Tumor-Targeted Accumulation of Ligand-Installed Polymeric Micelles

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Tumor-targeted accumulation of ligand-installed polymeric micelles influenced by surface PEGylation crowdedness Xi Yang, Qixian Chen, Jinjun Yang, Sudong Wu, Jun Liu, Zhen Li, Deqiang Liu, Xiyi Chen, and Yongming Qiu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16764 • Publication Date (Web): 01 Dec 2017 Downloaded from http://pubs.acs.org on December 1, 2017

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ACS Applied Materials & Interfaces

Tumor-targeted accumulation of ligand-installed polymeric micelles influenced by surface PEGylation crowdedness Xi Yang,a Qixian Chen,b,* Jinjun Yang,c Sudong Wu,d Jun Liu,e Zhen Li,f Deqiang Liu,g Xiyi Chen,f Yongming Qiu,a,**

a

Department of Neurosurgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, No.

227 South Chongqing Road, Shanghai 200127, China b

School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road,

Dalian 116024, China c

School of Environmental Science and Safety Engineering, Tianjin University of Technology, Xiqing

District, Tianjin 300384, China d

Ningbo Institute of Materials Technology and Engineering, China Academy of Sciences, Ningbo

315201, China e

Ningbo Hygeia Medical Technology Co., Ltd, No.6 Jingyuan Road, High-Tech Zone, Ningbo 315040,

China f

Dalian Medical University, No. 9 West Section Lvshun South Road, Dalian 116044, China

g

The No. 2 People's Hospital of Tongxiang, No. 18 Qingyangdong Road, Congfu Town, Tongxiang

314511, China

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ABSTRACT In respect to the intriguing biocompatibility and the stealthy functions of poly (ethylene glycol) (PEG), PEGylated nanoparticulates have been intensively engineered for utilities as drug delivery vehicles. To advocate the targeted drug transportation, targeting ligands were strategically installed onto the surface of PEGylated nanoparticulates. The previous in vitro investigations revealed that the ligand-specified cell endocytosis of nanoparticulates was pronounced for the nanoparticulates with adequately high PEG crowdedness. The present study aims to explore insight into the impact of PEGylation degree on in vivo tumor-targeted accumulation activities of cRGD-installed nanoparticulates. The subsequent investigations verified the importance of the PEGylation crowdedness in pursuit of prolonged retention in the blood circulation post intravenous administration. Unprecedentedly, the PEGylation crowdedness was also identified as a crucial important parameter to pursue the tumor-targeted accumulation. A plausible reason is the elevated PEGylation crowdedness eliciting the restricted involvement in non-specific protein adsorption of nanoparticulates in the biological milieu and consequently pronouncing the ligand-receptor-mediated binding for the nanoparticulates. Noteworthy was the distinctive performance of the class of the proposed systems once utilized for transportation of the mRNA payload to the tumors. The protein expression in the targeted tumors appeared to follow a clear PEGylation crowdedness dependence manner, where merely twofold PEGylation crowdedness led to remarkably tenfold augmentation in protein expression in tumors. Hence, the results provided important information and implications for design of active-targeting PEGylated nanomaterials to fulfill the targeting strategies in systemic applications.

KEYWORDS: polymeric micelle, PEGylation, cyclic RGD, tumor accumulation, drug delivery


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1. INTRODUCTION The intriguing potential of nanomedicine has sparkled the excitement of pharmaceutical scientists in pursuit of targeted therapeutics for treatment of intractable diseases.1-3 The ensuing decades of groundbreaking researches, particularly pertaining to tumor therapy, have validated the benefits of the nanoscaled (more specifically sub-100 nm nanoparticles) pharmaceutical formulations following intravenous administration capable of prompting tumor-preferential accumulation relying on the enhanced permeation and retention (EPR) characters of the tumors.4,5 The stimulated angiogenesis of tumors characterized with defective alignment of endothelial cells with wide fenestrations in the architecture of neovasculature results in enhancement of the extravasation and accumulation of 100 nm or sub-100 nm nanoparticulates in the tumors. This tumor-preferential accumulation of the nanoscaled formulations, once utilized for transport of chemotherapeutic drugs, conduced to pronounced therapeutic potency to the targeted tumors and minimized toxicity to the vital organs owing to the reduced non-specific biodistribution.6,7 The prerequisite for nanoparticle-mediated EPR effect lies on the appreciable stealthy functionality and adequate retention period of the nanoparticles in the bloodstream, capability in evading non-specific reactions with the biological species (or structures) and recognition by immune systems (e.g. circulating phagocytes, reticuloendothelial systems: RES).8,9 Aiming for stealthy functionality in the blood circulation, poly(glycol ethylene) (PEG) surface modification (PEGylation) onto the nanoparticles has acknowledged as a valid strategy in manufacture of a spatially stealthy shell.10,11 The external shell of PEG affords excellent biocompatibility and passivation behaviors in the physiological milieu by means of minimizing adverse bio-interfacial interaction, consequently accounting for improved persistence in the bloodstream and thus improved tumor accumulation based on EPR effect.12,13 These intriguing characters eventually encouraged translation of the PEGylated nanomedicine into clinical trials. Nevertheless, EPR effect was skeptical in clinical trials in respect to the markedly disparity between the experimental tumor models and the spontaneous tumors of the patients (where the spontaneous



tumors were diversified with distinctive pathophysiology and heterogeneous histology).14 This fact motivates the researchers to explore alternative strategies by installing the active targeting moieties (e.g. antibodies, peptides) onto the PEGylated nanoparticles for pursuit of active tumor-targeting strategies through ligand-receptor-mediated tumor accumulation.15,16 Nevertheless, the impact of strategic installment of active targeting moieties onto the nanoscaled delivery systems in pursuit of the active targeting efficiency was rarely been systematically studied. The promotion index of the active targeting strategies also appeared markedly inconsistency among the delivery systems in spite of the same targeting moieties. Apparently, the specific ligand-receptor affinity should be influenced by the physiochemical characters of nanoparticles,17,18 particularly their surface chemistry including the surface charge, the species of surface stealthy materials, the density of the surface stealthy materials. In the current study, we aim to explore the insight into the correlation of PEGylation density to the ligand-specified tumor-targeted accumulation activity. Recently, we attempted a ligand (cyclic Arg-Gyl-Asp: cRGD)-installed PEGylated polymeric nanoformulation with precise modulation of surface PEGylation crowdedness.19 The subsequent in vitro investigations revealed that the ligand-receptor mediated affinity highly depended on the PEGylation degree of the nanoformulation. Learning from this important information, we are motivated to investigate in vivo tumor accumulation efficiency of ligand-installed nanoparticles as a function of the tethered numbers of PEG chains on the polyplex micelle, more specifically PEG crowdedness. The subsequent investigations are postulated to provide important information and implications for design of active-targeting PEGylated nanomaterials to fulfill the targeting strategies towards practical applications.

2. MATERIALS AND METHODS 2.1. Materials and equipments. α-Methoxy-ω-amino-PEG (Mw 12,000) and acetal-PEG-NH2 was purchased from Nippon Oil and Fats Co., Ltd. (Tokyo, Japan). β-Benzyl-L-aspartate N-carboxyanhydride (BLA-NCA) was purchased from Chuo Kaseihin Co., Inc. (Tokyo, Japan).


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Diethylenetriamine (DET), N, N-dimethylformamide (DMF), n-butylamine, dichloromethane, benzene, and trifluoroacetic acid were purchased from Wako Pure Chemical Industries, Ltd. 3,3'-Dithiodipropionic acid di(N-hydroxysuccinimide ester) (NHS-SS-NHS) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 3-Dimethylaminopropyl-3-ethylcarbodiimide hydrochloride was purchased from Fisher Scientific International, Inc. (Pittsburgh, PA). Alexa Fluor 488 succinimidyl ester was purchased from Invitrogen, Life Technology (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Dainippon Sumitomo Parma Co., Ltd. (Osaka, Japan). Cell culture lysis buffer and luciferase Assay System Kit was purchased from Promega Co. (Madison, WI). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Sigma–Aldrich (St. Louis, MO). In pertinent to the cellular uptake and intracellular distribution assay, mRNA was labeled with Cy3 using a Label IT Nucleic Acid Labeling Kit from Mirus Bio Corporation (Madison, WI) according to the manufacturer’s protocol. For aqueous phase SEC, LC-2000 system (JASCO, Tokyo, Japan) equipped with Superdex200 10/300 GL (GE Healthcare, Tokyo, Japan) and a UV detector was used for characterizations. All animal experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals as stated by the guidelines of Renji Hospital (Shanghai Jiao Tong University School of Medicine). 2.2. DTT-responsive cleavage of PEG-SS-PAsp(DET). The stock solution of PEG-SS-PAsp(DET) was dissolved in PBS (10 mM, pH 7.4) at a concentration of 1 mg/mL, which was subsequently resorted into two equal fractions, either supplemented with DTT-containing PBS solution or blank PBS solution to have the final DTT concentration of 50 mM or 0 mM and final polymer concentration of 0.5 mg/mL. The reaction solution was transferred to aqueous GPC measurement. 2.3. Preparation of polymeric micelles. The stock solution of cRGD-PEG-PAsp(DET) together with PEG-SS-PAsp(DET) and PEG-PAsp(DET) was dissolved in PBS buffer (10 mM, pH 7.4) at a concentration of 10 mg/mL. Meanwhile, powder of PAsp was dissolved in PBS buffer (10 mM, pH 7.4) at a concentration of 10 mg/mL. Furthermore, aliquot of stock solution was mixed at an equal charge



ratio under vortex for 30 s. The complexation solution was transferred at an ice bath for incubation for physiological characterizations. 2.4. Crosslinking for polymeric micelles. The prepared polymeric micelles in PBS buffer (pH 7.4) were supplemented with EDC solution. Note the molar ratio of EDC and carboxyl group of PAsp was 1:5 with the aim of coupling reaction. The reaction was kept at 4 °C for 3 h, followed by dialysis in PBS buffer (pH 7.4) for three times at 4 °C to remove unreacted EDC. 2.5. DLS measurement. The hydrodynamic diameter and polydispersity index (PDI) of polymeric micelles were measured by DLS using a Zetasizer Nanoseries instrument (Malvern Instruments Ltd., UK). The measurement was performed for three times at 25 °C. The rate of decay in the photon correlation function was analyzed according to a cumulant method, and the corresponding diameter was calculated using the Stokes-Einstein equation. 2.6. ς-potential measurement. The ζ-potential of the polymeric micelles was measured by Nano ZS (ZEN3600, Malvern Instruments, Ltd., UK). The polymeric micelle solution, either with DTT (50 mM) treatment or no treatment was injected into folded capillary cells (Malvern Instruments, Ltd.). The ς-potential was determined from the laser-doppler electrophoresis using the Zetasizer nanoseries (Malvern Instruments Ltd., UK). From the obtained electrophoretic mobility, the ς-potential was calculated by using the Smoluchowski equation: ς = 4πην/ε in which η is the electrophoretic mobility, ν is the viscosity of the solvent, and ε is the dielectric constant of the solvent. The results are expressed as the average of three experiments. 2.7 Protein adsorption by Isothermal titration calorimetry (ITC). The ITC investigations were carried out at 37 °C, using a VP-ITC microcalorimeter (GE MicroCal Inc., USA). The protein adsorption reactions were performed by injecting 20 µM solution of BSA in 1 × PBS into a 2 mL sample cuvette containing polymeric micelles at a concentration of 2 mg/mL in 1 × PBS under a stirring speed of 300. A total of 50 injections were conducted with an interval of 240 s and a reference power of 10 µcal/s. Titration volumes of BSA were programmed as follows: an initial injection of 2 µL,


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followed by thirty-four injections of 5 µL, and fourteen injections of 10 µL. Binding isotherms were plotted and analyzed using Origin Software (MicroCal Inc., Worcestershire, UK), where the ITC measurements were fit into a one-site binding model. 2.8. Fluorescence correlation spectroscopy (FCS) measurements. FCS measurements were performed using a Confocol 3 module (Carl Zeiss, Jena, Germany) equipped with a Zeiss C-Apochromat 40 × water objective. Samples were measured with the excitation at 488 nm. The Alexa Fluor 488-labeled samples (100 µl for each) were placed into 8-well Lab-Tek chambered borosilicate cover-glass slides (Nalge Nunc International, Rochester, NY). Each analysis consisted of 10 measurements with a sampling time of 20 seconds. The measured autocorrelation curves were fitted with the Zeiss Confocol 3 software package to obtain the diffusion time. The diffusion time obtained from the Zeiss Confocol 3 software fitting was converted to diffusion coefficient as a reference of molecular Alexa Fluor 488. 2.9. Analytical ultracentrifuge. Molecular weights (MWs) of polymeric micelles were determined based on a sedimentation equilibrium method in analytical ultracentrifuge instrument equipped with absorbance optics (Beckman Coulter, Brea, CA) according to a method as described previously. In brief, the polymeric solution was prepared according to the Method 2.3 with treatment of the Method 2.4, followed by sedimentation in ultracentrifuge for 48 h. The absorbance at 260 nm along the centrifugal radius was recorded and analyzed using ORIGIN software (Beckman Coulter). 2.10. Fluid atomic force microscopy (AFM) characterization. Atomic force microscopy (AFM) imaging of the polymeric formulation was performed using a MMAFM, Nanoscope IIIa (Veeco, USA) in tapping mode with standard silicon probes. Imaging was conducted under aqueous conditions on a highly orientated pyrolytic graphite substrate. The obtained images were processed with flattening treatment with the aim of removing the background slope of the substrate surface. 2.11. Tumor accumulation activities. Balb/c nude mice were inoculated subcutaneously with U87 cells (107 cells in 100 mL of PBS). Tumors were allowed to grow for three weeks (tumor size was



approximately 300 mm3). Mice were randomly selected for two cohorts (n = 5). Solution of polymeric micelles (0.2 mL) were intravenously injected into the blood stream through the tail vein to quantify the tumor accumulation efficiency. Mice were sacrificed 24 h after injection. The tumor tissue was excised. The lysed solution of tumor tissue was transferred to the wells in a 96-well plate for IVIS measurement. (IVIS, Xenogen Co. Alameda, CA). 2.12. Biodistribution. A class of polymeric micelles was intravenously injected into BALB/c mice. Organs including spleens, liver, tumors, lung and kidneys from these BALB/c mice were harvested at 24 h post injection after euthanization in a CO2 chamber. The organs were homogenized. The lysed solution of tumor tissue was transferred to the wells in a 96-well plate for IVIS measurement. 2.13. Intravital confocal laser scanning microscopy. Balb/c nude mice were subjected to anesthesia under the flow of isoflurane and placed on top of a thermal confocal microscope stage. Prior to imaging, the mouse ear was attached to a glass coverslip with aids of microscope immersion fluid. Intravital microscopy was performed using a Nikon A1R confocal laser scanning microscope system IVRTCLSM equipped with a 40× objective lens post intravenous administration of polymeric micelles labeled by Alexa Fluor 488 (Excitation wavelength: 488 nm, Emission filter range: 520/50 nm). For image analysis, initial background fluorescence was subtracted, and circular regions of interest were highlighted within the mouse ear vessels. Fluorescence from these regions of interest was quantified and background fluorescence was subtracted. Intensity values were normalized to initial peak intensity. 2.14. Preparation of polymeric micelles containing the mRNA payload. The stock solution of cRGD-PEG-PAsp(DET) together with PEG-SS-PAsp(DET) and PEG-PAsp(DET) was dissolved in PBS buffer (10 mM, pH 7.4) at a concentration of 10 mg/mL. Meanwhile, mRNA (Luc) was dissolved in PBS buffer (10 mM, pH 7.4) at a concentration of 100 ng/µL. Polymeric micelles were prepared by mixing 1-unit volume of the PEG-PLys solution with 2-unit volume of mRNA solution at an N/P ratio of 1 under vortex for 10 s. The N/P ratio is defined as the residual molar ratio of amine (N) groups of


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PEG-PLys to the phosphate (P) groups of mRNA. The crosslinking for mRNA polymeric micelles was conducted according to a similar procedure with the crosslinker of NHS-SS-NHS. Note that the molar ratio of the NHS moieties in NHS-SS-NHS and the Asp(DET) units was 1:5 with the aim of crosslinking of amine groups of PAsp(DET) segments. 2.15. Gene expression in tumors. Polymeric formulation containing LUC (10 µg in 200 µl of 10 mM HEPES containing 150 mM NaCl) were intravenously injected once into the tumor-bearing mice via the tail vein. The mice were sacrificed after 48 h of injection and xenografted tumors were excised. The tumor tissue was homogenized and subjected in a cell lysis buffer. Furthermore, the cell lysate (20 µl) was transferred to a 96-well luminometry plate, followed by the addition of 100 µl of Luciferase Assay Reagent (Promega, Madison, WI) to each well. The Luc expression was then measured for 10 s from the photoluminescence intensity using Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany). The amount of total protein in the cell lysate was quantified using the Micro BCA™ Protein Assay Kit (Pierce, Rockford, IL), and the obtained Luc activity was normalized against the corresponding amount of total protein in the cell lysates. The data were expressed as the relative light units (RLU) per mg of protein (RLU/mg protein) (n = 6).

3. RESULTS AND DISCUSSION 3.1. Preparation and characterizations of a class of polymeric micelles with constant number of ligand moieties and varied PEGylation degree. Given that the PEGylation crowdedness should play a crucial role as a critical parameter in affecting bioavailability and circulation profile of nanoparticulates in bloodstream, it affects the extent of non-specific interactions in the biological milieu, particular protein adsorption, phagocytosis, opsonization, etc.20 Here, we attempted a polymeric micelle system, characterized to possess a constant number of ligand moieties on the surface of the polymeric micelles but varied PEGylation crowdedness. Specifically, a template polymeric micelle was manufactured based on electrostatic complexation of opposite-charged polycationic block



copolymer

PEG-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}

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PAsp(DET)

[(PEG-PAsp(DET)) and polyanionic poly(aspartic acids) (PAsp) (chemical descriptions were summarized in Table S1). Aiming to modulate the PEGylation degree of polymeric micelles, PEG-SS-PAsp(DET) characterized to cleavage into PEG and PAsp(DET) by virtue of disulfide breakage in a reducing milieu, was proposed to substitute a varied composition of PEG-PAsp(DET). Moreover, cRGD-PEG-PAsp(DET) was proposed to substitute a constant composition (10%) of PEG-PAsp(DET) to functionalize the polymeric micelles, aiming for active target affinity to the tumors. Note that cRGD was determined to have selective affinity to the αVβ3 and αVβ5 integrins, which are overexpressed on the surface of a variety of cancerous cell lines including the angiogenic endothelial cells.21,22 Herein, cRGD as the targeting moiety was utilized to study the impact of PEGylation degree on the targeting activities of the polymeric micelles to the tumors. In the current study, PEG-SS-PAsp(DET) characterized with facile disulfide linkage between the polymeric blocks was harnessed to modulate the PEGylation degree of the polymeric micelles. The degree of PEGylation degree was defined as the molar percentage of non-cleavable cationic PEG block copolymer compositions [sum-up of PEG-PAsp(DET) and cRGD-PEG-PAsp(DET)] to the total cationic PEG block copolymer [sum-up of PEG-PAsp(DET), cRGD-PEG-PAsp(DET) and PEG-SS-PAsp(DET)]. For instance, the cationic components of the system, consisting of the constant composition of 10% cRGD-PEG-PAsp(DET)], and varied compositions of 50% PEG-PAsp(DET) and 40% PEG-SS-PAsp(DET)] in terms of molar percentage, was referred to hereafter PEG60 (standing for 60% PEGylation remaining after PEG detachment treatment). The cleavage of disulfide linkage in the diblock copolymer of PEG-SS-PAsp(DET) into PEG and PAsp(DET) segments responsive to a reducing milieu was carried out under incubation in a dithiothreitol (DTT)-containing PBS solution (50 mM DTT, pH 7.4) for 1 h. This strategic approach enables the facile compositions of PEG-SS-PAsp(DET) and PEG-PAsp(DET) to obtain a precise modulation of the PEGylation density for a given polymeric micelle. To verify this disulfide cleavage, Alexa Fluor 488 fluorophore was


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conjugated at the distal end of PEG in the block copolymer of PEG-SS-PAsp(DET), namely Alexa Fluor 488-PEG-SS-PAsp(DET). The diffusion time of the following samples was characterized: Alexa Fluor 488-PEG-SS-PAsp(DET) block copolymer, the polymeric micelles of Alexa Fluor 488-PEG-SS-PAsp(DET) and PAsp prior to DTT treatment and post DTT treatment, and the control sample of the polymeric micelle of PEG-SS-PAsp(DET) [Alexa Fluor 488 fluorophore was conjugated at the side chain of PAsp(DET), namely, PEG-SS-PAsp(DET-Alexa Fluor 488)] and PAsp post DTT treatment. Fluorescence correlation spectroscopy (FCS) was employed to investigate the cleavage of PEG from the polymeric micelle (Fig. 1). As opposed to the diffusion time of Alexa Fluor 488-labeled PEG-SS-PAsp(DET) being approximate 136 µsec (orange dashed line in Fig. 1), the pronounced jump in diffusion time was observed once PEG-SS-PAsp(DET) was complexed with PAsp (approximate 2,630 µsec in terms of diffusion time) (orange solid line in Fig. 1). This jump in diffusion time approved the successful complexation of PEG-SS-PAsp(DET) and PAsp. Furthermore, the complex of PEG-SS-PAsp(DET)/PAsp (where PEG-SS-PAsp(DET) was labeled with Alexa Fluor 488 at the distal end of PEG segment and side chain of PAsp(DET) segment, respectively) was transferred for DTT treatment (50 mM DTT in PBS solution, pH 7.4). The diffusion of Alexa Fluor 488 tethered components was accordingly recorded by FCS measurement. The sample of Alexa Fluor 488-labeling in PAsp(DET) segment was determined to have a resembled diffusion time (2,613 µsec) (green solid line in Fig. 1) as compared to PEG-SS-PAsp(DET)/PAsp(DET) complex (orange solid line in Fig. 1), indicating the PAsp(DET) was preserved in the complex formation, in stark contrast to the sample of Alexa Fluor 488-labeling at the distal end of PEG segment where its diffusion time (92 µsec, green dashed line in Fig. 1) is even lower than that of PEG-SS-PAsp(DET) block copolymer (orange dashed line in Fig. 1). This result is consistent with the previous GPC study and affirmed the complete detachment of PEG from the complex formation,19 consequently liberating the PEG segments as the molecular form.



On the other hand, AFM measurement for the aqueous solution of PEG-SS-PAsp(DET)/PAsp complex prior to and post DTT treatment. As shown in Fig. 2, the complex of PEG-SS-PAsp(DET)/PAsp(DET) was observed as uniform spherical structures with average diameter of approximate 51.2 nm based on AFM measurement. On the other hand, distinctive reduced sized was confirmed for the same sample with DTT treatment (50 mM DTT, pH 7.4), whose average diameter of approximate 29.4 nm. This result is consistent with the previous dynamic light scattering measurement for the sample of PEG-SS-PAsp(DET)/PAsp complex prior to and post DTT treatment,19 thereby confirming the facile sheddable PEG shell from the proposed PEGylated formation by means of the disulfide bond cleavage. The facile detachment function of the proposed disulfide cleavage strategy was confirmed by FCS and AFM measurement, which approved the utility of modulating the compositions of PEG-SS-PAsp(DET) and PEG-PAsp(DET) for a controlled PEGylation degree of the polymeric micelles. Following this scheme, a class of polymeric micelles with varied PEGylation degrees was prepared. The physiochemical characterizations, particular for the calculation of PEG degree, were summarized in Table 1. In consistent with the previous research, a class of polymeric micelles was determined to have comparable dynamic laser scattering (DLS) size and zeta-potential despite varied PEGylation degree. In attempt for characterization of the PEG conformation in the class of polymeric micelles, the PEG crowdedness of diverse polymeric micelles was calculated as follows: the aggregation number of the tethered PEG chains per particles was determined by the analytical ultracentrifuge measurement for estimation of molecular weight of the sample of PEG100 and the subsequent calculation on the aggregation molecular numbers of PEG-PAsp(DET) derivatives and PAsp on a basis of stoichiometric complex. Furthermore, the number of the tethered PEG chains per particles for the rest samples was calculated accordingly by considering the composition of PEG cleavable PEG-SS-PAsp(DET) composition and PEG non-cleavable cRGD-PEG-PAsp(DET) and PEG-PAsp(DET) compositions.


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The diameter of the complex core (l) was estimated based on TEM measurement, given that PEG shell is invisible under TEM due to its minimal affinity with uranyl acetate (UA) as compared to that of poly(amino acids) components [including PAsp(DET) and PAsp]. Thus, the contours of PAsp(DET)/PAsp complex core were selectively visualized without interference from the external PEG shell.23 The obtained TEM image [Fig. S1] was further analyzed by analytical software of Image J 1.44 to measure the diameter of the complex cores in each sample, and 100 individual nanoparticles were measured to give the number average diameter (l) of the complex cores. To this end, the surface area of the complex core was calculated by the following formula of 2

4πl . The number-average PEG density (σ) was obtained in terms of the number of PEG chains in unit 2

2

area (chains/nm ). Furthermore, σ was converted to the index of the reduced tethering density πRg σ (which is an important index for insight into PEG crowdedness and conformation), defined as the 2

number of chains that occupies an area covered by an isolated polymer chain (πRg ).24 Given that Rg = 0.58

[0.181 × (Mn of PEG/44.06) 2

(in nm)] for PEG in water is 4.7 nm for Mn 12 kDa, and thus πRg is 69

2

nm , the πRg σ for a class of polymeric micelles was determined.25 As listed in Table 1, the conformation of PEG in the present class of polymeric micelles was determined to range from the isolated mushroom conformation to the overlapping mushroom conformation,26 which has been documented as a representative range that are subjected to protein adsorption to a significantly varied extend.27 3.2. Prolonged blood retention and promoted tumor accumulation by polymeric micelles with high PEGylation degree. PEGylation has been documented as a valid strategically approach to diminish the activity of protein adsorption to the bio-interface due to its intrinsic biocompatibility and stealthiness.28 A recent study by T. Tockary and K. Kataoka, et al,26 reported that the critical importance of PEG crowdedness in reducing the non-specific interaction in the biological milieu, wherein high PEGylation crowdedness afforded appreciable stealthiness function and consequently



prolonged retention in the blood circulation. To this respect, it is readily speculated that the polymeric micelles post PEG detachment should induce an increased susceptibility to the adsorption of protein in the biological milieu. To verify this speculation, a class of polymeric micelles with varied PEGylation degree was incubated with serum albumins and the protein adsorption was quantified by ITC measurement. In agree with our speculation, the protein adsorption activity was observed to be markedly high for the polymeric micelles at low PEGylation degrees (Fig. 3). The protein adsorption activity was considerably reduced for the polymeric micelle along a rising PEGylation degree. Particularly, very limited protein adsorption was confirmed for the polymeric degree exceeding 80% (Fig. 3). The elicited formation of protein corona was speculated to trigger blood clearance by RES systems, thereby leading to reduced blood circulation.29 In this respect, the retention in the blood circulation was studied for a class of polymeric micelles with varied PEGylation degrees. As shown in Fig. 4, improved longevity in the blood circulation appeared for the polymeric micelles with high PEGylation degree. For instance, the half-life of PEG100 was determined to exceed 200 min whereas approximate 30 min was determined for the half-life of PEG20. Close observation recognized substantial of polymeric micelles with low PEGylation degree (e.g. PEG20 and PEG40) being cleared from the blood circulation immediate post intravenous administration. A plausible reason for this rapid clearance should be as a result of their poor stealthy function, which induced them susceptible to the determined protein adsorption (including IgG, opsonins, Fig.3) and triggered the subsequent immune machinery (e.g. RES) for elimination. In consistence, the liver and spleen characterized to be the major RES organs appeared to have markedly high accumulation of polymeric micelles with low PEGylation degree, particularly for PEG10, PEG20 and PEG40. As summarized in Table 1, the PEG chains in these polymeric formations possess an isolated mushroom conformation, which is apparently susceptible to substantial protein adsorption, including opsonins. Hence, the opsonized polymeric micelles could be readily harvested by the livers and spleens and eliminated from the blood circulation.


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As determined by the previous study, the polymeric micelles with relatively low PEGylation crowdedness were subjected to intensive protein adsorption, accordingly conducing to reduced ligand-specific recognition and binding activity between the ligands and the receptors in the targeted cells [17]. In this regard, the tumor accumulation efficiency was assumed to exhibit a PEGylation degree-dependent manner where ligand-installed nanoparticulates with relative high PEGylation crowdedness was believed to exert high tumor accumulation activity. Herein, the tumor accumulation efficiency was investigated for a class of polymeric micelles with varied PEGylation degrees by intravenous administration of Alexa Fluor 488-labeled polymeric micelles to the bloodstream of U87 xenografted Balb/c nude mice. Clear PEGylation crowdedness dependence manner was obtained for a class of polymeric micelles, where the marked tumor accumulation was obtained for the polymeric micelle with high PEGylation degree (Fig. 5). Noteworthy was the tumor accumulation of PEG100, which was estimated to mediate approximate 12-fold higher tumor accumulation efficiency than that of PEG10 despite an identical number of targeting ligands (cRGD) on the surface of a class of polymeric micelles. 3.3. Promoted gene expression of polymeric micelle containing mRNA with high PEGylation degree. To claim the practical importance of advocating PEGylation degree for the performance of the ligand-installed polymeric micelles, we attempted to load the polyanionic LUC mRNA into a class of polymeric micelles with varied PEGylation degrees [where mRNA was used as an alternative to PAsp for complexation with PEG-PAsp(DET) derivatives, followed by crosslinking treatment with NHS-SS-NHS. In vivo expression of the LUC mRNA in tumors was studied. Of note, the polymeric micelles were intravenously injected to the mice bearing subcutaneous xenografted U87 tumors. At 48 h post administration, the tumors were dissected and transferred for homogenize with aids of trypsin, aiming for quantification of LUC expression based on a luminescence assay. Distinct gene expression of LUC mRNA was confirmed in the tumors, especially for the polymeric micelles with high PEGylation degree (Fig. 6). Of note, PEG100 mediated approximate 30-fold higher in vivo gene



expression efficiency than PEG10, which recapitulated the merits of high PEGylation degree for not only targeted accumulation of mRNA into tumors but also favorable cRGD-mediated gene transfection activities. These results verified the importance of engineering high PEGylation crowdedness in pursuit of minimized non-specific reaction in the physiological milieu and effectively harnessing the ligand motif for accumulation into the tumor sites.

4. CONCLUSIONS The PEGylation degree was facilely tailored for the nanoscaled PEGylated polymeric micelles with a constant number of ligands on their surface, and the PEG density and the corresponding PEG conformation of the tailored formation has also been successfully estimated. This opens the avenue of detailed insight into the impact of PEGylation density on the retention time in the bloodstream, ligand-mediated targeted accumulation efficiency and biodistribution. The subsequent investigations underlie a deep correlation of PEGylation degree with systemic tumor-targeting activity through ligand-mediated pathway. Higher PEGylation crowdedness was capable of reducing the non-specific interactions in the biological milieu, e.g. avoiding the formation of protein corona as a result of protein adsorption to the nanoparticulates. The reduced protein adsorption not only benefited for pursuit of prolong blood circulation but also appeared to facilitate the binding affinity of ligand to the receptors of the targeted cells. Hence, the effort in elevating PEGylation density from the isolated mushroom to the range of overlapped mushroom or scalable polymerbrush was observed to prompt one-order magnitude higher tumor-targeted accumulation activity and induce marked expression once the payload of mRNA was encapsulated. Hence, these results validated the crucial importance of PEGylation in advocating the ligand-mediated targeted delivery of nanoparticulates for development of targeted-nanomedicine.

Acknowledgement


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This research was funded by National Natural Science Foundation of China (No. 81671203 and No. 21304070), Science and Technology Commission of Shanghai Municipality (No. 16140902900), and Natural Science Foundation of Tianjin City (No. 15JCYBJC47300). Q. C. acknowledges the funding support from the Fundamental Research Funds for the Central Universities [No. DUT17RC(3)059].

Supporting Information The process of calculating PEG density, and TEM morphologies of polymeric micelles. This material is available free of charge via the Internet at http://pub.acs.org.

Corresponding Authors *E-mail: [email protected] (Q. Chen). *E-mail: [email protected] (Y. Qiu).

Notes The authors declare no competing financial interest.

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Fig. 1. The autocorrelation curves of a variety of Alexa Fluor 488-labeled samples by FCS measurement. Alexa Fluor 488-PEG-SS-PAsp(DET) polymer (orange dashed line), Alexa Fluor 488-PEG-SS-PAsp(DET)/PAsp prior to DTT treatment (orange solid line), PEG-SS-PAsp(DET-Alexa Fluor 488)/PAsp post DTT treatment (green solid line), Alexa Fluor 488-PEG-SS-PAsp(DET)/PAsp post DTT treatment (green dashed line).

Fig. 2. AFM morphologies of polymeric micelles of PEG-SS-PAsp(DET)/PAsp prior to DTT treatment (left image, the number average diameter was calculated to be 51.2 nm based on measurement of 100 individual nanoparticles) or post DTT treatment (right image, the number average diameter was calculated to be 29.4 nm based on measurement of 100 individual nanoparticles). Scale bar: 100 nm.


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Fig. 3. The protein adsorption of a class of cRGD-installed polymeric micelles with varying PEGylation degrees. The plasma adsorption as a function of PEGylation degree was summarized into the bar diagram (*p < 0.05, **p < 0.01, Student 2

t. test). The PEGylation degree was calculated into the absolute PEG density in term of πRg σ, as the point diagram.



Fig. 4. Blood circulation profile of a class of cRGD-installed polymeric micelles with vary PEGylation degree.

Fig. 5. Biodistribution profiles of a class of cRGD-installed polymeric micelles with varying PEGylation degrees.

Fig. 6. Gene expression of the payload of LUC mRNA in tumors via intravenous administration of a variety of polymeric *

micelles. ( p < 0.05,

**

p < 0.01, Student T. Test.)


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Table 1 Physiochemical characterizations of a class of polymeric micelles as a function of PEGylation degree. No. of the tethered PEG chains per particle

Diameter of complex core (nm)

PEG density (No. of PEG chains / nm2 surface of complex core)

PEG density (πRg2σ)

DLS size (nm in diameter)

ζ (mV)

PEG10

10.1

30.3

0.0035

0.24

51.4 ± 1.3

3.67 ± 0.03

PEG20

20.2

30.8

0.0068

0.47

51.9 ± 2.4

3.14 ± 0.03

PEG40

40.4

31.5

0.013

0.89

52.6 ± 0.9

2.67 ± 0.04

PEG60

60.6

29.8

0.023

1.5

50.9 ± 0.6

2.12 ± 0.03

PEG80

80.8

31.3

0.0262

1.81

52.4 ± 2.3

1.67 ± 0.05

PEG100

101

31

0.0335

2.31

52.1 ± 1.2

1.51 ± 0.06

Table of Contents


Tumor-Targeted Accumulation of Ligand-Installed Polymeric Micelles

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