Design and Fabrication of Streptavidin-Functionalized, Fluorescently

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Design and Fabrication of Streptavidin-Functionalized, Fluorescently-Labeled Polymeric Nanocarriers Ami Jo, Rui Zhang, Irving Coy Allen, Judy S. Riffle, and Richey M. Davis Langmuir, Just Accepted Manuscript • Publication Date (Web): 04 Nov 2018 Downloaded from http://pubs.acs.org on November 4, 2018

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Design and Fabrication of StreptavidinFunctionalized, Fluorescently-Labeled Polymeric Nanocarriers Ami Jo1,4, Rui Zhang2,4, Irving C. Allen3,4, Judy S. Riffle2,4, Richey M. Davis*1,4

1Department

of Chemical Engineering, Virginia Tech, Blacksburg, VA 24061

2Department

3Department

of Chemistry, Virginia Tech, Blacksburg, VA 24061

of Biomedical Sciences & Pathobiology, Virginia Tech, Blacksburg, VA 24061

4Macromolecules

Innovation Institute, Virginia Tech, Blacksburg, VA 24061

KEYWORDS polymer nanoparticles, flash nanoprecipitation, streptavidin, antibody, fluorescence

ABSTRACT

Targeted drug delivery has great potential for improving therapeutic outcomes for many diseases. Polymeric nanocarriers can improve the targeted delivery of insoluble and toxic drugs

ACS Paragon Plus Environment

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but, to achieve this, it is important to tailor the particle properties. In this study, nanoparticles comprised of poly(ethylene oxide)-b-poly(D,L-lactic acid) (PEO-b-PDLLA) were made by flash nanoprecipitation while varying the compositions of the additives poly(L-lactic acid) (PLLA), a fluorophore 6,13-Bis(triisopropylsylylethynyl) pentacene (TIPS), and poly(acrylic acid)-bpoly(D,L-lactic acid) (PAA-b-PDLLA) to characterize their effects on size, zeta potential, fluorescence, and surface functionalization. The particle size was readily increased by addition of PLLA homopolymer up to ~ 40 wt% without significant change to the zeta potential. The maximum nanoparticle fluorescence was at 0.5 wt% TIPS based on the PDLLA core and exhibited quenching that could be described by Forster resonant energy transfer. The cores of the particles

were

coupled

with

streptavidin

through

1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimide coupling chemistry. Even without the added carboxylate groups from the PAA, the base PEO-b-PDLLA nanoparticles were conjugated with streptavidin at comparable levels while retaining the functionality of streptavidin for further biotinylated ligand binding.

INTRODUCTION Polymeric nanoparticles have received much attention as delivery systems for therapeutics including small molecule drugs

1-2,

peptides3, and nucleic acids.4-5 For targeted delivery to be

effective, it is important to control the nanoparticle features that affect how they interact with cells, are transported in vivo, and can be imaged. This study is an investigation of the effects of composition on the size, zeta potential, and fluorescence of particles made using flash nanoprecipitation (FNP). Moreover, we explore a method for functionalizing the nanoparticles with biotinylated targeting and imaging ligands by attaching streptavidin (SA) to the particle surface.


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FNP is a method where an organic stream containing the polymer and therapeutic payload is mixed with an aqueous antisolvent at a time scale much shorter than that used in conventional precipitation processes, producing conditions of high supersaturation leading to high drug loadings and controlled particle sizes.6-8 Most of the work using flash nanoprecipitation has been done with amphiphilic block copolymers due to the steric stabilization provided by a poly(ethylene glycol) (PEG) block. However, poly(lactic-co-glycolic acid) (PLGA) alone has also been used to make nanoparticles containing curcumin at loadings as high as 47% for oral administration.9 Multiple methods have been used to further increase drug loadings in FNP such as increasing the hydrophobicity of the drug by covalent conjugation

10-12

or ion pairing.13-14

Compared to other methods of fabrication such as emulsion or traditional nanoprecipitation, FNP has the advantage of being scalable and partially continuous for materials that can withstand high shear conditions. Control of particle size is critical for drug delivery. The recent model by Pagels, et. al. that predicts the size of particles made by FNP as a function of the core and block copolymer concentrations is an important advance.15 Additional tests of this model would help validate it as an important tool for designing particle size. While there is a large body of literature using imaging agents to track particles made with methods other than FNP

16,

there is a growing number of studies on the encapsulation of

hydrophobic imaging agents into particles made with FNP. Metal oxides 17, fluorescent dyes 1819,

and quantum dots

20

have been successfully incorporated into polymer matrices by FNP.

Optical imaging using small molecule fluorescent dyes has particular advantages such as the ease of use, biocompatibility, and lack of ionizing radiation. FNP also allows for ease of combining imaging agents and therapeutics into particles such as beta-carotene (a model compound for a


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therapeutic) as well as an inorganic imaging agent (gold) simultaneously into a PCL-b-PEG nanoparticle.21 To address the poor photostability of typical small molecule fluorophores and insufficient distribution of high molecular weight fluorophores, Pansare et al. encapsulated a customsynthesized pentacene derivative fluorophore, Et-TP5, in particles comprised of blends of the diblock of poly(ethylene oxide)-b-poly(styrene) (PEO-b-PS) and polystyrene homopolymer.18 The nanoparticle exhibited maximum fluorescence at 2.3 wt% Et-TP5 loadings with respect to the poly(styrene) core. At higher loadings, the fluorescence began to drop rapidly due to selfquenching effects from Forster Resonance Energy Transfer (FRET). A model for calculating the Forster radius for a hydrophobic fluorophore dispersed in a polystyrene core was tested and shown to predict well the optimum dye loading. Further tests of this model with a different polymer system would be useful to show this is a general approach for designing particle fluorescence. The lack of availability of the custom-synthesized Et-TP5 motivated our investigation of a related commercially available pentacene derivative. Pentacene derivatives have largely been studied in the electro-optical physics field as an organic semiconductor alternative to typical inorganic semiconductors. These molecules not only have high charge carrier mobility but also high photoconductivity and luminescence. By functionalizing side groups, different properties can be manipulated, making these materials useful in opto-electronic devices.22 Pansare et al. used a homopolymer of polystyrene to keep the mass ratio of polymer diblock to hydrophobic components (PS + EtTP5) constant at 50:50 during their studies. They varied the total solids concentration of their organic phase injected into a 4-jet multi-inlet vortex mixer and found that the particle size increased with increasing solids concentration. More recently, Pagels et al.


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developed a model for nanoparticle formation using FNP and tested it with blends of poly(lactic acid) (PLA) homopolymer and PEO-b-PLA diblocks and blends of homopolymer polystyrene (PS) and the diblock PEO-b-PS. 15 In previous work by our group, TIPS pentacene was incorporated into nanoparticles of the diblock poly(ethylene oxide)-b-poly(D,L-lactic acid) (PEO-b-PDLLA) made using FNP.23 PEOb-PDLLA nanoparticles loaded with 1 wt% TIPS pentacene were used to study particle uptake in M1 and M2 macrophages. After 24 hours of exposure, ~70-80% of both M1 and M2 macrophages had taken up nanoparticles without exhibiting immunogenicity and cytotoxicity. Although the TIPS loading in the nanoparticles was not optimized for maximum fluorescence, these nanoparticles were successfully visualized using imaging flow cytometry. Unlike the PS used previously for the work done with Et-TP5, PDLLA is biodegradable. Surface functionalization of NPs is important for improved circulation time and targeted drug delivery.24 Many studies have investigated attaching different moieties on the surface of polymeric nanoparticles ranging from drug molecules to targeting peptides or antibodies. The methods of attachment have included covalent conjugation and protein-protein interactions both before and after particle fabrication.25 Gindy et al. demonstrated that PEG-b-PCL NPs could be functionalized by using diblock copolymers with reactive maleimide groups at the end of the PEG chains.26 The particles were made by FNP with different ratios of diblocks containing methoxy-PEG and maleimide-PEG blocks to vary the density of maleimide groups which were then conjugated to bovine serum albumin molecules after particle fabrication. Mannosideterminated PEG chains were optimized by D’Addio et al. to make particles by FNP for a more effective treatment for tuberculosis.27


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Park et al. used the emulsion method to produce palmitic acid-avidin conjugates during fabrication of poly(lactic-co-glycolic acid) (PLGA) nanoparticles to incorporate the palmitic acid into the hydrophobic core and expose the avidin protein onto the surface.28 The avidin then provided an anchoring point to attach biotinylated PEG intended to prolong circulation time. Avidin and streptavidin are versatile given their strong affinity for biotin with dissociation constants of KD ≈ 10-15 M and ≈ 10-14 M, respectively,29 – and given the large number of commercially available biotinylated proteins and peptides. Because FNP involves turbulent mixing at high shear stresses, protein-containing complexes such as palmitic acid-avidin conjugates might not retain their function if they were incorporated into the nanoparticles during the process. However, if functional streptavidin could be attached to the surface of the particles after fabrication, the result could be a versatile method for rapidly changing the surface functionalities of nanoparticles to address multiple biological targets from a common starting batch of particles. Because the polymer cores in this work are based on polylactic acid, free carboxylate groups on the surface of the cores can be coupled with amine groups on streptavidin using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) coupling chemistry. It was hypothesized that blending different amounts of the diblock poly(acrylic acid)-b-poly(D,L-lactic acid) (PAA-b-PDLLA) into the nanoparticles would increase the number density of carboxylate groups at the core-shell interface and thus lead to more effective conjugation with streptavidin. Coupling streptavidin to the nanoparticle provides a platform for any biotinylated ligand of interest, resulting in a class of nanoparticles that can be readily functionalized for targeted drug delivery applications. Prior studies illustrate the importance of understanding how particle design parameters affect drug delivery systems. This work explores the effects of particle composition on particle size,


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zeta potential, core composition, and shell chemistry using FNP where the following questions are addressed: 

When the total solids concentration of the organic phase is kept constant, does the addition of PLLA homopolymer increase the size and change the zeta potential?



Does TIPS pentacene fluorescence quenching occur in the nanoparticles via the Forster resonance energy transfer process? If so, this could provide guidance in determining the optimal dye loading.



Will the zeta potential become more negative with increased PAA-b-PDLLA content due to the increased carboxylate end groups present on the surface? If so, this could offer an approach for functionalizing the nanoparticles.



Can streptavidin be coupled to the PDLLA core using carboxylate groups arising from chain hydrolysis? Would streptavidin more effectively couple to particles when the abundance of carboxylates is increased using a combination of PAA-b-PDLLA along with PEO-b-PDLLA?



Is the streptavidin still functional once immobilized to the surface, capable of binding with biotinylated targeting ligands? If so, this may be a facile method for changing the surface chemistry of particles so that a common particle platform can be quickly directed to a variety of cell or tissue targets.

An overarching problem in the therapeutic applications of these nanoparticles is the difficulty in fabricating a system with the combination of size control, imaging, and targeting functionalities needed for effective performance. It is this combination of functionality that is the focus of this work.


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EXPERIMENTAL SECTION Materials Anhydrous

uninhibited

tetrahydrofuran

(THF)

(anhydrous

>99.9%),

6,13-

Bis(triisopropylsilylethynyl)pentacene (TIPS pentacene), Streptavidin-Cy3 (from Streptomyces avidinii in buffered aqueous solution w/ 1% bovine serum albumin), unlabeled Streptavidin (dry powder), and biotinylated Atto 488 bioreagent were purchased from Sigma Aldrich and used as received. Acetone was used as received from Fisher Scientific. Dialysis membrane tubing (MWCO 12-14kDa, dry standard cellulose 16 mm flat width) was purchased from Spectrum and used as received. Methoxy-poly(ethylene oxide)-b-poly(D,L lactic acid) (PEO(5kDa)-bPDLLA(8.9kDa); DPPEO = 114, DPPDLLA = 123) and benzyl-poly(L lactic acid) (PLLA (9.4kDa); DP = 131) was synthesized by Rui Zhang using methods described previously

17, 30

and used as

stored as a dry powder in a desiccator at room temperature (Figure S2). Poly(acrylic acid)-bpoly(D,L lactic acid) (PAA(1.3kDa)-b-PDLLA(3.7kDa); DPPDLLA = 51) was used as synthesized (see Supplementary Materials (S.1)) and stored as a dry powder in a room temperature desiccator. GE Hyclone Phosphate Buffered Saline (1X, 0.0067M PO4, without Calcium or Magnesium) (PBS) was purchased from Fisher Scientific and used as received. Polysciences Bead Coupling Buffer (pH 4.5) and DEPC-Carbodiimide (EDAC) were purchased from Polysciences, Inc. and used as received. The Pierce BCA Protein Assay Kit was purchased from ThermoScientific and used as received. Bovine serum albumin (BSA), used as a calibration standard for the BCA assay, was provided by Thermo Scientific as part of the Pierce BCA Protein Assay Kit and used as received. Milli-Q deionized water (resistivity ~ 18 M-cm produced from a Millipore Synergy Ultrapure Water system) was used in all experiments. The biotin anti-mouse PD-L1 antibody was purchased from BioLegend Inc and used as received.


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Fabrication of PEO-b-PDLLA nanoparticles with varying PLLA homopolymer loadings for increased core size PEO-b-PDLLA diblock copolymer, and PLLA were dissolved separately in uninhibited THF at 33 mg/mL. The PEO-b-PDLLA diblock copolymer was dissolved by vortex mixing followed by 30 minutes sonication (Fisherbrand CPXH Series Heated Ultrasonic Cleaning Bath). The PLLA homopolymer was dissolved using vortex mixing followed by sonicating for 30 minutes at 40oC. The diblock solution and the homopolymer were then combined for different wt% loadings, defined as the mass of PLLA divided by the total mass (PLLA+PEO-b-PDLLA) of material (eq. (S5)). The final solids concentration was kept constant at 33 mg/mL for all cases. The mixture was then filtered (Fisher Scientific, 0.2 µm Nylon) and vortex mixed. Using a 5 mL glass syringe, the organic solution was fed into a 4-jet multi-inlet vortex mixer (MIVM, Figure 1) at a flow rate of 9.99 mL/min using a syringe pump (New Era Pump Systems, Farmingdale, NY). Deionized (DI) water was fed simultaneously by three separate 60 mL plastic syringes at flow rates of 33.3 mL/min using a manually programed pump (Harvard apparatus PHD 4000, Holliston, MA). These flow rates correspond to a Reynolds number of ~ 13,000 and a final THF to water ratio of 1:10 (v/v) in the mixer. The exiting nanoparticle suspension was collected and dialyzed (12-14k MWCO) in 4 L of water for 24 hours, during which the dialysis water was changed 4 times. The sample was frozen either in a -80oC freezer or in a dry ice and acetone bath then lyophilized (FreeZone6, LABCONCO) for 3-6 days at conditions < 0.09 mBar and ~ -50°C. The freeze-dried powder was transferred to sealed glass vials for storage at 4°C and reconstituted at desired concentrations as needed.


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Figure 1. Flash Nanoprecipitation makes kinetically arrested particles by mixing a polymer and hydrophobic compound dissolved in an organic solvent with an aqueous anti-solvent. (adapted from figure in Gindy, et al. 21, Copyright American Chemical Society, 2008)

Fabrication of PEO-b-PDLLA nanoparticles with TIPS pentacene and PAA-b-PDLLA To make the PEO-b-PDLLA nanoparticles with different compositions, the fabrication steps were very similar to the method described above. In brief, the different components were dissolved in THF separately by sonication, or vortex mixing in the case of TIPS pentacene. The solutions were combined at ratios to yield the desired particle compositions and injected as above. If TIPS pentacene was present, the particles were protected from light during dialysis and freeze drying to minimize fluorophore degradation. More details can be found in the Supporting Information (S.3-S.4).


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Size and zeta potential analysis The hydrodynamic diameter and zeta potential were measured using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Software version 7.11 and 7.12). Freeze-dried nanoparticles were re-dispersed in DI water at an approximate concentration of 0.1 mg/mL. The suspension was vortex mixed and sonicated for 30 minutes at room temperature. After multiple rounds of sonication, the bath temperature sometimes increased to ~27-28oC. Unless otherwise noted, the temperature was kept below 30oC. Dynamic light scattering (DLS) measurements were done at 25oC with an equilibration time of 120 seconds in a disposable polystyrene (PS) cuvette. The reported values were averages of 5 measurements taken of 3 independent dispersions of each sample. Each measurement reported from the software is itself an average of 12-16 subruns. Zeta potential measurements were taken using the same samples for DLS measurements in DI water. A folded PS capillary cell was used and the measurements were conducted at 25oC with an equilibration time of 120 seconds.

Absorbance and fluorescence spectra The absorbance and fluorescence spectra of TIPS pentacene dissolved in THF were measured using an Evolution 300 UV-Visible Spectrophotometer (ThermoScientific) scanned from 400700 nm and a Cary Eclipse Fluorescence Spectrophotometer (Agilent) from 600-800 nm respectively. Both spectra were normalized to the maximum peak intensities. For the Cary fluorescence spectrophotometer, the excitation was done at 591 nm and emission was measured in the range 605-850 nm. The target TIPS loading with respect to the core, cTIPS,core, is defined as as:


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cTIPS,core(%) =

cTIPS ∗ 100 MWPDLLA block

cTIPS + cdiblock ∗ (MW

PDLLA ― b ― PEO

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(1) )

where cTIPS and cdiblock were the concentrations of TIPS and the diblock copolymer in the THF solution injected into the multi-inlet vortex mixer.

Functionalization of nanoparticles with streptavidin and PD-L1 antibody The particles were conjugated with streptavidin, streptavidin-Cy3, and bovine serum albumin (BSA) using EDC coupling and then washed by centrifugation before further incubation with a biotinylated PD-L1 antibody. The detailed procedure is outlined in Supporting Information S.8. Since each washing step during this process resulted in some particle loss, several cases - (1) uncoupled particles, (2) particles coupled with streptavidin-Cy3/BSA, and (3) particles coupled with streptavidin/BSA - were each washed 4 times and characterized to ensure comparable concentrations of nanoparticles were assayed for the different cases. The mass of protein per mass nanoparticles was calculated based on measurements of NP mass retained during centrifugation, and protein concentration measurements from the BCA assay (done in replicates of 10), (Supporting Information (S.7)). To test whether streptavidin was conjugated to the NP surface and still functional in its immobilized configuration, the amount of protein was measured at each step of the surface functionalization process by the bicinchoninic acid (BCA) protein assay (Supporting Information S.8).

Functionalization of nanoparticles with a biotinylated fluorophore The nanoparticles for flow cytometry were prepared in a manner similar to that used in the BCA assay. The freeze-dried particles were first suspended in DI water at a concentration of 1 mg/mL. They were then spun down at 100k x g for 30 minutes (25oC) and then resuspended in


12

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coupling buffer that had been titrated up from pH 4.5 to pH 5.2 by pipetting up and down followed by vortex mixing (~5 sec). EDAC and streptavidin and, in some cases, streptavidinCy3, were added and the mixture was mixed covered in foil on an end-to-end mixer for 3 hours. After incubation, the particles, now with attached streptavidin/BSA, were spun down again at 100k x g for 30 minutes and resuspended as described above in PBS and biotinylated-Atto 488 dye in place of the biotinylated-antibody from previous experiments. The mixture of NPs, streptavidin, and Atto 488 were incubated at room temperature for an hour on an end-to-end mixer (FisherScientific, Model 88861049) before being spun down, resuspended in PBS, and spun down again to remove all unbound fluorophore. At each step, aliquots were taken and washed by centrifugation to recover samples of NP only, Streptavidin/BSA coated NPs, and finally Atto 488+streptavidin/BSA coated NPs for imaging. Because Atto 488 and Cy3 have overlapping signals, particles containing just Cy3 and particles containing just Atto 488 were prepared. To prepare the samples with just Atto 488, streptavidin without Cy3 was used. The latter was received as a freeze-dried powder that was dissolved with BSA at a 1:10 ratio of streptavidin:BSA in PBS to mimic the composition of the suspension of as-received streptavidinCy3. Images were taken using an Amnis ImageStream Mark II Imaging Cytometer at 60x magnification. TIPS pentacene was excited at 642 nm and detected with a 702/86 bandpass emission filter. Cy3 and Atto 488 were both excited at 488 nm and detected with 577/35 and 527/65 bandpass emission filters, respectively. Of the 5000 particles detected by ImageStream, the >95% by number that were positive for the red fluorescence signal from TIPS pentacene were used in the subsequent analysis.


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RESULTS AND DISCUSSION Effect of particle composition on size and zeta potential In order to design and fabricate surface functionalized nanoparticles for targeted delivery, it is essential to learn how to control the size and zeta potential by varying the composition and processing conditions. The total solids concentration in the organic stream was kept constant at 33 mg/mL by systematically varying the concentrations of PLLA, TIPS pentacene, and PAA-bPDLLA and adjusting the PEO-b-PDLLA concentration. First, the wt% loading of PLLA homopolymer was varied. Secondly, the wt% loading of TIPS pentacene was varied and, finally, the wt% percent of PAA-b-PDLLA was varied. Particle sizes reported in Figure 2 were measured after freeze-drying and redispersion in DI water. No cryoprotectant was needed or used and no changes in particle composition upon freeze-drying were likely. Particle sizes were measured after dialysis but before freeze drying and, for some cases, the redispersed freeze-dried particles were larger than the size before freezedrying by 10-30 nm but still monomodal. In other cases, the freeze-dried particles were smaller, which can likely be attributed to the presence of loose aggregates that were dispersed during the sonication step after freeze drying. In most cases, sonication for 30 minutes was sufficient to achieve monomodal distributions but additional sonication for 15 minutes was needed in some cases. The focus in this work was on the size of the particles redispersed after freeze-drying; the resulting particle sizes were in the range relevant for drug delivery. Further investigation of the effect of freeze-drying on size could be investigated in future work to better define the size distributions. Sonication was done at room temperature with the bath temperature initially at ~


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21oC but, after multiple rounds of sonication, it sometimes increased to ~27-28oC. Unless otherwise noted, the temperature was kept below 30oC. Effect of PLLA homopolymer composition Because the PLLA homopolymer was not acid end capped, we hypothesized that the size may increase with added PLLA but the zeta potential could vary due to the changing density of the PEO surface layer. As the PLLA homopolymer content in the NPs increased, the particle size increased with intensity-average diameters as large as 420 nm at 75 wt% PLLA (Figure 2a, Table S1) The size distributions were monomodal for all PLLA compositions except for the cases of 50 and 75 wt% PLLA, where the size distributions were bimodal, most likely due to incomplete surface stabilization from the PEO. The reported diameters are those for the mass dominant peak where ~76% and ~74% of the mass were found at this size. As more homopolymer was added, there was less PEO to sterically stabilize the particles resulting in aggregation. The density of the PEO brush can be roughly quantified by a parameter, RF/DPEO, comparing the Flory radius (RF) to the estimated spacing between tether points for PEO chains on the PDLLA core surface (DPEO).The Flory radius is defined as RF ~aN3/5 where a is the effective monomer length, 0.35 nm for PEO

31,

and N is the degree of polymerization. If the spacing

between the tether points of neighboring PEO chains becomes smaller than the Flory radius (RF/DPEO > 1), the PEO chains extend to form a brush. A dense brush can be defined roughly when RF/DPEO > 2.8.31 For the 5k MW PEO used in the diblock for the nanoparticles, RF = 6 nm. Assuming that the thickness of the PEO brush on the NP core is ~15 nm 18, then 30 nm for the PEO brush was subtracted from the hydrodynamic diameter measured by DLS to approximate the PDLLA core size, Dcore. Using the approximate core size and polymer composition of the


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particles, values of DPEO were estimated for each PLLA composition. For PLLA compositions ranging from 0-40 wt% PLLA, RF/DPEO  4.5 (Table S1), well above the limit of 2.8 needed for a dense brush. However, because the size distributions of particles with 50 and 75 wt% PLLA were bimodal, the RF/DPEO model was not applicable due to the difficulty of estimating DPEO. Pagels et al. developed a scaling model that predicts the size of particles formed by FNP. depends on primarily on the mass concentration of solids in the mixer and the weight% core:15

(

𝑘𝐵𝑇𝑐𝑐𝑜𝑟𝑒

1 5 3 3

D = 2 K 𝜋𝜇𝜌𝑐𝐵𝐶𝑃

)

(2)

where kB is the Boltzmann’s constant, T is absolute temperature, ccore is the mass concentration of the hydrophobic additive component in the mixer,  is the solvent viscosity,  is the bulk density of the core, and cBCP is the mass concentration of the block copolymer. K is a parameter (= 253 mg*g1/3m-1) that depends on the composition and geometry of the stabilizer which was 5kDa MW PEO. Since the PEO block molecular weight was also 5kDa in our experiments, we used this value for K to analyze our data for the effect of PLLA loading on particle size (Figure 2c). In our work, the mass concentration of solids in the mixer was kept constant at 33 mg/mL while the weight% PLLA was varied. As originally defined in the model, ccore was the mass concentration of the hydrophobic additive component not counting the hydrophobic part of the diblock. With that definition for ccore, our data for the effect of PLLA weight % on size in Figure 2c (closed squares) has a slope of 1636 nm/(m1/3/g1/9) while equation (2) (solid line) predicts a slope of 1311 nm/(m1/3/g1/9). However, the absolute magnitudes of the experimental diameters were ~ 2X larger than those predicted by the theory. The 2 lowest weight% PLLA loadings of 0 and 5% were not included because these fell within the lower range specified by Pagels et al. where equation (2) is not valid. The 2 highest loadings of 50 and 75 wt% were not included in


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the figure due to the bimodal size distributions which would also make equation (2) not valid. The reason for the offset in the values is not clear but we note that the molecular weight of the PDLLA block in our diblock was 8.9 kDa whereas the molecular weights of the diblocks used by Pagels et al. were at least ~ 2X smaller. Moreover, the model does not account for the contribution of the hydrophobic block from the diblock to the volume of the core. Since the larger PDLLA block length in the present work could contribute significantly to the volume of the core, we redefined ccore to include the PDLLA block from the diblock along with any added PLLA homopolymer, while keeping all other parameters constant. The results in Figure 2c (open squares) that now include the PLLA compositions of 0 and 5 wt%, show better agreement with the theory, but we note this is a semi-empirical modification to the theory and thus further analysis is needed. The PEO brush conformation can also be a major factor in how the zeta potential, , varies with PLLA loading (Figure 2b). The zeta potential measurements were conducted in DI water to maximize differences between samples. As the wt% PLLA increased, there was less PEO to stabilize the particles and thus more poly(lactic acid) chains exposed to the aqueous phase, increasing the likelihood of hydrolysis that could result in carboxylate groups on the surface. Moreover, as the PEO layer becomes less dense with increasing PLLA content, the slip plane shifts closer to the NP which, for a constant surface charge density, would result in a larger, more negative zeta potential.32 However, the differences in zeta potential were statistically insignificant and therefore it is difficult to conclude whether the PEO is densely packed on the surface. Still, in the middle range between 10-40 wt% PLLA, the zeta potential was nearly constant, most likely due to the denser PEO layer on the surface that protected the core from hydrolysis. The addition of PLLA homopolymer provides a method to increase the


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hydrodynamic diameter and core size without significant modification to the state of charge on the PLLA core up to about 40 wt% PLLA. The increased core volume could possibly be used for increased drug payload per nanoparticle.

Figure 2. (a) Intensity-average hydrodynamic diameter from DLS and (b) zeta potential of nanoparticles as a function of wt% PLLA homopolymer. (c) Hydrodynamic diameter measurements of PLLA homopolymer-containing nanoparticles compared to model by Pagels et al. 15; the solid squares represent the data points plotted to x-values using the original definition for ccore; open squares represent modification of the ccore definition to include the hydrophobic block of the diblock. (d) Hydrodynamic diameter and (e) zeta potential of particles as a function


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of wt% TIPS pentacene. (f) Hydrodynamic diameter and (g) zeta potential of particles as a function of wt% PAA-b-PDLLA diblock. Total constant injected organic concentration kept constant at 33 mg/mL. Effect of TIPS pentacene composition TIPS pentacene is a much smaller molecule than PLLA with a MW of 639.07 g/mol as compared to ~11,000 g/mol MW for PLLA. The fluorophore has a partition coefficient (logP) of 9.28 as estimated using Molinspiration.33 Thus, when present in the MIVM at concentrations needed for the same wt% loading, the number of TIPS pentacene molecules that can serve as a nucleating agent for particle growth is much larger than that for PLLA. Thus, the TIPS pentacene-loaded particles were fabricated over a range of 0-10 weight percent to compare size and zeta potential effects to those particles made by varying the much larger PLLA loadings. There was a statistically insignificant increase in size with increased TIPS loading (Figure 2d). Similar to the trends seen for the mid-range 10-30 wt% loadings of PLLA, the zeta potential stayed essentially constant for TIPS-loaded particles as expected since the fluorophore is nonionic and very hydrophobic (Figure 2e).

Effect of PAA-b-PDLLA diblock composition Another goal of this work was to investigate conditions needed to covalently couple streptavidin to the PDLLA cores so that the particles could be further functionalized with biotinylated ligands. Although a PDLLA core could provide some carboxylate groups due to hydrolysis of PDLLA chains as evidenced by the negative zeta potentials typically measured, we hypothesized that the streptavidin binding could be increased by increasing the number of carboxylate groups in the core which would likely segregate near the core surface. Thus, we


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incorporated the diblock PAA-b-PDLLA into the nanoparticles with 4 different compositions ranging from 0-20 wt% PAA-b-PDLLA while the TIPS composition was maintained at 0.5 wt% in the core. The particle size did not change significantly for the PAA-b-PDLLA content ranging from 0-10 wt% but increased sharply at 20 wt% (Figure 2f) due to the lower molecular weight of the PAA(1.3k)-b-PDLLA(3.7k) diblock compared to the PEO(5k)-b-PDLLA(8.9k) diblock. Thus, for 20 wt% loading of the PAA-b-PDLLA in the NPs, the ratio of the number of PEO-bPDLLA to PAA-b-PDLLA chains was 58:42. As with the PLLA wt% variations, the largest particle size occurred at the highest PAA-b-PDLLA loading due to the reduced PEO content. The zeta potential also was essentially constant for all the tested compositions suggesting some amount of PEO brush shielding (Figure 2g). For all compositions of PAA-b-PDLLA wt% loadings, RF/D  4, well above the limit of 2.8 needed for a dense brush. The resulting sizes for the 20 wt% PAA-b-PDLLA were found to be stable albeit poly-dispersed. The size distribution was a bimodal peak where the two peaks overlapped and, in some measurements, resulted in a single broad peak. Because the calculation of RF/D assumes that all of the PEO is found on the surface, the brush density would be overestimated if some of the PEO chains were buried in the core. It is possible some PEO complexed with PAA chains via hydrogen bonding, resulting in both types of chains being buried in the PDLLA core or forming relatively thin layers on the core surface.34 Either form of complexation would lead to less PEO available to form an extended brush for steric stabilization.

Fluorescence of TIPS pentacene-loaded PEO-b-PDLLA based nanoparticles The absorbance and fluorescence spectra of TIPS pentacene dissolved in THF overlap in the range of 600-700 nm (Figure 3a). The difference in wavelength between the maximum


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absorbance and maximum emission peak - the Stokes shift - is small enough that one TIPS pentacene molecule can absorb the emission of another TIPS pentacene molecule to result in self quenching by means of Forster Resonance Energy Transfer (FRET).35 More quenching occurs when the molecules are in closer proximity to each other as they are when confined in the core of the nanoparticles. The Forster radius is the distance at which 50% of the energy from the donor is transferred to the acceptor rather than being emitted and is defined by: 18 R60 =

(9ln10)κ2QD ∞ ∫ F (λ)ε(λ)λ4 128π5NAn6 0 D

(3)

dλ

where 2 is the orientation factor (2/3 for randomly oriented molecules), QD is the quantum yield of the dye, NA is Avogadro’s number, n is the refractive index of the material the dye is in (PLA; 1.44 36),  is the wavelength (nm), FD is the fluorescence intensity of the dye where the area is normalized to 1 (because fluorescence intensity are typically represented in arbitrary units) and  is the molar extinction spectrum of the dye (nm2 mol-1)

18

(see Supporting Information (S.6) for

more details). The Forster radius therefore depends on the area of overlap between the fluorescence and molar extinction spectra.


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Figure 3. (a) Absorbance and Emission spectra of TIPS pentacene in THF normalized to the maximum intensity peak found at 640 and 646 nm respectively (b) Calibration curve of absorbance versus TIPS pentacene concentration in THF. From calculations based on the absorbance and emission spectra of TIPS pentacene in THF and quantum yield data, the calculated Forster Radius from equation (3) for the fluorescently labeled nanoparticles was ~ 5.3 nm. The highest fluorescence occurred at a loading of 0.5 wt% TIPS pentacene of the PDLLA core (Figure 4). The estimated distance between TIPS pentacene molecules when encapsulated in the core was based on a cubic lattice model: 18 RTIPS =

3

CTIPS + CPLLA + CPDLLA block (PAA ― PDLLA) + CPDLLA block (PEO ― PDLLA) nTIPSNAVρPDLLA

(4)

where Ci is the mass concentration of component i injected into the mixer, nTIPS is the molar concentration of TIPS pentacene injected into the mixer, NAV is Avogadro’s number and PDLLA is the density of the core which was assumed to be the density of bulk PDLLA (1.25 g/cm3).37 From eq. (4), for a core TIPS pentacene loading of ~ 0.5 wt% and assuming 100% encapsulation efficiency, RTIPS = 5.6 nm, which agrees well with the calculated Forster radius which also corresponds to the loading with the highest observed fluorescence. The normalized fluorescence is reported per mass of NP rather than per individual particle because the estimation of the latter is a sensitive function of the size as measured by DLS which can introduce error through estimations of PEO layer thickness and is thus problematic whereas calculating the fluorescence per mass of NP is more accurate. Moreover, nanoparticle dosages for cell and animal studies are typically described in mass concentration for injections or mass dosages for oral delivery. The TIPS dye loading is specified as a weight% with respect to the PDLLA core and not the entire nanoparticle because the core is where the fluorophore is encapsulated. As calculated in equation (4), the cubic lattice model only accounts for the volume of the core. When the composition of


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the nanoparticle changes with the addition of different polymers such as the PLLA homopolymer or the PAA-b-PDLLA diblock, the core mass can be estimated accurately by assuming 100% encapsulation efficiency. The encapsulation efficiency was calculated from absorbance measurements of nanoparticles with various loadings of TIPS pentacene compared to a calibration curve of TIPS pentacene concentrations in THF (Figure 3b) and averaged ~ 85%. This value was lower than expected but could be due to thermo-oxidative degradation of TIPS that can occur after the particles were formed and processed. When this reduced encapsulation efficiency is taken into account by reducing the concentration CTIPS in equation (4), the resulting value of RTIPS is 5.3 nm, ~ 6% lower than the value estimated with 100% encapsulation efficiency. Thus, this reduction in encapsulation efficiency did not result in a significant error in RTIPS.

Figure 4. Normalized fluorescence per mass of nanoparticles indicating peak fluorescence occurs at 0.5 wt% TIPS in the NP core. This corresponds to a TIPS molecular spacing of ~5.6 nm as shown in the table. Lines connecting the data points are to guide the eye. Excitation at 591


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nm and emission measured at 644 nm. The error bars represent a standard deviation calculated using a propagation of errors analysis of the fluorescent intensity and the mass concentration of the particles in suspension. As a comparison, particles encapsulating the pentacene derivative Et-TP5 used by Pansare et al. were maximally fluorescent at a loading of 2.3 wt% of the core corresponding to a fluorophore separation of 3.9 nm whereas the Forster radius was R0 ~ 4.1 nm.18 As mentioned in that study, there are opposing effects that influence the fluorescence of the resulting nanoparticles. With increasing loading, there are more molecules to absorb and fluoresce but the molecules are closer together to cause self-quenching. For TIPS pentacene, there is an optimum loading where the fluorescence is maximized due to a balance of both of these factors, as was the case for the Et-TP5. Our calculations confirm that the Forster radius calculation is a useful modeling tool for understanding optimum fluorophore loading in particles made by Flash Nanoprecipitation.

Surface functionalization of NPs with streptavidin and biotinylated antibodies EDC coupling chemistry was used to attach streptavidin to the nanoparticle surface by reacting the carboxylate groups on the particle cores with amine groups on the protein to form amide bonds. The nearly constant zeta potential seen in all 4 PAA-b-PDLLA/PEO-b-PDLLA nanoparticles samples (Figure 2f) suggested that the number density of carboxylate groups available for streptavidin coupling might be very similar amongst the 4 different compositions. The PEO brush on the surface could provide additional steric inhibition and thus hinders the protein’s access to the carboxylate groups needed for efficient coupling.


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Figure 5a shows the mass of protein per mass of NP as measured by the BCA assay for the streptavidin/BSA case as well as when those particles were functionalized with the antibody. The final mass concentration of NP, CNP4, used for the BCA assay is defined by eq. (S13) in the Supporting Information. From the assay, the mass of coupled protein per mass of NP did not vary significantly as the PAA-b-PDLLA content varied from 0-20 wt%. BSA was added along with the streptavidin at a ratio of BSA:streptavidin of 10:1 because the streptavidin-Cy3 solution as received that was also conjugated to the nanoparticles had BSA in the ratio of 10:1. Because EDC coupling is non-specific, we presume that BSA was coupled to the NP surface along with the streptavidin. Because BSA and streptavidin have comparable characteristics such as molecular weight (56 kDa for SA 29; 66 kDa for BSA 38), diameter (~ 4.6 nm for SA 39; ~ 7 nm for BSA

40)

and isoelectric points (5-6 for SA

29;

4.9 for BSA

38),

the kinetics and extent of

coupling are likely to be similar. To test this, the protein concentration of the pelleted NPs and the supernatant were measured by the BCA assay. Assuming this ratio coupled to the particle surface is the same as in solution during incubation, the protein measured on the NP and in the supernatant after incubation and centrifugation was divided by 11 to estimate the streptavidin mass concentrations. The resulting mass of streptavidin on the particle surfaces and in the supernatant summed to ~ 88-92% of the total amount of streptavidin initially added to the system for coupling, closing the mass balance as shown by Figure 5b and outlined in Supporting Information S.8. The high degree of pegylation of the particles (RF/D > 4.5) which formed a dense brush tends to suppress nonspecific protein adsorption and so the protein binding for the “NP+SA/BSA” case in Fig. 5(a) is due mainly to the covalent attachment of the streptavidin and BSA to the particle cores, especially since the particles were subjected to 2 centrifugal washing steps prior to measuring the protein loading by the BCA assay.


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Figure 5. (a) Mass protein per mass of NPs as measured by BCA assay for the 4 different particle compositions at different stages in the surface functionalization process – NP only before streptavidin conjugation, after streptavidin coupling but before addition of biotinylated antibody, and after full functionalization with the PD-L1 antibody. (b) Mass balance of total streptavidin added during surface functionalization as compared to the 4 cases with masses calculated from BCA of both the streptavidin conjugated to the nanoparticles and that measured from the supernatant of the washing step. The masses were normalized to the total mass added during the coupling step.

Although the 20 wt% PAA-b-PDLLA case resulted in larger particles, on a per mass basis the streptavidin loadings were all comparable (Figure 5b and Table 1). The lack of difference between the cases could be attributed to the hydrogen bonding between PAA and PEO mentioned previously causing some of the PAA blocks from the PAA-b-PDLLA chains to be


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buried beneath the surface of the PDLLA core, thus making them unavailable for protein functionalization. For all 4 PAA-b-PDLLA compositions, there were ~ 2-3 antibodies attached per bound streptavidin. Although streptavidin has 4 binding sites for biotin, they may not all be accessible given its conjugation to the particle surface as well as steric barriers from the bulkiness of the large antibody and nearby PEO brushes. The use of BSA could prove beneficial as a blocking protein reducing the number of attached streptavidin and therefore the number of active binding sites. More antibodies are not necessarily better if they are too closely packed to be functional but this could depend on the specific application. Changing the ratio of BSA to streptavidin may be a facile way to tune the number of targeting ligands on the nanoparticles surface. With regards to the limit of detection in terms of nanoparticle and protein concentrations, the lowest detectable protein concentration limit for the BCA microplate assay used in this work is 25 g protein/mL. Referring to Fig. 5(a), the average protein composition for the case “NP+SA/BSA” (the nanoparticles to which streptavidin and BSA were coupled) was ~ 50 g protein/mg NP. In view of the detectability limit of 25 g/mL, the lowest nanoparticle concentration for this case for which protein could be detected was (25 g protein/mL  50 g protein/mg NP =) 0.5 mg NP/mL. By comparison, the typical NP concentrations used for the BCA assays were ~ 1.3 mg/mL. Thus, the assays were conducted at nanoparticle and protein concentrations sufficient for accurate detection.


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Table 1. Measured hydrodynamic diameter and mass or # of protein per mass NP with varying PAA content. + values are standard deviations as calculated by the propagation of error analysis.

Sample

PEO-bPDLLA

5 wt% PAA-b-PDLLA 95 wt% PEO-b-PDLLA



NP diameter (from DLS*) (nm)

98 + 33

112 + 29

99 + 33

201 + 56

Polydispersity index, PDI from DLS

0.19

0.24

0.15

N/A

Total mass SA** + BSA***/mass NP

0.07 + 0.01

0.05 + 0.01

0.05 + 0.01

0.06 + 0.01

Mass SA/mass NP

0.006

0.004

0.005

0.006

Mass BSA/mass NP

0.064

0.044

0.045

0.055

Total Mass SA + BSA + PD-L1 antibody/mass NP

0.10 + 0.01

0.09 + 0.01

0.09 + 0.01

0.11 + 0.01

Mass PD-L1 antibody/mass NP

0.028

0.033

0.031

0.041

# SA/mg NP

7.3 x 1013

5.0 x 1013

5.2 x 1013

6.3 x 1013

#PD-L1 antibodies/mg NP

1.3 x 1014

1.6 x 1014

1.5 x 1014

2.0 x 1014

#PD-L1 antibodies/# SA

1.8

3.1

2.9

3.1

*Intensity average hydrodynamic diameter from dynamic light scattering; ** Streptavidin; ***Bovine Serum Albumin


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The # proteins per # of NPs can be roughly estimated by assuming a 15 nm thickness for the PEO brush to estimate an NP core diameter from the DLS measurements. The core diameter Dcore is then used to calculate the NP mass as Dcore3/6. Assuming a brush thickness of 15 nm, the estimated # SA/NP ranged from ~ 10 for the PEO-b-PDLLA case to ~ 130 for 20 wt% PAAb-PDLLA while the # antibodies/NP ranged from ~ 80-420.

Visualization of retained functionality of NP surface-bound streptavidin In order visually depict the functionalization of streptavidin and biotinylated moieties, the particles were coupled with streptavidin and then a biotinylated dye Atto 488 was attached to the streptavidin. For particles that were coupled with functional streptavidin-Cy3, this system would result in 3 separate and distinct signals: red from TIPS pentacene, orange/yellow from Cy3 and green from Atto 488. Imaging cytometry made it possible to rapidly image ~ 5000 individual particles per sample. Figure 6 shows representative images of TIPS pentacene-loaded nanoparticles with varying surface functionalities. NPs containing just TIPS showed a strong fluorescent signal at the red emission peak for the fluorophore whereas NPs made with TIPS and then conjugated with streptavidin-Cy3 showed emission signals for both TIPS and Cy3. By using streptavidin that was not conjugated with Cy3, particles coated just in Atto 488 could be made as well and these showed a green emission from the Atto 488 as well as the red emission from the TIPS. This experiment was a secondary check to confirm the functionality of streptavidin conjugated to the NP surface for binding with biotinylated compounds.


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Figure 6. Representative fluorescence images of TIPS pentacene particles with varying surface functionalization as imaged by cytometry.

CONCLUSIONS This study investigated the design, fabrication, and characterization of PEO-b-PDLLA-based nanoparticles fluorescently labeled with TIPS pentacene and functionalized with streptavidin and biotinylated moieties - steps needed for the design of a targeted drug delivery system. The results outline a methodology for the functionalization of nanoparticles comprised of commercially available, biodegradable polyesters with streptavidin and the subsequent attachment of biotinylated ligands. The addition of hydrophobic components such as PLLA and TIPS pentacene increased the particle size with increased loading but with minimal effects of zeta potential at the ranges tested. Once above ~ 40 wt% PLLA, the particles became destabilized as the PEO brush became too sparse, exposing the surface to increased hydrolysis and therefore increased acid groups. The semi-quantitative agreement between the particle size results and the model by Pagels et al.15 is very promising and suggests this model is a valuable tool for designing polymer nanoparticles made by FNP.


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Fluorescence measured from various TIPS loaded nanoparticles showed that the optimum loading was ~ 0.5 wt% of the PLA core which corresponded to the loading where the calculated distance between TIPS pentacene molecules within the particle core was similar to the Forster radius. The combination of the Forster radius and the cubic lattice spacing calculations provide a valuable tool for the optimizing the fluorophore loading which can be translated to other fluorophores that exhibit this quenching mechanism. Furthermore, the surfaces of these particles could be readily modified post-fabrication using EDC coupling chemistry to coat the surface with streptavidin. Although it was hypothesized that by adding more carboxylate groups to the surface by adding PAA-b-PDLLA to the nanoparticles more streptavidin could be attached to the surface, the study found that the base PEO-b-PDLLA nanoparticles had sufficient carboxylate groups to conjugate streptavidin at comparable levels. The attached streptavidin then becomes a ready platform for targeting ligands using biotinylated antibodies as shown by fluorescence images. In future work, a more detailed understanding of the core-shell structure of the nanoparticles is needed, including the core size, brush thickness, and aggregation number, in order to better design the particles for drug delivery. SAXS is a powerful tool for making these measurements as described in a recent study of polymer micelles.41 In addition to SAXS, the density of free PEO chains at the particle surface could be quantitated using NMR42 or a colorimetric method.43 These studies could be used to test more fully the particle formation model by Pagels et al..15 The partitioning of the PAA block in the particle (free in the core or at the surface; complexed with PEO in the core or at the surface) is a relatively unexplored issue, particularly since particle formation by FNP is kinetically controlled rather than thermodynamically controlled. In a previous study, 1H NMR was used to show that PEG chains were located at the surfaces and not


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in the cores of nanoparticles made by FNP.15 In future work, NMR experiments with labelled PAA-b-PDLLA copolymers could possibly be used to probe the locations of the PAA groups. Further tuning of the conjugation of the nanoparticles with streptavidin is possible by varying the ratio of streptavidin/BSA in the coupling step. A promising application for this work is to use particles functionalized with anti-PD-1 or anti- PD-L1 (B7-H1, CD 274) antibody with the aim of improving checkpoint inhibition and T-cell mediated anti-tumor immune responses in cancer treatment. Checkpoint inhibitors are a promising class of antibody-based cancer therapeutics and it is possible that the NPs described here could significantly improve their functionality. In the tumor microenvironment, cancer cells can present high levels of PD-L1 protein on the cell surface that functions as an inhibitor of T-cell mediated cytotoxic effects. The PD-L1 ligand is a type I protein and therefore interacts with the PD-1 receptor on CD8+ T-cells (or cytotoxic Tcells)

44.

When the PD-1 receptor is engaged, T-cell activation and cytotoxicity is averted.

However, if nanoparticles could be used to inhibit this checkpoint, then T-cell mediated antitumor immune responses should significantly improve in tumors expressing high levels of PDL1. Having a streptavidin-coated platform on the surface of nanoparticles allows for facile surface functionalization. With the large number of biotinylated antibodies, ligands, proteins, and peptides, this one family of particles could be readily tailored for a variety of different treatments and applications.

ASSOCIATED CONTENT Supporting Information. Chemical structures of compounds used in this work; Synthesis of poly(acrylic acid)-b-poly(D,L-lactic acid) copolymer; Effect of PLLA homopolymer composition; Fabrication of PEO-b-PDLLA nanoparticles with varying TIPS pentacene loading to determine optimal loading for fluorescence; Fabrication of fluorescently labeled PEO-b-


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PDLLA/PAA-b-PDLLA NPs; Encapsulation efficiency of TIPS pentacene in NPs; Fluorescence; Testing NP mass retention after centrifugation; Functionalization of NP with streptavidin and the PD-L1 antibody.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] ORCID: (0000-0002-4838-2541)

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGEMENT Funding for this work is gratefully acknowledged from the Institute for Critical Technology and Applied Science and the Virginia Tech Department of Chemical Engineering. The authors thank Prof. Amanda Morris, Veronica Ringel-Scaia, Dr. Dylan K. McDaniel, and Melissa Makris for their help with the experiments.


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Table of Contents Graphic


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Design and Fabrication of Streptavidin-Functionalized, Fluorescently

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