Molecular and Biocompatibility Characterization of ... - ACS Publications

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Molecular and biocompatibility characterization of red blood cell membrane targeted and cell penetrating peptide-modified polymeric nanoparticles Kaustuv Sahoo, Sriharsha Karumuri, Rangika S. Hikkaduwa Koralege, Nicholas H. Flynn, Steve Hartson, Jing Liu, Joshua D. Ramsey, Kaan Kalkan, Carey Pope, and Ashish Ranjan Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 15 May 2017 Downloaded from http://pubs.acs.org on May 15, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

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

Molecular Pharmaceutics

Molecular and biocompatibility characterization of red blood cell membrane targeted and cell penetrating peptide-modified polymeric nanoparticles

Kaustuv Sahoo1, Sriharsha Karumuri2, Rangika S Hikkaduwa Koralege3, Nicholas H. Flynn3, Steve Hartson4, Jing Liu1, Joshua D. Ramsey3, Kaan Kalkan2, Carey Pope1, and Ashish Ranjan1* 1. Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078 2. Mechanical and Aerospace Engineering, Oklahoma State University, Stillwater, OK 74078 3. School of Chemical Engineering, Oklahoma State University, Stillwater, OK 74078 4. Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078

Correspondence Dr. Ashish Ranjan, B.V.Sc., Ph.D. Associate Professor Laboratory of Nanomedicine and Targeted Therapy 169 McElroy Hall, Center for Veterinary Health Sciences Oklahoma State University, Stillwater, Oklahoma-74074 Phone: 4057446292; Fax: 4057448263 Email: [email protected]

ACS Paragon Plus Environment


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

Abstract Red blood cells (RBCs) express a variety of immunomodulatory markers that enable the body to recognize them as self. We have shown that RBC membrane glycophorin A (GPA) receptor can mediate membrane attachment of protein therapeutics. A critical knowledge gap is whether attaching drug encapsulated nanoparticles (NPs) to GPA, and modification with cell penetration peptide (CPP) will impact binding, oxygenation and induce cellular stress. The objective of this study was to formulate copolymer-based NPs containing model fluorescent tagged bovine serum albumin (BSA) with GPA-specific targeting ligands such as ERY1 (ENPs), single chain variable antibody (scFv TER-119, SNPs), and low molecular weight protamine based CPP (LNPs) and determine their biocompatibility using a variety of complementary high-throughput in vitro assays. Experiments were conducted by co-incubating NPs with RBCs at body temperature, and biocompatibility was evaluated by Raman spectroscopy, hemolysis, complement lysis, and oxidative stress assays. Data suggested that LNPs effectively targeted RBCs, conferring 2-fold greater uptake in RBCs compared to ENPs and SNPs. Raman spectroscopy results indicated no alteration adverse effect of NP attachment or internalization on the oxygenation status of RBCs. Cellular stress markers such as glutathione, malondialdehyde, and catalase were within normal limits, and complement-mediated lysis due to NPs was negligible in RBCs. Under the conditions tested, our data demonstrates that molecular targeting of the RBC membrane is a feasible translational strategy for improving drug pharmacokinetics and that the proposed high-throughput assays can pre-screen diverse NPs for preclinical and clinical biocompatibility. Keywords: Glycophorin A, Red blood cell, Raman spectroscopy, Cell penetrating peptide, Single chain variable fraction of peptide, Complement, Biocompatibility, Oxidative stress


Page 2 of 34

Page 3 of 34

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


Abbreviations: NP

nanoparticle

RBCs

red blood cells

GPA

glycophorin A

PEG

polyethylene glycol

BSA

bovine serum albumin

PLL

poly L-lysine

BNP

BSA nanoparticles

ENP

ERY1-BSA nanoparticles

LNP

LMWP-BSA nanoparticles

scFv-TER119 single chain variable region of TER-119 antibody against GPA DLS

dynamic light scattering

GSH

glutathione

MDA

malondialdehyde

TBARS

thiobarbituric acid reactive substances

RES

reticuloendothelial system



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

1. Introduction Therapeutic application of proteins and small molecule drugs are limited by degradation by serum enzymes, rapid uptake by the reticuloendothelial system (RES), and clearance by the renal system1-5. This leads to reduced half-life, insufficient delivery to tissues, and reduced in vivo activities, thereby often requiring frequent and increased dosing for therapeutic benefit4,6. To overcome such limitations and improve the circulatory half-life properties relative to conventional therapy7, drug encapsulation in micro- and nanoparticles (NPs) with hydrophilic polyethylene glycol (PEG) polymer coating has been reported8,9. PEGylation of drug/NPs is promising but can induce an immune response following repeated injection, and this has been shown to decrease tissue-specific drug delivery and targeting10. Previously, surface modification and coating of NPs and drugs ex vivo with red blood cell (RBC) membranes protected them from macrophage uptake and systemic clearance by the RES (liver, spleen)11, improved particle retention in circulation relative to PEGylate NPs, and accumulation especially in endothelium rich organs like lung over 24h12-16. Although ex vivo coating of drugs and NPs with RBC membranes has merits, this procedure still requires RBC collection and chemical modification and manipulation of cell membranes. This may impact biocompatibility, complexity of treatment, and translatability for clinical use17,18. RBCs have a lifespan of ~120 days and express a variety of immunomodulatory markers that enable the body to recognize them as self. Thus, in contrast to ex vivo manipulation, direct in vivo targeting of NPs to RBCs in blood vessels can prolong circulation time of NPs regardless of variability of inter-patient pharmacokinetics19-21. Recently, we and others found that targeting of the ERY1-peptide and single chain variable fragment (scFv) of Ter119 to glycophorin A22,23, a transmembrane protein that represents approximately 2% of the total RBC membrane proteins, significantly improved RBC membrane binding and targeting of polymeric NPs24,25. Related studies reported that bioavailability of bio-scavengers and anti-thrombotic drug could be increased by 15–85% of the injected dose following RBC membrane attachment for 24-48h, thereby enhancing the long-term prophylaxis26,27. These results are promising, but we do not know whether attaching ligands and NP drug carriers to the RBC membrane affects cellular function, hemoglobin (Hb) characteristics, and their oxygen carrying ability. Additionally, the


Page 4 of 34

Page 5 of 34

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


impact of cell penetrating peptides28 such as a low molecular weight protamine (LMWP) that achieve effective intracellular protein delivery on RBC function is yet to be determined29. The objective of this study was to investigate the RBC biocompatibility of NPs composed of grafted copolymers encapsulating a model protein (bovine serum albumin or BSA, BNP), and decorated with scFv-TER119 (SNPs) and ERY1 (ENPs) against GPA receptor, and LMWP (LNPs) for RBC internalization in vitro. Molecular targeting and biocompatibility was characterized by designing a set of complementary high-throughput in vitro assays (hemolysis, complement lysis, oxidative stress, and Raman spectroscopy) to compare the binding rates of the synthesized NPs to RBCs and evaluate their impact on various cellular functions. Our data suggest that the proposed high-throughput assays, especially Raman spectroscopy, can be used to measure the NP uptake rate, evaluate the oxygenation and spin state dynamics of the heme in different oxygenation states30-33, and provide an innovative way to screen RBC-directed novel nano-formulations for preclinical and clinical investigations. 2. Materials and Methods 2.1 Chemicals Fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) was purchased from Invitrogen (Carlsbad, CA, USA) and used as the model encapsulated drug agent because its molecular weight is similar to that of thrombolytics (e.g thrombokinase). Poly-L-lysine•HBr (PLL) (molecular weight 15–30 kDa), PEG-2000, and p-nitrophenyl caprylate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Heterobifunctional PEG (MAL (Maleimide)-PEG-NHS) and mPEG-NHS (molecular weights of 2 and 5 kDa, respectively) were purchased from Creative PEGWorks (Winston Salem, NC, USA). Glutaraldehyde (50%), acrylamide/bisacrylamide (37.5:1), and other polyacrylamide gel casting and running materials were purchased from Fisher Scientific (Pittsburgh, PA, USA). ERY1 targeting peptide with an additional C-terminus linker containing a cysteine residue (WMVLPWLPGTLDGGSGCR) and LMWP cell penetrating peptide with the sequence CVSRRRRRRGGRRRR were custom synthesized by EZBiolab (Carmel, IN, USA). scFvTER119 was synthesized as previously described by Spritzer et al.22. Twelve mm diameter microwell of 35 mm Petri dishes were obtained from MatTek Corporation (Ashland, MA, USA). Catalase (CAT), thiobarbituric acid reactive substance (TBARS), and glutathione (GSH) assay kits were purchased from Cayman Laboratory (Ann Arbor, MI, USA). The peristaltic pump was purchased from Watson-Marlow Inc. (Wilmington, MA, USA). Streptavidin (SA) and the long arm biotin ester BxNHS were purchased from VWR (Radnor, PA, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively.



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

2.2 Synthesis of BNP, ENP, LNP, and SNP The grafted copolymer synthesized for this study was composed of neutrally charged PEG grafted onto a cationic PLL backbone via the amine-reactive NHS group on the PEG polymer and the lysine residues that make up the PLL25,34. In a typical synthesis reaction, 15 mg of 15– 30 kDa PLL was dissolved in 200 µL of phosphate buffered saline (PBS) (pH 7.4) to which 20 mg of 2 kDa mPEG-NHS and 20 mg of 5 kDa MAL-PEG-NHS were added. The final mixture was incubated for 1 h at room temperature. Following incubation, the polymer product was purified using a 10 kDa centrifugal concentrator. The sample was washed three times with a 50/50 mixture of PBS and ethanol and a final time with pure ethanol. The sample was then air dried before being stored at –20 °C. NPs were prepared by forming an ionomer complex with the cationic PLL-g-PEG copolymer and negatively charged BSA. The PLL-g-PEG copolymer was dissolved in PBS at a concentration of 6 mg/mL, and a working solution of BSA protein in PBS was prepared at a concentration of 0.266 mg/mL. The copolymer (7.5 µL) was then added to the protein solution (25 µL) dropwise while gently vortexing. Following incubation of the mixture for 1 h at room temperature, the sample was cross-linked with 5 µL of a glutaraldehyde solution (5.0 µL of 0.025% in PBS) and incubated for another 3 h at room temperature. To create targeted NPs (ENP, LNP, and SNP), the ERY1 targeting ligand, LMWP, and scFvTER119 were subsequently conjugated to the NPs through a reaction of the thiol-reactive maleimide group on the distal end of the 5 kDa PEG. The ERY1 peptide was dissolved in a 50/50 mixture of dimethyl sulfoxide and PBS at a concentration of 0.03 mg/mL. LMWP and scfvTER119 were dissolved in PBS at a concentration of 0.03 mg/mL. Twenty microliters of each sample were added dropwise to the NPs while gently vortexing. The sample was incubated for 30 min at room temperature. Following incubation, the sample was washed three times with PBS using a 100 kDa centrifugal concentrator. 2.3 Physicochemical characterization of NPs and RBC bound NPs The average hydrodynamic diameter of the NPs was determined by dynamic light scattering (DLS) (Brookhaven Instruments Corporation, Holtsville, NY, USA). Five measurements lasting 3 min per measurement at a 90° angle were used to determine the average size of the NPs. The zeta (ζ)-potential, determined using phase analysis light scattering, was calculated from the electrophoretic mobility using Smoluchowski’s equation. Ten measurements, each for 30 sec, were used to determine the average ζ-potential of the NPs. Prior to DLS or ζ-potential


Page 6 of 34

Page 7 of 34

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


measurements, samples were diluted in PBS to a total volume of 750 µL or 1.5 mL, respectively, and passed through a 0.2 µm syringe filter directly into a disposable cuvette. Similarly, size and zeta potential measurements of the RBCs attached NPs encapsulating 25 µg BSA were determined at 6 h. 2.4 Gel retardation assay BSA encapsulation into NPs was confirmed using a gel retardation assay. Eight percent SDSPAGE gels loaded with approximately 2 µg of BSA, all types of NPs, and BSA-LMWP conjugate were run at a voltage of 200 V using a BIO RAD Mini-PROTEAN® Tetra Cell apparatus until the dye front reached the bottom of the gel35. The SDS-PAGE gels were stained with Coomassie G250 before imaging. 2.5 Assessment of NP binding and uptake by flow cytometry and confocal microscopy Animal-related procedures were approved and carried out as per the guidelines of the Oklahoma State University Animal Care and Use Committee. The terminal procedure to collect blood by intracardiac puncture in mice was performed under deep general anesthesia36,37. To estimate binding efficiency of BNP, ENP, LNP, and SNP, NPs containing 25 µg of FITC-BSA were added to 3 x 107 RBCs in 200 µL buffer X (PBS containing 150 mM NaCl and 6 mM glucose) in 1.5 ml centrifuge tube and incubated for 24 h at 37 °C in the dark under shaking25. At specified time points (1, 3, 6, and 24 h), 10 µL of RBC suspension were withdrawn, redispersed in 500 µL of buffer X, and transferred to flow cytometry tubes. The fluorescence signal of the FITC–BSA was measured using a FACS Aria flow sorter (BD Biosciences, Franklin Lakes, NJ, USA) with an excitation wavelength of 488 nm and a 530/30-nm emission filter using BD FACS Diva 8.0.1 software. The percentage of cells positive for binding was computed from the histogram plot calculated from triplicate measurements of each sample. Ten thousand events were counted for each sample. To confirm cellular attachment of NPs, the sorted cells were examined under confocal microscopy. Briefly, the RBCs were seeded at 1 x105 cells/well in 10 mm diameter microwells within 35 mm MatTek Petri dishes. All imaging was performed with constant acquisition and display parameters using an inverted microscope (Olympus IX81-ZDC2) equipped with a color CCD camera, cooled monochrome CCD camera, motorized scanning stage, and mosaic stitching software (Metamorph) with a 60x oil-immersion objective. The FITC channel (excitation



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

Page 8 of 34

480/15 nm, emission 520/15 nm) was used for gating to quantify the percentage of cells positive for FITC signal after excitation with a mercury lamp-based monochromator. 2.6 Raman investigation of NP interaction with RBCs Cell sorting of RBCs positive for FITC fluorescence was conducted using a FACS Aria flow sorter (BD Biosciences) with 4-way purity as described in 2.5. The sorted RBCs were then analyzed by Raman spectroscopy to confirm the attachment of NPs on RBCs and investigate the influence on oxygenation/deoxygenation of RBCs. For spectral acquisition, blood samples were diluted 100X in PBS. Next, a 2 µL aliquot was spotted on the cover glass of a Mattek dish well to prevent drying during spectral acquisition. We employed a WITec alpha300 R Raman system with a 532 nm. Raman spectra were acquired with incident power of 0.8 mW, laser spot size of 2 µm, integration time of 10 s, and objective lens of 20× magnification and 0.4 numerical aperture. Based on the video images of the RBCs captured at the laser spot during measurement, the spectral signal was continuously collected from an immobilized single cell. For determination of cellular oxygenation, the peaks at 1640 and 1608 cm−1 were attributed to asymmetric stretching of vibration between Cα─Cm (represented as ν10) in Hb of oxygenated and deoxygenated RBCs, respectively

32,38,39 40,41

. The peaks at 1586 and 1552 cm−1 were assigned

to asymmetric stretching of vibration of Cβ─ Cβ (represented as ν19) in Hb of oxygenated and deoxygenated RBCs, respectively. The oxygenation and deoxygenation of Hb typically results in a shift of the ν10 band from 1608 to 1640 cm−1, whereas the ν19 band shifts from 1586 to 1552 cm−1. We conducted Raman spectroscopy to: i) confirm the attachment of NPs on RBCs; and ii) investigate the influence of NPs on the oxygenation state of RBCs.

Prior to a spectral

acquisition, the sorted RBCs were diluted in PBS by 100×. Subsequently, a 2 µL aliquot was spotted on the cover glass of a Mattek dish well to prevent drying during the acquisition. We employed a WITec alpha300 R Raman system with 532 nm laser excitation. Raman spectra were acquired with incident laser power of 0.8 mW and spot size of 2 µm. An objective lens of 20× magnification and 0.4 numerical aperture were employed. The signal was accumulated for 10 s.

Based on the video images of the RBCs captured at the laser spot during the

measurement, we are assured that the signal was continuously collected from an immobilized single cell. We acquired Raman spectra from at least 5 different RBCs for each case (i.e., RBCs as received and attached with 4 different NPs). For acquiring Raman spectrum from


Page 9 of 34

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


deoxygenated cells, the cover glass with RBCs was sealed inside an optical cell and then purged by argon gas. 2.7 Compatibility of NPs with RBCs using hemolysis assay The hemolysis assessment was conducted using freshly obtained RBCs at 10% hematocrit in 100 µL volume. Briefly, RBCs were diluted in buffer X and then mixed with BSA, BNP, ENP, LNP, and SNP, all containing 25 µg BSA. The mixture was gently vortexed and incubated at 37 °C for 6 h and 24 h. The volume was adjusted to 200 µl with buffer X (to have sufficient supernatant volume), followed by centrifugation (350 x g, 5 min). Absorbance of the supernatant at 541 nm was measured in a 96-well plate using a UV-visible spectrometer. PBS and water were used instead of the NP preparation as negative and positive controls (100% hemolysis), respectively. The percent hemolysis of RBCs was calculated using the following formula: Percent hemolysis = ((sample absorbance − negative control absorbance)/(positive control absorbance − negative control absorbance)) × 100 2.8 Effect of NPs on RBC Osmotic Fragility Osmotic fragility assay was performed at 10% hematocrit as previously described by D Pan et al.42. Briefly, RBCs were washed three times with ice cold PBS and re-suspended in ice cold PBS, followed by incubation with NPs at 25 and 50 µg BSA for 60 min at 4°C. RBCs were washed three times with ice cold 4% DPBS to wash off unbound NPs. Hemoglobin release was measured by incubating cells at salt concentrations of 0, 37.5, 50, 75, 125 and 150mM NaCl. RBC suspensions were centrifuged at 13,400g for 4 min and immediately removed from salt solution at 37°C. Absorbance of the supernatant at 541 nm was measured in a 96-well plate using a UV-visible spectrometer. Each sample in water was taken as 100% hemolysis. 2.9 Evaluation of oxidative stress in RBCs 2.9.1 Lipid peroxidation (MDA) assay To assess lipid peroxidation, we measured TBARS as a surrogate for malondialdehyde (MDA) production according to the manufacturer’s instructions for the TBARS assay kit. Briefly, RBCs were treated with NPs encapsulating 25 µg of BSA or 6h and 24h as described in 2.2. Cells were then collected in 1 ml of PBS and sonicated three times for 5 s. Lysates (100 µl; n = 3/group) were mixed with 100 µl sodium dodecyl sulfate solution and added to 4 ml of the TBARS color reagent (provided by the manufacturer). The final mixture was boiled for 1 h,



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

Page 10 of 34

followed by incubation on ice for 10 min. The samples were kept at 4 °C for 6 h to increase the sensitivity of detection. Finally, 150 µl of the prepared sample was loaded into a 96-well plate and the absorbance at 530 nm was recorded using a 96-well Elisa plate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA) and analyzed for MDA production. 2.9.2 GSH activity GSH is a nucleophilic co-substrate for glutathione transferases, and it plays an important role in prevention of oxidative stress. For the GSH assay, cells were treated as described in section 2.8.1 and lysed with ice-cold HPLC water. They were centrifuged at 10,000 g for 15 min at 4 °C, and the supernatant then was collected. The supernatant was deproteinated by adding 1.22 M ice-cold trichloroacetic acid to the cell lysate. The samples were kept on ice for 5 min followed by centrifugation at 10,000 g for 15 min at 4 °C43. Fifty microliters of the supernatant in 96 wells were then quickly mixed with 150 µL of the freshly prepared assay cocktail buffer provided by the manufacturer. Absorbance at 405 nm was recorded using the 96-well Elisa plate reader and analyzed for GSH activity. 2.9.3 CAT activity CAT is widely present in aerobic cells and is important for detoxifying hydrogen peroxide. For the CAT assay, NP-treated RBCs were lysed as described in section 2.8.1. The assay was performed by mixing 100 µL of diluted assay buffer, 30 µL of methanol, and 20 µL of cell lysate. The reaction was initiated by adding 20 µL of diluted hydrogen peroxide, and the mixture was vigorously shaken for 20 min at room temperature. The reaction was terminated using 30 µL of potassium hydroxide. Next, 30 µL of Catalase Purpald (chromogen provided with the kit) were added to the mix, which was then incubated for 10 min at room temperature. Finally, 10 µL of catalase potassium periodate were added and the mixture was incubated for 5 min at room temperature. Absorbance at 540 nm was recorded using the 96-well Elisa plate reader to calculate CAT activity. 2.10 RBC complement lysis Coupling the RBC membrane to drugs, proteins, and NPs might sensitize the RBCs to complement-mediated lysis12,44,45. To study such a phenomenon in the present investigation, freshly isolated RBCs were washed three times with buffer X before the addition of the long arm biotin ester BxNHS. Biotin dissolved in dimethylformamide (DMF,100 mM) was added to the RBC suspension at 10% hematocrit (1 ml) to obtain a final concentration of 1000 µM biotin, and


Page 11 of 34

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


the sample was incubated at room temperature for 30 min. Unreacted biotin was removed by washing three times with PBS containing 2 mg/ml of BSA. Five microliters of SA (5 mg/mL in DPBS) were then added to 100 µl of 10% biotinylated RBCs and incubated at room temperature for 30 min. Unbound SA was removed by centrifugation three times with PBS containing 2 mg/ml of BSA. Samples of biotinylated RBCs (100 µl) incubated with or without different types of NPs at 1.0% hematocrit in DPBS containing Ca2+ and Mg2+ were further incubated with the addition of 200 µl of fresh homologous serum at 37 °C for 4 h on a shaker. Biotin-streptavidin to RBC suspension was used as the control. RBCs in water and PBS were used as the positive and negative controls, respectively. The absorbance of the Hb released from the RBCs was recorded at 541 nm. The percent hemolysis of RBCs was calculated using the following formula: Percent hemolysis = ((sample absorbance − negative control absorbance)/(positive control absorbance − negative control absorbance)) × 100 2.11 Effect of NPs on RBC Oxidative Fragility This assay was performed as described by D Pan et al.42. Briefly, 3mM H2O2 in DPBS was added to RBC and RBC-NP suspensions of 1.0% hematocrit and rotated 360° at 24 rpm at 37°C for different time periods. The control samples were not subjected to H2O2. The hemoglobin released from the RBCs during rotation was immediately assayed, as was the free hemoglobin in the control samples. 2.12 Evaluation of protein markers in modified Red Blood Cell The retention of surface proteins in modified red blood cells was analyzed using polyacrylamide gel electrophoresis using a slightly modified procedure as previously described46. Briefly, the control RBCs, and RBC-NPs (BNP, ENP, LNP, and SNP) were heated in a boiling water bath for 5min and loaded in a 8% polyacrylamide gel. Finally, coomassie brilliant blue staining was performed to identify the protein bands in the gel. 2.13. Statistical analysis Treatment groups were compared for differences using analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test. All analyses were performed using GraphPad Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA). All p-values were two-sided, and a pvalue < 0.05 indicated statistical significance. Values are reported as mean ± SEM unless otherwise indicated.



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

Page 12 of 34

3. Results 3.1 Characterization of ligand bound NPs The gel retardation assay indicated that complete encapsulation of BSA protein occurred, as shown in the SDS-PAGE gel image (Fig. 1). Un-encapsulated BSA in the first lane was able to freely migrate into the gel. However, upon addition of the PLL-g-PEG copolymer to BSA, migration of BSA into the gel was retarded (2nd lane), indicating complete encapsulation. Similarly, BSA BNPs modified with ERY1, LMWPs, and scFvTER119 showed complete gel retardation before and after centrifugation (3rd, 4th, and 5th lanes). DLS measurement indicated that the average sizes of the resulting BNPs, ENPs, LNPs, and SNPs were 31.5 ± 3.6, 57.1 ± 2.5, 38.8 ± 3.9, and 52.3 ± 5.4 nm, respectively. ζ-potential values of the BNPs, ENPs, LNPs, and SNPs were, -3.10±1.74, -6.78±0.19, -5.29±0.73, and -3.39±1.24 respectively (Table 1). Similarly, RBCs incubated with NPs demonstrated a size increase with time, indicating NP interaction with the cells (Table 2). Importantly, incubation with LNP resulted in a relatively smaller increase in size compared to membrane targeted BNP, ENP, SNPs. 3.2 NP binding kinetics Confocal microscopy confirmed cellular attachment in the sorted RBCs, as shown by green fluorescence on the cell membrane (Fig. 2). Additionally, no visible damage to RBC membranes was noted. For LNPs, membrane attachment and internalization could not be distinguished. Quantitative estimation using flow cytometry suggested that binding of ENPs and SNPs to RBCs at 6 and 24 h was 30–40% greater than that of BNPs. At these time points, LNPs achieved 2-fold greater RBC uptake compared to both ENPs and SNPs (Fig. 3). These findings were supported by a similar increase in fluorescence intensity shown by Raman spectroscopy, with relatively greater spectra for LNPs relative to other NPs (Fig. 4). 3.3. Impact of NP binding on RBC oxygenation NP treatment resulted in no new peaks or bands, which was directly attributable to the membrane attachment or uptake. In addition, the relative intensity of peaks ν10 and ν19 of oxygenated and deoxygenated Hb with respect to untreated RBCs were similar (Fig. 5). The average increase intensity ratio of oxygenated-state to deoxygenated-state marker (Roxy/deoxy) in Hb with BSA, LNP, SNP, ENP, and BNP was 11, 200, 77, 25, and 15 %, respectively.


Page 13 of 34

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


Prior to investigating the influence of NPs on oxygenation state of RBCs, we had to confirm the binding of NPs to RBCs. To this end, we first inspected the Raman spectra of NP-targeted RBCs (Figs. 4a-4d) for the major vibrational peaks of poly-L-lysine (PLL) and polyethylene glycol (PEG). However, those peaks are apparently masked by the FITC fluorescence. FITC, which is tagged to the BSA (and therefore encapsulated in the polymer NP with BSA), has a broad excitation range (with a maximum at 494 nm) extending up to 540 nm.25 Therefore, during a Raman acquisition with 532 nm excitation, we observe a significant fluorescence background due to FITC which likely masks the Raman spectrum of the polymer. Therefore, binding of polymer NPs to a RBC, is validated by the broad FITC fluorescence baseline in the Raman spectrum of that RBC (in Figs. 4a-4d). The intensity of this baseline is a measure of the density of fluorophore-tagged NPs attached to the RBC. Vibrational spectrum of a RBC is dominated by heme porphyrins of hemoglobin (Hb) owing to ultrahigh Raman cross section of the heme. Each Hb protein constitutes 4 heme porphyrin groups, which govern the protein structure to be in either relaxed, R, or tense, T, state.38,39,47 The transitions between R and T states of Hb occur through oxidation and reduction of the heme porphyrins, respectively.48 The transition of Hb from R to T state occurs because of deoxygenation, that alters the oxidation state of iron in heme from Fe3+ to Fe2+.38,55 Likewise, oxygenation leads to transition of Hb from T to R state, where iron in heme undergoes oxidation from Fe2+ to Fe3+.38,55 Vibrational spectroscopy enables monitoring of these transitions in Hb owing to discernable peak shifts resulting from changes in electron distribution over the heme porphyrin as well as its structure.38,39,45,55

Figure 5a shows transformation of a RBC from oxygenated to deoxygenated state through argon purging in an optical cell. The peaks at 1640 and 1608 cm−1 are assigned to asymmetric stretching of Cα─Cm (i.e., ν10) for oxygenated and deoxygenated heme, respectively.32,38,39,40,41 Whereas, the peaks at 1586 and 1552 cm−1 are assigned to asymmetric stretching of Cβ─Cβ (i.e., ν19) in oxygenated and deoxygenated heme, respectively.32,38,39,40,41 Therefore, transition of Hb from oxygenated to deoxygenated state (i.e., from R to T) results in the lower wavenumber shift of ν10 mode from 1640 to 1608 cm−1 and ν19 mode from 1586 to 1552 cm−1, respectively.32,38,39,40,41

Figures 5b and 5c compare the representative Raman spectra of NP-attached (after 6 and 24 h of incubation) versus as-received RBCs (6 and 24 h after collection). As observed from the



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

Page 14 of 34

spectra, as-received RBCs show the Raman markers, indicative of both oxygenated and deoxygenated Hb in them. The RBCs exhibit essentially the same spectrum 6 and 24 h after collection. In the present work, we define Roxy/deoxy as the intensity ratio of oxygenated to deoxygenated state Raman markers.

Raman spectra of RBCs with attached NPs as well as RBCs treated with BSA (Figs. 5a and 5b) show higher Roxy/deoxy compared with RBCs. The average increase of Roxy/deoxy in Hb with BSA is 11%. Whereas, it is 200, 77, 25, and 15 % for RBCs targeted with LNP, SNP, ENP, and BNP, respectively. 3.4 Effect of NPs on membrane integrity of RBCs No hemolysis at the BSA concentration of 20 µg was noted for BNPs, ENPs, and LNPs (Fig. 6). This is in accordance with our earlier findings, where we observed significant hemolysis at 24 h for NPs containing 50 µg BSA25. For SNPs, mild hemolysis was observed (0.56 ± 0.004%) at 24 h (p < 0.05, Tukey’s multiple comparison), but this was well below the clinical limits (< 5%)49. 3.5 NP-RBC stability under osmotic stress Incubation of RBCs with NPs at 25 and 50 µg total BSA had no significant effect on the hemolysis of RBC in sub-physiological osmotic conditions (75 mM NaCl) with BNP, ENP, and SNP. A slight increase in hemoglobin release with LNP at higher concentration was noted (Fig. 6 c&d). Most likely, the cell penetrating peptides in LNP affected the membrane stability of the RBC at higher concentration. 3.6 Oxidative stress upon NP interaction with RBCs Oxidative damage has been shown to contribute to the adverse effect of NP interaction with RBCs, leading to hemolysis50. Therefore, we evaluated the oxidative damage to RBCs by measuring their GSH and CAT activities and MDA content. MDA assay Treatment with NPs did not alter the MDA levels in the RBCs (Fig. 7a & d). There was no significant difference among the NP groups at 6 h and 24 h, which is consistent with the hemolysis data.

GSH activity


Page 15 of 34

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


No differences in GSH levels were observed in RBCs upon incubation with NPs containing 20 µg BSA at both 6 and 24 h (Fig. 7b & e)25. CAT activity A slight increase in CAT activity in RBCs treated with ENPs and SNPs was observed compared to LNPs at 6h however, there was non-significant increase in enzyme activity for LNP treated cells at 24 h. This suggests that the CAT levels in the RBCs were able to counteract the oxidative stress (Fig. 7c & f). 3.7 RBC susceptibility to complement-mediated lysis Biotinylated RBCs upon reaction with SA (positive control) induced lysis via the complement pathway. In contrast, NPs did not cause hemolysis (Fig. 8). Because surface attachment of NPs to RBCs can induce immune responses by a complement-activated alternative pathway to result in phagocytosis and clearance, these data indicate that such effects were not present in the NP groups12,45. 3.8 Stability of RBC-NPs to combined oxidative and shear stress Hemolysis of RBCs happens at NP concentration of 20 µg was increased for SNP and LNP in a time-dependent manner when subjected to exposed to 3 mM H2O2 in the presence of low level of shear stress25 (Fig. 8b). 3.6. Retention of surface protein in NP modified RBCs RBC control and NP-RBC samples demonstrate the presence of similar surface markers and proteins, thereby suggesting that the RBC protein markers were intact after incubation with NPs (Fig.9). 4. Discussion Efficient long-acting and targeted nanotherapeutics are increasingly being utilized to deliver therapeutics throughout the body51. As we move forward with therapeutic use, it is critical that the potential interaction with tissues and cells and associated drug toxicity be studied in greater detail52,53. In this study, we characterized the effects of core-shell polymeric NPs on RBCs. In recent years, RBC-based drug delivery has been shown to prolong the half-life of protein therapeutics54. RBCs come directly in contact with intravenously administered substances, thus comprehensive investigations of bio-compatibility of diverse NPs with variable physico-chemical



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

Page 16 of 34

properties of RBCs are needed to better understand in vivo toxicity. To investigate such impacts, we used simple yet relatively sensitive assays based on release of Hb, oxygenation status of RBCs, and oxidative stress markers to evaluate the impact of ligand-mediated attachment of NPs to RBCs.

As a candidate NP, we used a copolymer composed of neutrally charged PEG grafted onto a cationic PLL backbone34,55,56 to encapsulate a fluorescent protein. PEG-PLL is a widely used polymer for drug delivery, and its use follows that of polymeric polystyrene NPs that were previously utilized as a model nanosystem for biocompatibility assessment of untargeted NPs with RBCs15. As a model protein, BSA mimics the molecular weight of many therapeutic proteins (e.g., thrombokinase). To provide precise cellular targeting, the PEG molecules were functionalized by covalent labeling with the RBC-targeting peptides ERY1, scFvTER119, and CPPs such as LMWP. This functionalization resulted in a slight increase in NP size (from 30 to 50 nm). Functionalizing the particle surface with high molecular weight single chain antibody fragment of TER119 in SNPs may make them larger compared to non-functionalized and peptide functionalized particles. Additionally, the larger size of this single chain antibody fragment of TER119 might be favoring an extended form of PEG conformation resulting a larger particle. ERY1 present in ENPs is a hydrophobic peptide compared to LMWP present in LNPs which is a very hydrophilic peptide. The presence of hydrophobic ERY1 on the particle surface may cause the PEG to be in an extended conformation in aqueous medium causing the ENPs to be larger than LNPs and BNPs. This phenomenonc can increase macrophage uptake in vivo. However, this scenario was not investigated because the main study goals were to understand the impacts of cellular attachment and internalization into RBCs. Interestingly, LNPs internalized RBCs demonstrated relatively smaller size compared to RBC attached NPs, and the zeta potential also showed a reduction in the negative charge. Because poly-l-lysine polymers reduces the negative charge of the cells, our result suggest that enhanced cellular binding of LNPs and SNPs may be mediating such changes in the membrane potential57 . Another important observation from our study was the slow binding rates of NPs relative to previous publication from Zaitsev et al. Zaitsev et al. expressed scFv TER119 in mammalian tissue culture, while we expressed it in insect cells, perhaps resulting in glycosylation or folding of the single-chain scFv molecules that is slowly restored during the course of binding assay23,26. Alternatively, maybe it could have been due to NP polymer component, however, this will need to be investigated further in future studies. Cellular NP binding performed using Raman spectroscopy revealed a strong emission by FITC that masked the peaks of polymer


Page 17 of 34

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


NPs. Because the background intensity in the Raman spectra of RBCs with polymer NPs depended on the density of fluorophore-tagged NPs bound to the RBCs as well as possible fluorescence quenching due to resonance energy transfer between emission of FITC and conjugated peptides, we could detect distinct differences in peak intensity suggestive of relative uptake. In general, LNPs showed the greatest peak intensity relative to other NPs, and these results matched quantitative flow cytometry estimations (Fig. 4). To the best of our knowledge, Raman spectroscopy has not been used previously to detect differences in NP uptake by cells, and our results show that this technique serves as a good high-throughput method for understanding attachment and intracellular trafficking. Notably, the vibration spectrum of RBCs was largely dominated by the heme porphyrin of Hb, and this allowed us to monitor transitions in Hb peak shifts associated with the corresponding changes in spin states and heme porphyrin structure38,39,47. In general, each Hb protein with four heme porphyrin groups allowed either relaxed R or tensed T states48. The transition between R– T states of Hb occurred only when the oxygenation and deoxygenation of heme porphyrin changed58. As in prior research, we observed that the transition of Hb from the R to the T state occurs because of deoxygenation, which alters the oxidation of iron in heme from Fe3+ to Fe2+ and the spin state from S = ½ to 238,58. Likewise, oxygenation leads to transition of Hb from the T to the R state and correspondingly to oxidation (i.e., from Fe2+ to Fe3+) and a spin state change (from S = 2 to ½) of heme porphyrin38,58. As seen in Figure 5a-b, such significant changes in the intensity of oxygenation and deoxygenation markers in RBCs with and without NPs were not observed with our candidate NPs. This result confirmed that the NPs did not adversely impact the oxygenation status of the RBCs, even 24 h after membrane attachment in vitro. Regarding the LNP- and SNP-targeted RBCs, we observe a significant increase in the relative intensity of oxygenated markers (Roxy/deoxy) indicative of oxygenation of heme porphyrins. We infer that this increase is an artifact due to laser excitation (532 nm) during our Raman acquisitions. The increase of Roxy/deoxy is also observed for the BSA, ENP and BNP treated RBCs as well as untreated RBCs (i.e., control) for extended signal integration times, such as 100 s.; however, it is slow enough in these cases to allow for an artifact-free acquisition in the first 10 seconds. On the other hand, for RBCs targeted with LNP and SNP, the increase of Roxy/deoxy is significant during the first 10 seconds (i.e., 200% and 77%). Yet, we also employed an exposure/accumulation time of 10 s for these two cases to maintain an acceptable signal to



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

Page 18 of 34

noise. Accordingly, we have adopted an exposure/accumulation time of 10 seconds for all the measurements. Recently, the laser-induced increase of Roxy/deoxy in untreated RBCs has also been reported by Ramser et al., who attributed it to photo-induced transformation of Fe from Fe2+ to Fe3+ (i.e., deoxy-Hb protein transforms to meth-Hb).41 Although the deoxygenated heme is not necessarily attached to the oxygen by this process, the increase of oxidation number of Fe from +2 to +3 makes the heme behave like oxygenated and the Raman markers change accordingly. The laser-induced increase of Roxy/deoxy in RBCs has been reported only once in the literature and it is not fully elucidated. We realize the effect is faster with higher density of NPs attached to the RBCs. Our FACS results indicate the affinity of NPs to the RBCs increases in the following order: BNP, ENP, SNP, and LNP. Roxy/deoxy also increases in this order. Hence, we conjecture photo-excitation of the heme also increases in this order. Additional and enhanced excitation of the heme is possible through resonant energy transfer (RET) from the dye molecules (FITC here) carried by the nanoparticles. Because hemoglobin resides close to the cell membrane and the Raman laser is focused on the cell membrane, the Raman signal of the heme is essentially collected from the membrane. RET is possible through the cell membrane even though the NPs are not internalized. Therefore, we anticipate the LNP and SNP cases yield the most intense RET to the heme groups due to the highest density of FITC (i.e., RET donors) on the cell membrane. Mechanisms such as oxidative stress following a pathological insult due to the NP–RBC interaction can lead to membrane lipid peroxidation. This may destabilize RBC functions and ultimately cause hemolysis59,60. For instance, interaction of silica NPs was shown to generate reactive oxygen species and free oxygen radicals from RBCs61,62. A fundamental mechanism driving such outcomes is related to the presence of polyunsaturated fatty acids (PUFAs) on the RBC membranes that are highly vulnerable to peroxidation and changes in micro-rheological properties63. A key oxidative damage biomarker of lipid peroxidation of PUFAs is MDA production64. In contrast, GSH (a biomarker of cell function and viability) and CAT are intracellular antioxidants that prevent such oxidative damage to RBCs65. We studied the effect of different ligand-conjugated NPs on these oxidative stress markers. Our data shows that incubation of different NPs at 20 µg total BSA did not cause increased MDA and GSH, and the hemolysis data corroborated this result. However, a slight increase in CAT levels with ENPs and SNPs were noted. We believe that this might be an adaptive response to counterbalance the


Page 19 of 34

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


oxidative effect of NPs (Fig. 7). Finally, multivalent antibody conjugation to RBC membranes can induce binding of complement-regulating proteins to RBCs. In contrast, small monovalent fragments of antibodies, such as scFv, that are devoid of Fc-mediated side effects are unlikely to induce such activation12,22,66. Our studies suggest that NPs decorated with scFV, ERY1, and LNPs did not cause any RBC damage, lending credence to the theory that peptides with low molecular weight have fewer hemolytic effects (Fig.8). In summary, our data suggest that ligand-mediated interaction of NPs with mouse RBCs enhances the binding of NPs without adversely impacting RBC function. 4. Conclusions Our previous studies described the specificity of RBC-binding peptides to attach NPs to RBCs25. Our current data suggest that bio-interaction of NPs with RBCs has no negative impact on their oxygen carrying capacity24,67. Additionally, the ligand-mediated targeting of RBCs did not induce oxidative stress at the concentration tested. This study also showed that Raman spectroscopy has the potential to serve as a point-of-care testing modality for measurement of RBC oxygenation and uptake rate following intravenous therapy longitudinally in patients. Thus, the proposed complementary high-throughput assays can serve as important benchmarks for assessment of polymeric NPs targeted to RBCs in preclinical and clinical settings. 5. Acknowledgement and Disclosures We thank Marie Montelongo and the Oklahoma Center for Respiratory and Infectious Diseases (GM103648) for providing core facility support and technical expertise. This study was supported by the Defense Threat Reduction Agency under Award Number HDTRA1-13-1-0021, The OSU Interdisciplinary Toxicology Program, and Kerr Endowed Chair award. A.R., J.L, C.P, J.R, and S.H. have patent interests in NP-mediated erythrocyte technology according to Oklahoma State University policies. K.S., N.F., R.H, P.C, and S.K. declare no competing financial interests.



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

Page 20 of 34

Figure legends:

Figure 1: Encapsulation Efficiency of BSA in NPs. Lane 1: BSA; Lane 2: BNP; Lane 3: ENP; Lane 4: LNP; Lane 5: SNP


Page 21 of 34

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


Figure 2: NP interaction with RBCs using confocal microscopy of flow sorted RBCs at 6 h. Merged images of FITC-BSA fluorescence (green) and bright field optical images showing the attachment of NPs to RBCs. Membrane damage was not noted. Magnification 60X



70 Percent RBC binding

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

BNP

ENP

Page 22 of 34

LNP

SNP

60 50 40 30 20 10 0 -10

0

1

3 Time (hrs)

6

24

Figure 3: RBC binding kinetics of BNP, ENP, LNP, and SNP from 0 to 24 h. FITC-positive RBCs were significantly more abundant in the LNP treatment compared to the other engineered NP treatments. (p < 0.05, Tukey’s multiple comparison).


BNP RBC-BNP

2.0

5 1.5

4 3

1.0

1.0

1.4 0.8

1.2 1.0

0.6

0.6

0.5

0.4

500 1000 1500 2000 2500 3000 3500

500 1000 1500 2000 2500 3000 3500

Raman Shift (cm-1)

Raman Shift (cm-1)

(d)

x 103

0.8 1.8 0.7 1.5 0.6 1.2 0.5

0.9

x 103 1.4

0.9

Intensity (a.u.)

LNP RBC-LNP

Intensity (a.u.)

2.1

ENP RBC-ENP

0.8

1

x 103 2.4

x 103

1.6

2

(c)

x 103 1.8

SNP RBC-SNP

x 103 1.2

1.2

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

0.6

0.4 500 1000 1500 2000 2500 3000 3500

500 1000 1500 2000 2500 3000 3500

-1

Raman Shift (cm )

Raman Shift (cm-1)

Figure 4: Raman spectra of NP-targeted RBCs: (a) BNP, (b) ENP, (c) LNP, and (d) SNP. For comparison, Raman spectra of the polymer NPs are provided, too.


Intensity (a.u.)

Intensity (a.u.)

6

(b)

x 103

Intensity (a.u.)

x 103

Intensity (a.u.)

(a)

Intensity (a.u.)

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


Intensity (a.u.)

Page 23 of 34


(a) 1588 1552 1605 10 counts 1640

1174 Offset Y values

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

675

750

Deoxygenated (Ar-purged)

As received

800

1000

1200

1400

1600

Raman Shift (cm-1)

(b) 1588 1552 1605 40 counts 1640

1174 675

750 BSA LNP SNP ENP BNP RBCs

800

1000

1200

1400

1600

Raman Shift (cm-1)

(c) 1588 1605 1552

30 counts 675

1640

1174

750 BSA LNP SNP ENP BNP RBCs

800

1000

1200

1400

1600

Raman Shift (cm-1)

Figure 5: Raman spectra of single RBCs: a) as received and after deoxygenation (purged with Ar); b) after 6 h of incubation with NPs; c) after 24 h of incubation with NPs.


Page 24 of 34

Page 25 of 34

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


Figure 6. Hemolytic effect of BNP, ENP, LNP and SNP on RBCs at 6 (a) and 24h (b). Hemoglobin release was not observed with NPs containing 25 µg of BSA at 541 nm wavelength. (c&d) Osmotic fragility of red blood cells- Hemolytic curves were determined at 25 µg (c) and 50 µg (d) equivalent BSA (p

Molecular and Biocompatibility Characterization of ... - ACS Publications

Recommend Documents