieved, to which 500 uL of nanopure water was added to resuspend the solution.Pulse vortexing and sonication (3 minutes at power level 9) were used on occasion to help promote dissolution of the pellet, followed by another round of rotary evaporation to remove any residual organic solvent and drive all QDs into aqueous phase.The ABQD solution should appear transparent.The solution is then stirred for 1 hr at ~400 rpm before undergoing purification via: (i) filtration using a 0.1 μm syringe filter, (ii) ultracentrifugation of the eluent at 100k rpm, 4 o C, 45 minutes, acceleration 5 (Beckman Coulter; Optima MAX), (iii) resuspension of the pellet in 100 uL for another round of centrifugation at 16,000 rcf for 30 minutes (Beckman Couter; Microfuge 22R), (iv) the supernatant collected and passed through a size exclusion spin column (Cytiva; cat: 27513001) to remove any unencapsulated QDs or free DSPE-PEG2k-Aβ.The eluent is the purified ABQD solution that is then characterized via absorbance, photoluminescence, dynamic light scattering (DLS), and transmission electron microscopy (TEM).
DLS Analysis: Samples were spotted in duplicate into a 384-well plate and analyzed using a DynaPro system controlled using v7.10 of the DynaPro software (Wyatt).Each well of interest was measured using ten 3 sec acquisitions per run over three runs.The pooled results over analyzed using the "Legacy" settings on DynaPro for fitting.The fitted results were then imported into MATLAB for plotting as the Gaussian-fitted histogram in Figure 1.
TEM Analysis: Samples were prepared by spotting 10 uL of the ABQD solution onto an ultrathin lacey carbon-supported copper grids (mesh size 400, Ted Pella; cat: 01824).The solution was allowed to sit for 10 minutes, followed by wicking off using a Whatman #1 filter paper at a 45 o angle.This was followed by addition of 10 μL of 1% uranyl acetate as a counterstain.The counterstain was allowed to react for 1 to 2 minutes before wicking off with the same filter paper and drying under a gentle stream of inert gas (N2).The grid was then imaged on an FEI TECNAI F-20 field electron microscope with an accelerating voltage of 200 kV.

Primary Culture of Hippocampal Neurons and Astroglia
The hippocampal cells were harvested from Sprague-Dawley rats at embryonic day 18.The care and use of these animals abided by the Guide for the Care and Use of Laboratory Animals and followed protocols approved by the University Committee on Animal Resources at the University of Rochester.Hippocampi were dissected and dissociated in 0.25% trypsin (ThermoFisher; cat: 15050065) followed by seeding of the cells at either a density of 45k cells/well on poly-D-lysine coated 12 mm coverslips (Neuvitro; cat: GG-12-15H) in a 24-well plate for general immunocytochemical experiments, or at a density of 120k cells/well on poly-D-lysine coated 25mm coverslips (Structure Probe, Inc.; cat: 01002-AB) for calcium signaling experiments.Cells were first cultured in a supplemented neurobasal media (NBM, ThermoFisher; cat: 21103049) containing 2% B-27 TM supplement (ThermoFisher; cat: 17504044), 1% GlutaMAX (ThermoFisher; cat: 35050061), 25 μM glutamic acid, and 5% fetal bovine serum (FBS, Atlas Biologicals; cat: F-0500-D).The culture media was replenished every 3-4 days by aspirating off half of the conditioned media and replacing the aspirated volume with a reduced supplemented NBM containing B-27 TM without antioxidants (ThermoFisher; cat: 10889038) and 1% GlutaMAX.Cultures were kept incubated at 37 o C in a 5% CO2 environment.

Calcium Transients
Treatments: Primary rat hippocampal neuronal glial cultures were treated with 2.5 µM solution of Fluo-4 AM (ThermoFisher; cat: F14201) in NBM for 30 minutes to introduce the cell permeant calcium indicator dye.The Fluo-4 AM solution was made from a stock solution of 2.5 mM Fluo-4 AM in pure DMSO, such that the final 2.5 μM concentration in NBM results in a DMSO content of ≤0.1%.The cells were then washed with NBM for 5 minutes, followed by aspiration and 1 mL treatments of vehicle, 10 nM ABQDs, or 10 nM PEGQDs.These treatments were incubated for 10 minutes, followed by addition of 1 mL KCl in NBM, to result in a total sample volume of 2 mL and final KCl concentration of 30 mM.The KCl addition serves to induce neuronal depolarization and calcium influx.
Imaging: Coverslips were transferred to the stage of an inverted Olympus IX-70 microscope equipped with a metal halide arc lamp (X-Cite 120; Excelitas).Samples were imaged 20x magnification (Olympus UPlanApo 20x/0.70NA objective) on a CCD camera (Q imaging Retiga Exi Fast) using a GFP emission filter (488 TIRF C101581).Images were captured every 20 seconds for 12 minutes with 100 ms exposure times.Cells were placed onto the microscope right after the incubation period of the treatment groups and recordings began as frame 1 being t = 0 sec.After 10 background frames were collected, the 1 mL of KCl in NBM was added.Images were acquired using MetaMorph (Molecular Devices) and saved as .stkfiles.

BV-2 Microglial Cultures
Cell Line Maintenance: BV-2 cells were maintained similarly to as previously described in work from the Gelbard lab relevant to HIV-1 studies of microglial activation. 6Specifically, the immortalized murine microglial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, high glucose (+) sodium pyruvate (-) L-glutamine; ThermoFisher cat: 10313-021) supplemented with 1% GlutaMAX (ThermoFisher cat: 35050061), 10% fetal bovine serum (FBS; Atlas Biologicals cat: F-0500-D), 1% penicillin and streptomycin (ThermoFisher cat: 15140-122).The cells are plated onto either cell culture flasks, multi-well cell culture dishes, or glass coverslips that are coated with 100 μg/mL poly-D-lysine (PDL) in ddH2O via room temperature incubation for 1-3 hours, followed by three washes with ddH2O.For glass coverslips, 20 minutes of plasma cleaning is additionally done prior to PDL deposition to ensure sterility of the surface.The cultures are passaged between 80-90% confluency (roughly 2-3 days) by digestion of surface adhesion proteins via 0.25% trypsin-EDTA (ThermoFisher cat: 25200056) for ~5 minutes in a 37 o C + 5% CO2 cell culture incubator.Cell were passaged into fresh T-175 culture flasks at a density of ~3 million cells/flask, 24-well cell culture plates (with and without coverslips) at 50k cells/well, and 12-well cell cultures plates at 85k-100k cells/well.

S-7
Treatment of BV-2 cells: Prior to treatment of the BV-2 cultures at ~75% confluency, the cells were first serum deprived for 45 minutes in the same complete DMEM composition as for culturing, except at a reduced serum content of 1% FBS to prime the cells to be in a similar cell cycle to minimize heterogeneity within a single well.The reduced serum media was then gently aspirated off and replaced with the following treatment groups dissolved in the same reduced serum DMEM: vehicle (0.1% DMSO), 100 nM URMC-099 (from 100 μM stock in 100% DMSO), 50 nM ABQDs, 50 nM PEGQDs, 100 nM URMC-099 + 50 nM PEGQDs, 100 nM URMC-099 + 50 nM ABQDs.After ~18 hours, the treatments were aspirated off, briefly rinsed with ice cold Dulbecco's phosphate buffered saline (DPBS; ThermoFisher cat: 14040117) and processed either for RT-qPCR or immunofluorescent imaging following the protocols described in the following sections.

RT-qPCR
RNA isolation: RNA was isolated from BV-2 microglia using a PureLink TM RNA Mini Kit (Invitrogen cat: 12183018A), following the manufacturer recommended protocol with no modifications.After isolation, the purity (A260/A280 and A260/A230) and concentration (A260) of the extracted RNA was determined using a NanoDrop TM Spectrophotometer (Thermo Scientific).Only total RNA of acceptable quality (A260/A280 > 1.8, A260/A230 > 1.5) were then stored at -80 o C until cDNA synthesis for downstream applications.
cDNA synthesis: Total RNA from the BV-2 microglia was used to generate cDNA using the SuperScript III First Strand Synthesis Kit (Invitrogen cat: 18080051) following the manufacturer's recommended protocol without modifications.All cDNA syntheses included in the final qPCR analysis were synthesized using 1 μg of total RNA as a template.The final cDNA was measured for purity (A260/A280 > 2, A260/A230 > 1.8) and concentration (A260) on the same NanoDrop TM Spectrophotometer and stored at -20 o C until RT-qPCR analysis.
RT-qPCR measurement: RT-qPCR was performed using specific primers designed to target inflammation (CXCL10) and unfolded protein response (CHOP, XBP-1 splicing) pathways (Table S1) and the SYBR GreenER TM qPCR SuperMix Universal (Invitrogen cat: 11762100) kit.The manufacturer recommended protocol was followed, with the following modifications: (i) reaction volumes were scaled down to 20 μL in a 96-well format; (ii) 4 μL of cDNA synthesized from 1 μg of template total RNA was loaded in each well; and (iii) 250 nM final concentrations of paired primers in the combined solution.The 96-well plates were sealed (Microseal 'B' PCR plate sealing film; Bio Rad cat: MSB1001) to minimize evaporation from the reduced volume conditions and ran on an Agilent AriaMx RT-qPCR system.The reaction conditions were followed as prescribed from the SYBR GreenER TM manufacturer protocol.

Immunofluorescent Staining
The neuroglial co-cultures and BV-2 microglia cultures on coverslips were stained using an indirect immunofluorescent labeling protocol, with the associated pairings of primary and secondary antibodies outlined in Tables S1 and S2.Specifically, cultures were briefly rinsed with ice-cold DPBS, followed by fixation in 4 % paraformaldehyde (PFA) diluted in a 1x PBS solution for 12 minutes at room temperature and gentle rocking.The fixative is then removed and quenched with 100 mM glycine (Bio Rad cat: 1610718) in 1x PBS solution for 5 minutes at room temperature and rocking, followed by two washes with 1x PBS at the same conditions.The cells were then permeabilized using 0.25 % Triton X-100 (Millipore Sigma cat: T9284) in 1x PBS for 15 minutes at room temperature and gentle rocking, followed by one wash with 1x PBS in the same conditions.The cells were then blocked using 10 % BSA (Millipore Sigma cat: A3294) in ddH2O for 1 hr at room temperature and gentle rocking.This was followed by the primary antibody incubation step rocking overnight at 4 o C, with the primary antibodies diluted in a 3 % BSA with 0.01 % Triton X-100 in 1x PBS.The next day the primary antibody solution was aspirated off, followed by three washes with 1x PBS at room temperature rocking for 3 to 5 minutes.This was followed by incubation in the 2 o antibody solution rocking at room temperature for 1 hr protected from light (i.e.wrapping with foil).The secondary antibodies were diluted in the same composition as the primary antibodies were.This was followed by three rounds of washing in 0.1% Tween 20 (Millipore Sigma cat: P1379) in 1x PBS for 3 to 5 minutes at room temperature, rocking, and protected from light.After staining, the coverslips were mounted onto cover glass using ProLong TM Diamond Antifade Mountant with DAPI (Thermo Scientific cat: P36962 and stored protected from light until needed for imaging.was used to collect and collimate the emission through an OptiGrid structured illumination element to form a "grid" confocal image on the detector.Z-stacks were captured for all samples at either 1 μm (20x) or 0.5 μm (60x) step sizes and compressed into the extended focus view presented in the representative images used in this manuscript.The exposure time for each channel was optimized and kept the same between each sample.

OptiGrid Structured Illumination Imaging and Analysis
Volocity Image Analysis: Volocity 3D Image Analysis software (PerkinElmer) was used to analyze the collected image sets.The general workflow of A fine noise filter was used on all images before applying a set of measurement protocols.For the neuroglial cultures, a measurement protocol was designed to identify objects above a certain threshold corresponding to immunofluorescent labeled nuclei (λex = 350 nm, DAPI BPF), PSD-95 (λex = 405 nm, FITC BPF), MAP2 (λex = 488 nm, TRITC BPF), and GFAP (λex = 568 nm, Cy5 BPF), as well as CdSe/CdS QD emission (λex = 350 nm, TRITC BPF).Immunoreactive objects that were deemed as "positive" for the targets were determined using standard deviation thresholds that were at least 1 standard deviations larger than the mean intensity if the distribution was found to be normal.Intensity distributions that exhibited a skew were adapted to based on the degree of skew to ranges of 1.5 to 2.5 standard deviations above the mean.The sum of measured object intensities and spatial volume was extracted for the PSD-95, MAP-2, and GFAP objects.The MAP-2 objects were also further analyzed to extract the prevalence of dendritic beading by setting a cutoff for object volume and threshold for spheroidicity to identify true "beads."The total number of beads, nuclei, and CdSe/CdS objects were also exported for further analysis.

Statistical Analysis
All quantitative values were organized and pre-processed on Excel prior to importing the values onto GraphPad Prism 9.Each replicate value was imported.For the rescue experiments of the NVU co-culture experiments, a two-way ANOVA with Holm-Sidak post-hoc correction was used.For all other experiments, a one-way ANOVA with Holm-Sidak post-hoc correction was used.Statistical significance was defined as an adjusted p-value less than 0.05 for all analyses.Figure S4 represents a scheme for approximating the density of N-terminal Aβ42 residues exposed on the ABQD surface.A few assumptions: (i) there is approximately 100% surface coverage of the QD surface with native oleic acid ligands, (ii) the surface area can be modeled as a sphere, (iii) the intercalation of the oleic acid ligands with the lipid-PEG-Aβ(1-16) monomers during the micellar self-assembly process via hydrophobic interactions occurs at between a 1:1.5 and 1:3 ratio influenced by surface curvature and sterics.
Experimentally, we have shown via TEM (Figure 1d), that the ABQDs encapsulate 1 QD on average per micelle.Thus, we can perform an estimation of oleic acid density by approximating the number of cadmium atoms on the QD surface.Using the crystal lattice parameter for the shell CdS composition, we approximate the "surface monolayer" of CdS unit cells, and find the number of Cd atoms on the surface.Assuming a perfectly passivated system, we find the number of oleic acid ligands on the surface using a1:1 ratio of surface Cd atoms to oleic acid.This would be the ideal case and represents the maximum number of sites available for the lipid-PEG-Aβ(1-16) to intercalate and form the ABQD micellar structure.With the QDs used in this manuscript, the sizes of the ABQDs extracted from absorbance and TEM measurements results in a maximum coverage of ~166 Aβ(1-16) monomers at the surface (1:1 oleic acid to lipid-PEG-Aβ(1-16)) and a minimum of 83 Aβ(1-16) monomers at the surface (3:1 oleic acid to lipid-PEG-Aβ(1-16) ratio).
For spheroidal amyloid oligomers, the reported molar mass across the size distribution ranges from 158 kDa to 669 kDa (Noguchi, JBC 2009, PMC2781705).With an Aβ42 monomer mass of ~4.5 kDa, this results in 35 monomers to 149 monomers per oligomer.Given that structural studies via solid-state NMR have consistently shown the N-terminal residues as unstructured, these monomers are likely exposed outside of the highly structured core (Xiao, JBC 2020, PMC6956526) and implies that each monomer's Nterminal domain may be exposed.Thus, with these assumptions, our expected range falls within a similar range of what's known structurally.A representative image neuroglia treated with PEGQDs that resulted in notable binding of PEGQDs.Circled PEGQD puncta represent sites that overlap predominantly with GFAP (astrocyte) densities rather than neuronal features labeled by PSD95 and MAP2.These sites of PEGQD colocalization with GFAP likely arise from astrocyte recognition of PEGQDs akin to cellular debris and phagocytosis of such "debris" leading to some degree of astrogliosis.Nuclei are labeled in blue by DAPI.Scale bar is 22 μm.

Figure S4 .
Figure S4.Graphical presentation of QD surface coverage with DSPE:PEG2k:Aβ(1-16) based on intercalation density of phospholipids with native oleic acid ligands on QD surface.Estimation of Surface Density of N-terminal Aβ42 residues.

Figure S6 . 16 Figure S7 .
Figure S6.PEGQD treated neuroglia showing healthier post-synaptic density expression, lack of significant dendritic beading in neurons, and no aberrant astrogliosis.This results from the lack of significant binding by PEGQDs.Nuclei are labeled in blue by DAPI.Scale bar is 14 μm.
Fluorescent Imaging w/ "Grid" Confocal Microscope: The immunostained samples were imaged on an Olympus BX51 microscope excitation with a Prior Lumen 200 illumination source equipped with a Hg lamp (Prior cat: LM200B1-A) and detected with a Hamatsu ORCA-ER scientific camera.The following pairs of excitation and emission filters were used to selectively illuminate and detect biomolecular targets immunolabeled with DAPI, AlexaFluor488, AlexaFluor568, AlexaFluor647, and CdSe/CdS QD, respectively: