Intracellular Delivery of Charge-Converted ... - ACS Publications

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Intracellular Delivery of Charge-converted Monoclonal Antibodies by Combinatorial Design of Block/Homo Polyion Complex Micelles Ahram Kim, Yutaka Miura, Takehiko Ishii, Omer F. Mutaf, Nobuhiro Nishiyama, Horacio Cabral, and Kazunori Kataoka Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01335 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 28, 2015

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.

Biomacromolecules 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 30

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

Biomacromolecules

Intracellular Delivery of Charge-converted Monoclonal Antibodies by Combinatorial Design of Block/Homo Polyion Complex Micelles Ahram Kim,† Yutaka Miura,‡ Takehiko Ishii, † Omer F. Mutaf, ⊥ Nobuhiro Nishiyama,# Horacio Cabral,† and Kazunori Kataoka*,†,‡,⊥ †

Department of Bioengineering, Graduate School of Engineering, ‡Center for Disease

Biology and Integrative Medicine, Graduate School of Medicine, and ⊥Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan #

Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of

Technology, R1-11, 4529 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan

KEYWORDS: antibody delivery, charge-conversion, polymeric micelle, drug delivery systems

ACS Paragon Plus Environment

1

Biomacromolecules

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 2 of 30

ABSTRACT

Direct intracellular delivery of antibodies has gained much attention, although only a few agents have been developed and none of them have reached clinical stages. The main obstacles here are the insufficient characteristics of delivery systems, including stability and appropriate ability for intracellular antibody release. We tailored the structure of polyion complex (PIC) micelles by loading transiently charge-converted antibody derivatives for achieving enhanced stability, delivery to cytosol, and precise antigen recognition inside cells. Citraconic anhydride was used for the charge conversion of the antibody; the optimized degree of modification was identified to balance the stability of PIC micelles in the extracellular compartment and prompt pH-triggered disintegration after their translocation into the acidic endosomal compartment of target cells. The use of a mixture of homo- and block-catiomers in an appropriate ratio to construct PIC micelles substantially enhanced the endosomal escaping efficacy of the loaded antibody, leading to improved recognition of intracellular antigens.


2

Page 3 of 30

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

Biomacromolecules

INTRODUCTION Direct recognition and deactivation of intracellular antigens is a fascinating approach for the manipulation of subcellular organelles or pathways, which cause desired consequences, such as subcellular protein detection, enzyme inhibition, and apoptosis. However, the clinical application of antibodies against intracellular antigens has been hindered due to the lack of robust delivery systems capable of routing the antibodies to subcellular locations inside target cells. Therefore, safe and effective delivery systems are imperative for translating state-of-art antibody engineering technology to clinics. Nanocarriers prepared from biocompatible polymers have the potential for effectively delivering proteins to subcellular spaces by exploiting the advantages of polymer shielding [1-9], which can protect the antibodies from protein interaction, loss of affinity, and decomposition [10-14]. Previously, we demonstrated the validity of polyion complex (PIC) micelles for delivering immunoglobulin G (IgG) antibodies, whose cationic amino groups were modified to anionic carboxylates by conjugation with charge-conversional moieties of maleic anhydride derivatives. These included citraconic anhydride, cis-aconitic anhydride, and succinic anhydride, which generate negatively charged antibodies [4-5]. Among these moieties, citraconic acid amide (Cit) was confirmed to be fully reversible at endosomal pH (pH 5.5), restoring the original amino groups in the antibodies and subsequently dissociating the micelles. Thus, as shown in Scheme 1A, PIC micelles prepared by charge-converted IgG derivatives

and

poly(ethylene

glycol)-block-poly[N-{N’-(2-aminoethyl)-2-

aminoethyl}aspartamide] (PEG-PAsp(DET)) copolymers, whose flanking 1,2-diaminoethane units provide selective destabilization of endosomal membranes with an increase in the protonation degree at endosomal pH [15], facilitated endosomal escape and delivery of charge-restored IgG antibodies into the cytosol. Nevertheless, despite progress of the aforementioned concept systems, their formulation remains to be further clarified. For


3

Biomacromolecules

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 4 of 30

example, the degree of charge conversion of the antibodies could be expected to affect antigen recognition by over-modification of antigen-binding (Fab) fragments. Therefore, the number of Cit conjugations with lysine units in IgG derivatives needs to be precisely manipulated and compared for their biological potencies. Moreover, the addition of the homo-catiomer, PAsp(DET), into PEG-PAsp(DET)-based PIC micelles can further enable refinement of biologically relevant properties, such as stability, cellular uptake, and escapability from endosomes [16-17]. However, these chemical modifications have yet to be considered in previously reported antibody-incorporated PIC micelles.

Scheme 1. A. Pathways for successful intracellular antibody delivery with PIC micelles. B. Formation of PIC micelles incorporating charge-converted IgG antibody derivatives and strategies to engineer the systems in this study.


4

Page 5 of 30

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

Biomacromolecules

Herein, we defined the composition and assembly of antibody-loaded PIC micelles for optimal efficacy of intracellular antigen detection. As a model antibody, we used the antinuclear pore complex (NPC) IgG antibody, which recognizes a family of nucleoporins [18] and facilitates the direct visualization and quantification of the antigen targeting by following the signal of fluorescent-labeled antibodies on nuclear membranes. The degree of modification of antibodies was optimized by controlling the amount of Cit to obtain appropriately charge-converted IgG derivatives (Scheme 1B). Besides, the ratio of the total amount of polymers, i.e., PEG-PAsp(DET) and PAsp(DET) to the charge-converted IgG derivatives, was regulated for improving stability, cellular uptake, and endosomal escape of the PIC micelles (Scheme 1B). Our results demonstrate the relationships between compositional factors and biological efficiency of antibody-loaded PIC micelles for attaining successful cellular uptake, intracellular delivery and preservation of antibody specificity, and provide fundamental insights for further development of delivery systems for antibodies targeting intracellular antigens.


5

Biomacromolecules

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 6 of 30

MATERIALS AND METHODS Materials. Anti-nuclear pore complex (NPC) immunoglobulin G (IgG) (Clone 414), IgG1

κ from murine myeloma (Clone MOPC-31c), citraconic anhydride (Cit), 1-Methyl-2pyrrolidinone (NMP), and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Alexa Fluor 647-labeled (Fab’)2 fragment from goat anti-mouse IgG and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA, USA). α-Methoxy-ω-amino poly(ethylene glycol) (molecular weight = 12,000) and β-benzyl-L-aspartate-N-carboxyanhydride (BLA-NCA) were obtained from NOF Co., Ltd. (Tokyo, Japan). Vivaspin 6 (MWCO = 10,000) was purchased from Sartorius (Göttingen, Germany). NMP and diethylenetriamine (DET; bis(2-aminoethyl)amine) (Tokyo Chemical Industry Co. Ltd, Japan) were re-distilled before use. Murine colon adenocarcinoma (C26) cells were kindly supplied by the National Cancer Center (Tokyo, Japan). PAsp(DET) and PEG-PAsp(DET) were synthesized following the previously reported method [16, 19]. The degree of polymerization (DP) of the polyaspartamide segment was confirmed by 1H nuclear magnetic resonance to be 55 for PAsp(DET) and 64 for PEGPAsp(DET), respectively.

Charge-conversion of antibodies. IgG antibodies were purified with NaHCO3 buffer (0.1M, pH 9.0) by centrifugal filtration using Vivaspin 6 (MWCO = 10,000) for 4 times (30 min at 3,000 rpm for each centrifugation). Purified IgG solutions were diluted with NaHCO3 buffer (0.1M, pH 9.0) to have IgG concentration of 1 mg/mL, then stirred for 30 min at 4 °C. One, two, three, and fifty eq. mol. of citraconic anhydride were slowly added to the solution and further stirred for 3 h. The derivatives were purified by Vivaspin 6 for 4 times with 10 mM NaHCO3 buffer (pH 7.4). The degree of charge-conversion was determined by fluorescamine method, as follows: IgG derivatives (100 µL, 0.5 mg/mL, phosphate buffer)


6

Page 7 of 30

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

Biomacromolecules

were incubated with fluorescamine (10 µL, 5 mg/mL; DMF) for 10 min at room temperature and the fluorescent signal was measured by using a ND-3300 fluorospectrometer (Nanodrop, Wilmington, DE, USA). Obtained signals were translated to the concentration of primary amino groups based on a standard calibration curve of serially diluted bovine serum albumin (BSA). The degree of charge-conversion of the obtained IgG derivatives was estimated to be 25, 50, 75, and 95% (IgG-25, IgG-50, IgG-75 and IgG-95, respectively). As the primary structure of anti-NPC IgG antibody (clone 414) has not been clarified elsewhere, the percentage of converted amines was calculated based on the result of the fluorescamine method (Table 1). Accordingly, a total of 90.4 primary amino groups in the native anti-NPC IgG was found by this method and defined as 100%. Note that the buried inaccessible amino groups were not taken into the calculation. Thus, for example, for IgG-50, the fluorescamine method indicates 44 unconverted amine groups, which subtracted to the total amines (90.4) results in the number of amines converted to carboxylates (46.4; 48.7%).

Fluorescent labeling of antibodies. Native and charge-converted IgG antibodies were purified with NaHCO3 buffer with the same condition for the charge-conversion. Purified IgG solutions (1 mg/mL in 0.1M pH 9.0 NaHCO3 buffer) were added by either Alexa Flour 647 NHS ester (Molecular Probes, OR, USA) (10 mg/mL in DMSO) or Cy5-NHS ester (GE HealthCare, NJ, USA) solution. The reaction mixture was incubated for 1 h at room temperature, with pipetting every 10 min. After the incubation, the mixture was purified by gel filtration with PD MiniTrap G-25 (GE HealthCare, NJ, USA), followed by centrifugal filtration with Vivaspin 6 (MWCO = 10,000) for 4 times. The concentration of Alexa Fluor 647 or Cy5-labeled IgGs were measured by a Micro BCA Protein Assay Reagent Kit, then adjusted to be 2 mg/mL in phosphate buffer (10 mM, pH 7.4). The ratios between fluorescent dyes and antibody derivatives were calibrated to allow consistent fluorescent intensities


7

Biomacromolecules

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 30

throughout the formulations. The procedure for labeling the IgG derivatives involved adding of 1.2 eq. mol. of fluorescent dyes to the least number of residual primary amino groups of IgG, i.e. 4.4 amino groups as indicated in Table 1. Accordingly, all the labeled antibody derivatives were confirmed to have 3 - 4 fluorescent dyes and showed almost identical fluorescent intensities. The final product was stored at -80 °C before use. Alexa Fluor 647labeled IgGs were used to evaluate the biological performance in vitro, and Cy5-labeled IgGs were used to evaluate the stability of antibody-loaded PIC micelles in serum-containing media.

Acid triggered cleavage of charge-converted anti-NPC IgG derivatives.

Charge-

converted anti-NPC IgG derivatives (0.5 mg/mL) were incubated at 37 °C in acidic phosphate buffer (pH 5.5, 10 mM) or in neutral phosphate buffer (pH 7.4, 10 mM) for 1 - 24 h. At defined time points, aliquots of these samples were diluted 4 times with pH 7.4 phosphate buffer (25 mM) to neutralize the pH. Subsequently, the amount of amino groups in the samples was evaluated by using the aforementioned fluorescamine method, and the results were translated to the cleavage of the amide bonds in IgG derivatives (Figure 1).

Evaluation of biological activities of anti-NPC IgG derivatives upon incubation at pH 5.5. Biological activity of the samples collected in previous experiment were assessed against isolated nuclei of C26 cells, which were extracted from cultured C26 cells by using a Nuclear Extraction Kit (Millipore, MA, USA) following the manufacturer’s protocol. Extracted nuclei were suspended in PBS (5000 nuclei/mL) and anti-NPC IgG derivatives preincubated in acidic phosphate buffer or neutral phosphate buffer were added at a final IgG concentration of 20 µg/mL. After 1 h incubation, the samples were blocked with 1% BSA, stained with Alexa Fluor 647-labeled secondary antibody, and purified by centrifugation (150


8

Page 9 of 30

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

Biomacromolecules

g, 5 min). The binding of anti-NPC IgG derivatives to nuclei was evaluated by flow cytometry (BD LSR II Flow Cytometer system, BD Biosciences, CA, USA) equipped with 633 nm He-Ne laser for excitation and 670/LP and 695/40 filters for emission detection.

Measurement of the association number of polymers to antibodies. PEG-PAsp(DET) was labeled with Alexa Fluor 555-NHS ester (Molecular Probes, OR, USA). The labeling efficiency was determined to be 64%, confirmed by fluorescence spectrometry. These Alexa Fluor 555-labeled polymers were then mixed with anti-NPC IgG derivatives at different Polymer/Antibody molar ratios (P/A) and incubated for 1 h at room temperature to prepare PIC micelles. Aliquots of these micelles (500 µL) were injected into thick-wall polycarbonate tubes (Beckman Coulter, Inc., CA, USA), and ultracentrifuged for 4 h at 50,000g by using an Optima TLX Ultracentrifuge (Beckman Coulter, Inc., CA, USA). The supernatant containing unassociated polymers was then collected, and the fluorescence intensity was measured by using a ND-3300 fluorospectrometer. The concentration of unassociated polymers was determined based on a calibration curve from serially diluted solutions of Alexa Fluor 555labeled PEG-PAsp(DET). The fraction of polymers associated to the IgG derivatives was determined by subtracting the concentration of unassociated polymers from the total concentration of fed polymers. In addition, the association number of polymers to IgG derivatives was calculated by dividing the concentration of the associated polymers by the concentration of IgG derivatives.

Preparation of PIC micelles from PEG-PAsp(DET) and IgG derivatives. Typical procedure for preparation of PIC micelles prepared at the optimal P/A determined by saturation of polymer association, i.e. P/A = 4, involved mixing of PEG-PAsp(DET) (64.8

µM, 8.2 µL) and IgG derivatives (6.7 µM, 20 µL) in phosphate buffer (10 mM, 11.8 µL, pH


9

Biomacromolecules

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 30

7.4) at room temperature and incubated for 1 h. PIC micelles with different P/A were prepared in the same manner but with variable concentration of the polymer.

Integration of PAsp(DET) homo-catiomer into PIC micelles. To improve the stability, cellular uptake and endosomal escape, PIC micelles were equipped with different percentage of PAsp(DET) homo-catiomer content (Chomo), which was defined as the percentage of amino groups from PAsp(DET) in the total amino groups from both PEG-PAsp(DET) and PAsp(DET). Thus, PAsp(DET) and PEG-PAsp(DET) were mixed at Chomo 25%, 50%, 75% and 100%, and since the appropriate value of P/A was estimated to be 4 in previous experiment, all these formulations were prepared at P/A = 4.

Dynamic Light Scattering (DLS) and ζ-potential measurements.

Prepared IgG

derivatives and their PIC micelles were characterized at 25 °C through dynamic light scattering and ζ-potential measurements by a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) equipped with He-Ne laser (633 nm) light source. The micelle solutions were diluted with 10 mM phosphate buffer (pH 7.4) before the measurement to have final IgG concentration of 0.1 mg/mL. Scattered light signals were collected at detection angle of 173° and subsequently analyzed by the manufacturer’s software, utilizing the Stokes-Einstein equation to obtain hydrodynamic diameters of the samples. Correlation function was curve fitted by cumulant method to calculate average size and polydispersity index (PDI). The ζpotential was determined by electrophoretic light scattering measurement and following calculation based on Henry’s equation that relates electrophoretic mobility to ζ-potential:

µ=(2εε0ζ)/3ηf(κR) where µ is the measured electrophoretic mobility; ε0 is the dielectric permittivity of vacuum;

ε is the dielectric constant of the solvent; η is the viscosity of the solvent; ζ is the ζ-potential;


10

Page 11 of 30

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

Biomacromolecules

and f(κR) is the Henry’s function (Smoluchowski approximation was applied as f(κR) = 1.5). The diffusion barrier method was applied to measure small volumes (20 µL) of samples at maximum IgG concentration (2 mg/mL) for each measurement.

Confocal laser scanning microscopy. C26 cells (6 × 103 cells/well) were plated on 8-well glass based chambers (Nalge Nunc International, NY, USA), and observed by confocal laser scanning microscopy (CLSM; LSM780 (Carl Zeiss, Oberkochen, Germany)) equipped with C-Apochromat 63× oil objective and a 633 nm He-Ne laser with excitation/emission filters at 650/668 nm, respectively. Fluorescent and bright field images were acquired every 10 min during 48 h for tracing the intracellular distribution of the Alexa Fluor 647-labeled IgG derivatives.

Fluorescent correlation spectroscopy (FCS) measurement of PIC micelles. IgG PIC micelles loading the Cy5-labeled anti-NPC IgG derivatives were incubated for defined time periods in phosphate buffer (10 mM, pH 7.4) containing 10% FBS and 150 mM NaCl (final IgG concentration = 0.5 mg/mL). These micelles were then placed in 8-well chambers and observed by CLSM (LSM510 (Carl Zeiss, Jena, Germany)) equipped with Conforcor3 module and C-Apochromat 40× water objective. The samples were excited using a 633 nm He-Ne laser and the emission was detected through a 650 nm long pass filter. Diffusion coefficients of each micelle were obtained by the Zeiss Confocor3 software with using Rhodamine 6G as the reference.

Fluorescence microscopy and quantification of in vitro delivery efficiency. C26 cells were seeded on a 96-well plate (2,000 cells/well) and incubated for overnight in 100 µL of DMEM containing 10% FBS and 1% streptomycin/penicillin. IgG PIC micelles loading anti-


11

Biomacromolecules

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 30

NPC IgG derivatives (anti-NPC IgG/m) or IgG1 κ derivatives (control IgG/m) were prepared with various Chomo percentages and added to each well at IgG concentration of 20 µg/mL. At defined time points from 1- to 24-h of incubation, cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.2% Triton X-100. After washing the cells with PBS, 1% BSA solution was applied for 10 min to prevent non-specific adsorption. The cells were then treated with Alexa Fluor 647-labeled (Fab’)2 goat anti-mouse (10 µg/mL) in 1% BSA solution for 1 h. The cells and their nuclei were stained with Calcein AM (Invitrogen, CA, USA) for 30 min and Hoeschst 33258 (Dojindo, Kumamoto, Japan) for 5 min, respectively. Then, images of approximately 2,000 cells in a single well were obtained by using an InCell Analyzer 1000 (GE Healthcare, NJ) with 10 × 0.45 numerical aperture objective lens and a set of filters for 360/460, 475/535 and 620/700 for excitation/emission wavelengths. Obtained images were processed by Developer Toolbox software to determine subcellular localization and corresponding concentration of delivered IgG antibodies.


12

Page 13 of 30

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

Biomacromolecules

RESULTS AND DISCUSSION Charge conversion of anti-NPC IgG. The cationic amino groups in anti-NPC IgG were converted to citraconic amide (Cit) through the reaction with citraconic anhydride to generate an anti-NPC IgG derivative equipped with charge-conversional properties at endosomal pH [5]. To precisely assess the impact of charge conversion on IgG charge densities, chargeconversional rates and the number of unreacted –NH2 groups in IgG were fundamentally controlled by adjusting the feed ratios of citraconic anhydride in the reaction system. Approximately 1/4 of the citraconic anhydride was consumed during the reactions and conjugated to the lysine residues on anti-NPC IgG, producing ca. 25%-, 50%-, 75%-, and 95%-citraconic amide converted IgGs (Table 1). The ζ-potential values of the obtained antiNPC IgG derivatives decreased by increasing the charge-conversional rate, supporting our strategy to systematically modulate the charge of IgG antibodies through the chemical modification of lysine residues to citraconic amide.


13

Biomacromolecules

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 30

Table 1. Number of residual amines, ratio of converted amines, and ζ-potential at pH 7.4 of native anti-NPC IgG and anti-NPC IgG derivatives

a

Sample

Number of residual aminesa

Converted amines (%)b

ζ-potential (mV)c

Native IgG

90.4

-

−7.1 ± 3.1

IgG-25d

69.1

23.6

−15.7 ± 5.7

IgG-50 d

44.0

48.7

−26.4 ± 3.8

IgG-75 d

20.0

77.9

−33.2 ± 3.1

IgG-95 d

4.4

95.1

−44.1 ± 1.7

Number of residual amines per single IgG molecule.

b

Percentage of converted amines from the total amine groups in solution.

c

Calculated from Zetasizer software 6.20, based on Smoluchowski approximation and equation. d

The numbers in sample name indicate percentage of charge-converted amino groups in anti-NPC IgG derivatives.

pH-dependent cleavage and biological activity of charge-converted anti-NPC IgG derivatives. To confirm the acid-triggered cleavage of the amide bond in Cit-capped lysine units, each anti-NPC IgG derivative was incubated at endosomal pH conditions (pH = 5.5), and the exposed amino groups were quantified using the fluorescamine method. Approximately 95% of the original amines on IgG-25 and IgG-50 could be recovered to original lysines within 3 h (Figure 1), indicating that the charge reversal of IgG-25 and IgG50 may be completed within the time span of endosomal escape, which continues up to 24 h, as indicated in our previous reports [4, 5]. It is also reasonable to consider that the charge recovery of IgG-25 and IgG-50 may reduce the interaction between cationic block in PIC micelles, inducing micelle dissociation and contributing to the endosomal escape of charge-


14

Page 15 of 30

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

Biomacromolecules

recovered antibodies. In contrast, complete recovery was not achieved for IgG-75 and IgG-95, as less than 80% of original amines could be reverted even after 12 h incubation. These results indicate a potential restriction for highly charge-converted IgG derivatives, such as IgG-75 and IgG-95, which would not be expected to completely recover before translocating from the endosomal compartments into the cytoplasm.

Figure 1. Acid (pH 5.5) triggered cleavage kinetics of charge-converted IgG derivatives (log scale in time) Data are expressed as mean ± S.D. (n = 3).

We then investigated the biological activity of obtained IgG derivatives by flow cytometry. Accordingly, the nuclei were extracted from cultured C26 cells and subsequently incubated with anti-NPC IgG derivatives, followed by staining with Alexa Fluor 647-labeled secondary antibody. Then, the fluorescence intensities for each sample were analyzed. Compared with the positive control, i.e., native anti-NPC IgG antibody, the affinity to the antigenic site of IgG derivatives decreased according to the charge-conversional rate before acidic incubation (0 h in Figure 2). The biological activities of anti-NPC IgG derivatives were gradually recovered upon 1 h incubation at pH 5.5, as evidenced by an increase in the affinity (Figure 2). It is noteworthy that the biological activities of IgG-25 (gray bars) and IgG-50 (black bars) were completely recovered within 4 h incubation (Figure 2), and these results are consistent with the acid triggered cleavage kinetics (Figure 1). Therefore, IgG-25 and IgG-50


15

Biomacromolecules

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 30

are expected to achieve complete cleavage and activity restoration inside the cells, unless they are prematurely released from the acidic compartments before 4 h incubation. Previous reports on PAsp(DET)-based PIC micelles [16-17] have shown that the process of endosomal escape lasts for at least 24 h, suggesting that IgG-25 and IgG-50 would be able to recover their binding ability and recognize cytosolic antigens after being successfully internalized into the cells.

Figure 2. Evaluation of biological activities of anti-NPC IgG derivatives upon incubation at pH 5.5 (10 mM phosphate buffer). Isolated nuclei of C26 cells were treated with anti-NPC IgG derivatives and native anti-NPC IgG (positive control), following immunostaining by Alexa Fluor 647-labeled secondary antibodies and flow cytometry measurement. Data are expressed as mean ± S.D. (n = 3).

Formation of the anti-NPC IgG derivative-loaded PIC micelles. Polymer shielding of PIC micelles is regulated by the mixed molar ratio of cationic and anionic components, i.e., P/A in this study. To determine an optimal P/A, we prepared PIC micelles with different P/As, and their actual composition was determined by ultracentrifugation (Figure 3A). The association numbers, i.e., the molar ratio between polymers and IgG derivatives, were found to increase with the degree of charge conversion, suggesting that a higher negative charge


16

Page 17 of 30

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

Biomacromolecules

may facilitate polyion complex formation. Furthermore, for each anti-NPC IgG derivative, the association number of polymers reached a plateau when the P/A was 4, which suggests that the maximum shielding of PIC micelles can be obtained with this composition. At this inflection point, the amount of polymers associated with IgG derivatives increased with the degree of charge conversion (Figure 3B), indicating that polymer shielding was mainly driven by charge interaction. Therefore, we used P/A = 4 hereafter. The hydrodynamic radii of obtained PIC micelles were determined by DLS measurement. As shown in Table 2, the diameter of PIC micelles was controlled by the amount of –Cit conversion, with high-conversion giving the smaller sizes with narrow PDI. In addition, all micelles showed ζ-potential close to neutral pH, pH 7.4. The stability of these PIC micelles was confirmed in phosphate buffer containing 10% FBS and 150 mM NaCl by fluorescence correlation spectroscopy (FCS). The results demonstrated the long-term stability of IgG-75/m and IgG-95/m, and the facile dissociation of IgG-25/m (Figure 4). Because the balance between stability of micelle structure and the recovery in antigen-recognition capability is an important factor for biological application, IgG-50/m appears as the most suitable formulation for targeting of intracellular antigens.

Figure 3. A) Association numbers (mol/mol) between polymers and anti-NPC IgG derivatives, depending on P/A and degree of charge conversion. B) Association numbers at


17

Biomacromolecules

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 30

inflection point P/A = 4 (mol/mol), depending on the degree of charge conversion. Data are expressed as mean ± S.D. (n = 3).

Table 2. PIC micelles composed of anti-NPC IgG derivatives and PEG-PAsp(DET) PIC micelle

ζ-potential (mV)a

Average diameter (nm)a

PDI a

IgG-25/mb

0.7 ± 2.4

123 ± 5

0.12 ± 0.03

IgG-50/mb

1.2 ± 2.7

94 ± 5

0.08 ± 0.02

IgG-75/mb

2.8 ± 4.1

77 ± 2

0.07 ± 0.04

IgG-95/mb

1.9 ± 2.7

48 ± 3

0.06 ± 0.02

a

Determined by a Zetasizer Nano ZS. The ± symbol of ζ-potential indicates zeta deviation obtained by manufacturer’s software, and the ± symbol of both average diameter and PDI indicates the standard deviation of three replicates. b

IgG/m denotes anti-NPC IgG derivative-loaded PIC micelles, and the numbers in sample name indicate percentage of charge-converted amino groups in anti-NPC IgG derivatives.

Figure 4. Stability of anti-NPC IgG derivative-loaded PIC micelles in PBS containing 10% FBS and 150 mM NaCl. Data are expressed as mean ± S.D. (n = 3).

In vitro delivery of anti-NPC IgG derivative-loaded PIC/m to C26 murine colon carcinoma cells. To confirm the delivery efficiency of micelles, the time-lapse intracellular localization of fluorescence labeled anti-NPC IgG derivative in PIC micelles was evaluated


18

Page 19 of 30

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

Biomacromolecules

by confocal laser scanning microscopy (CLSM). Accordingly, both native anti-NPC IgG and IgG-25/m showed low cellular uptake in C26 cells even after 24 h incubation (Figure 5A and B). In contrast, IgG-50/m, IgG-75/m and IgG-95/m were rapidly internalized (Figure 5C, D, and E). However, only IgG-50/m exhibited intracellular recognition of the nuclear membrane. Presumably, smooth translocation of Cit-liberated antibodies may occur from acidic compartments of late endosomes into the cytoplasm due to impaired integrity of the endosomal membrane through the interaction with highly-protonated polycations (Scheme 2; Pathway A-C). These results indicate that ca. 50% modification of amino groups in the antiNPC IgG by citraconic anhydride is the most appropriate condition for the aim of this study.


19

Biomacromolecules

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

1h

4h

Page 20 of 30

24 h

A

B

C

D

E


20

Page 21 of 30

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

Biomacromolecules

Figure 5. Real-time observation of in vitro IgG PIC/m delivery to the C26 cells. A) Native IgG; B) IgG-25/m; C) IgG-50/m; D) IgG-75/m; and E) IgG-95/m. Scale bars (white) represent 20 µm.

Scheme 2. Schematic interpretation of Figure 5 showing the plausible intracellular pathways (A-D) of IgG PIC micelles loaded with 25%, 50%, 75% and 95% charge-converted IgG derivatives.

Effect of homo-catiomer integration on the characteristics of PIC micelles. An advantage of PIC micelles is their capability for refining their characteristics by modifying their structural components. Particularly, integration of homo-ionomer into PIC micelles prepared from block-ionomer occasionally causes a significant effect on their physicochemical and biological properties [16-17]. Accordingly, there may be a possibility to modulate the delivery efficiency of antibody-incorporated PIC micelles through the addition of homo-catiomers (PAsp(DET)). Thus, we evaluated the effect of PAsp(DET) addition on the properties of the micelles. A series of PAsp(DET) catiomers integrated anti-NPC IgG-


21

Biomacromolecules

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 22 of 30

50/m were prepared by the use of the mixed solution of PEG-PAsp(DET) and PAsp(DET) (P/A = 4) with varying mixing percentages (Chomo = 0, 25, 50, 75, and 100%). The size of PIC micelles were increased from 98 nm (Chomo = 0%) to 188 nm (Chomo = 75%) by increasing Chomo, while keeping the narrow size distribution. There was also a slight increase in ζpotentials with the addition of PAsp(DET). It was notable that the mixture of IgG-50 with 100% PAsp(DET) (Chomo = 100%) resulted in the formation of heterogeneous aggregates to precipitate, indicating the indispensable role of block copolymers in size regulation of PIC for maintaining micellar structure.

Table 3. IgG-50/m composed of various Chomo percentages. Sample namea IgG-50/m

0

1.2 ± 2.7

Average diameter (nm)b 94 ± 5

IgG-50/m+Chomo 25%

25

1.7 ± 2.4

128 ± 7

0.12 ± 0.05

IgG-50/m+Chomo 50%

50

2.8 ± 0.8

168 ± 4

0.09 ± 0.04

IgG-50/m+Chomo 75%

75

4.4 ± 1.2

189 ± 3

0.15 ± 0.05

IgG-50/m+Chomo 100%

100

N/A

precipitation

N/A

Chomo (%)

ζ-potential (mV)b

PDIb 0.08 ± 0.02

a

The subscript +Chomo indicates the integration of PAsp(DET) into the PIC micelle, and the percentage values e.g. 25%, correspond to the ratio of amino group concentration in PAsp(DET) to the total amines in the polymer mixture. The same manner was adopted for Chomo 50%, Chomo 75%, and Chomo 100%. b

Determined by a Zetasizer Nano ZS at pH 7.4 The ± symbol of ζ-potential indicates zeta deviation obtained by manufacturer’s software, and the ± symbol of both average diameter and PDI indicates the standard deviation of three replicates. To investigate the effects of homo-catiomer integration on the stability of the PIC micelles, The PIC micelles loaded with Cy5 labeled IgG derivatives and different Chomo were prepared, and the time-dependent changes in the diffusion coefficients (D) were evaluated by FCS (Figure 6). Initial D values of free IgG, IgG50/m, IgG50/m+Chomo 25%, IgG50/m+Chomo 50%, and IgG50/m+Chomo 75% were determined to be 18.4, 2.4, 2.2, 2.0 and 1.8 µm2/sec, respectively


22

Page 23 of 30

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

Biomacromolecules

(Figure 6, time = 0 h). The decrease in D of micelles with the integration of homo-catiomer was consistent with the diameter increase observed by DLS (Table 3). The time-lapse FCS measurement revealed that only IgG-50/m+Chomo

75%

was stable during 4 h, while IgG-

50/m+Chomo 0%, IgG-50/m+Chomo 25%, and IgG-50/m+Chomo 50% showed a gradual increase of D values, probably due to the dissociation of the micelles and/or the release of antibodies. By ultracentrifugation of these IgG PIC micelles, we observed that an increment of Chomo augmented the association efficiency and the number of associated polymers to single antibody (Table 4), which not only suggests that the plateau in the association number observed in Figure 3 could be associated with the steric hindrance of PEG, but also indicates improvement of stability with Chomo most likely due to higher polymer shielding.

Figure 6. Effect of PAsp(DET) integration on stability of anti-NPC IgG derivative-loaded PIC micelles composed at P/A ratio 4, in PBS with 10% FBS and 150 mM NaCl. Data are expressed as mean ± S.D. (n = 3).

Table 4. Association numbers between polymers and antibodies in IgG-50/m, depending on the P/A and Chomo. Sample

Association number of polymer per one IgG

Association


23

Biomacromolecules

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

a

Page 24 of 30

(mol/mol)a

efficiency (%)a,b

IgG-50/m

0.82

21

IgG-50/m+Chomo25%

1.88

47

IgG-50/m+Chomo50%

2.74

69

IgG-50/m+Chomo75%

3.60

90

Determined by ultracentrifugal analysis.

b

Calculated as the percentage of associated polymers from the total fed polymers.

In vitro evaluation of the PIC micelles containing anti-NPC IgG derivative and control IgG derivative. To precisely quantify the impact of PAsp(DET) integration, high-throughput analysis on fluorescent microscopy was applied to C26 cells treated with PIC micelles with various Chomo percentages. Cells were incubated with PIC micelles incorporating anti-NPC IgG derivative for 48 h, and they were further stained with Alexa Fluor 647-labeled secondary antibodies after fixation with 4% paraformaldehyde. IgG1 κ from murine myeloma was used for construction of control micelles (control IgG/m). Subcellular localization of control IgG and anti-NPC IgG was investigated by detecting Alexa Fluor 647 signals in the nuclei or cytosol by using InCell Analyzer (Figure 7). IgG-50/m+Chomo75% had a higher fluorescence intensity around nuclei than those with different Chomo percentages, indicating this micelle formulation could efficiently deliver active anti-NPC IgG to nuclei (Figure 7A, black bars). Moreover, total fluorescence intensity in nuclei and cytosol showed that the IgG50/m+Chomo 75% had the highest cellular uptake (Figure 7A, line, and Scheme 2; Pathway D), probably due to their stability in medium and slightly positive ζ-potential (Table 3). For the IgG-50/m+Chomo75%, at least 83% of the internalized antibody molecules could successfully escape from the endosomes, and subsequently recognize the cytosolic antigen (Supporting Information, Table S1). In contrast, the fluorescence intensities around nuclei in the cells treated with the control IgG/m were low and similar between the samples and independent on


24

Page 25 of 30

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

Biomacromolecules

Chomo percentages in micelle formation (Figure 7B). This demonstrated that nuclear targeting is related to specific recognition of anti-NPC IgG delivered by PIC micelles. These results indicate the potential for fine-tuning of PIC micelles for the development of novel therapeutic strategies using intracellular targeted antibodies.

Figure 7. In vitro evaluation of IgG PIC micelle by InCell Analyzer. Data are expressed as mean ± S.D. (n = 1,000). A) native anti-NPC IgG and anti-NPC IgG/m, B) native control IgG and control IgG/m


25

Biomacromolecules

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 26 of 30

CONCLUSIONS Our study provides valuable knowledge for the construction of antibody-incorporating polymeric micelles for efficient intracellular delivery by fine-tuning their characteristics through chemical modifications. Appropriate control of the charge-conversional rate enabled antibodies to effectively recover their affinity when passing through the endosomes. By adjusting the polymeric components, PIC micelles evolved as effective intracellular carriers of the charge-converted IgGs. Thus, the PIC micelle, which equipped the IgG derivative with 50% Cit-conversion and 75% PAsp(DET), with 4 of the polymer/antibody mixing molar ratio, exhibited a significantly higher performance for the intracellular delivery of antibodies. Given the importance of the controlled charge conversion and integration of the structural components in PIC micelles performed in this examination, we believe that these strategies will contribute to the future development of safe intracellular antibody delivery systems and can serve as a platform for the discovery and development of novel intracellular targeted antibody strategies.

Supporting Information Available The supporting information includes a table, showing the fraction of antibodies recognizing the nuclei, calculated based on the result of Figure 7. This material is available at free of charge via the Internet at http://pubs.acs.org


26

Page 27 of 30

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

Biomacromolecules

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected], Tel: +81-3-5841-7138; Fax: +81-3-5841-7139 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.


27

Biomacromolecules

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 28 of 30

ACKNOWLEDGMENT This research was financially supported by the Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and Development (AMED) (NN, KK), and Center of Innovation Program (COI) from Japan Science and Technology Agency (JST) (KK). This work was partially supported by Initiative for Accelerating Regulatory Science in Innovative Drug, Medical Device, and Regenerative Medicine (KK), Grants-in-Aid for Young Scientists (A; No. 24689051 to YM, B; 25750172 to HC) and Challenging Exploratory Research (No. 24659584 to YM). We thank Dr. Qixian Chen, Dr. Ahn Jooyeon, Dr. Hyun Jin Kim, Graduate School of Engineering, The University of Tokyo for technical assistance and useful discussion.

REFERENCES (1) Harada, A.; Kataoka, K. Macromolecules 1998, 31, 288-294. (2) Harada, A.; Kataoka, K. Langmuir 1999, 15, 4208-4212. (3) Lee, Y.; Kataoka, K. Soft Matter 2009, 5, 3810-3817. (4) Lee, Y.; Ishii, T.; Cabral, H.; Kim, H. J.; Seo, J. H.; Nishiyama, N.; Oshima, H.; Osada, K.; Kataoka, K. Angew. Chem., Int. Ed. 2009, 48, 5309-5312. (5) Lee, Y.; Ishii, T.; Kim, H. J.; Nishiyama, N.; Hayakawa, Y.; Itaka, K.; Kataoka, K. Angew. Chem., Int. Ed. 2010, 49, 2552-2555. (6) Lu, Y.; Sun, W.; Gu, Z.; J. Controlled Release 2014, 198, 1-19. (7) Postupalenko, V.; Sibler, A.; Desplancq, D.; Nominé, Y.; Spehner, D.; Schultz, P.; Weiss, E.; Zuber, G. J. Controlled Release 2014, 178, 86-94.


28

Page 29 of 30

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

Biomacromolecules

(8) Auger, A.; Park, M.; Nitschek, F.; Minassian, L.; Beilhartz, G.; Minassian, B.; Melnyk, R. Mol. Pharmaceutics, 2015, 12, 2962-2971. (9) Verdurmen, W.; Luginbühl, M.; Honegger, A.; Plückthun, A. J. Controlled Release 2015, 200, 13-22. (10) Dalkara, D.; Zuber, G.; Behr, J. P. Mol. Ther. 2004, 9, 964-969. (11) Didenko, V. V.; Ngo, H.; Baskin, D. S. Anal. Biochem. 2005, 344, 168-173. (12) Futami, J.; Kitazoe, M.; Maeda, T.; Nukui, E.; Sakaguchi, M.; Kosaka, J.; Miyazaki, M.; Kosaka, M.; Tada, H.; Seno, M.; Sasaki, J.; Huh, N. H.; Namba, M.; Yamada, H. J. Biosci. Bioeng. 2005, 99, 95-103. (13) Niikura, K.; Horisawa, K.; Doi. N. J. Controlled Release 2015, 212, 85-93. (14) Liao, X.; Rabideau, A.; Pentelute, B.; ChemBioChem 2014, 15, 2458-2466. (15) Miyata, K.; Oba, M.; Nakanishi, M.; Fukushima, S.; Yamasaki, Y.; Koyama, H.; Nishiyama, N.; Kataoka, K. J. Am. Chem. Soc. 2008, 130, 16287-16294. (16) Chen, Q.; Osada, K.; Ishii, T.; Oba, M.; Uchida, S.; Tockary, T. A.; Endo, T.; Ge, Z.; Kinoh, H.; Kano, M. R.; Itaka, K.; Kataoka, K. Biomaterials 2012, 33, 4722-4730. (17) Uchida, S.; Itaka, K.; Chen, Q.; Osada, K.; Ishii, T.; Shibata, M. A.; Harada-Shiba, M.; Kataoka, K. Mol. Ther. 2012, 20, 1196-1203. (18) Davis, L. I.; Blobel, G. Cell 1986, 45, 699-709. (19) Kanayama, N.; Fukushima, S.; Nishiyama, N.; Itaka, K.; Jang, W. D.; Miyata, K.; Yamasaki, Y.; Chung, U. I.; Kataoka, K. ChemMedChem 2006, 1, 439-444.


29

Biomacromolecules

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 30 of 30

Table of Contents Intracellular Delivery of Charge-converted Monoclonal Antibodies by Combinatorial Design of Block/Homo Polyion Complex Micelles Ahram Kim, Yutaka Miura, Takehiko Ishii, Omer F. Mutaf, Nobuhiro Nishiyama, Horacio Cabral, and Kazunori Kataoka


30

Intracellular Delivery of Charge-Converted ... - ACS Publications

Recommend Documents