Controlled PEGylation Crowdedness for Polymeric Micelles To Pursue

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Controlled PEGylation Crowdedness for Polymeric Micelles to Pursue Ligand-specified Privilege as Nucleic Acids Delivery Vehicles Xiyi Chen, Haifeng Gu, Jinjun Yang, Sudong Wu, Jun Liu, Xi Yang, and Qixian Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01045 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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ACS Applied Materials & Interfaces

Controlled PEGylation Crowdedness for Polymeric Micelles to Pursue Ligand-specified Privilege as Nucleic Acids Delivery Vehicles Xiyi Chen,a,* Haifeng Gu,b Jinjun Yang,c Sudong Wu,d Jun Liu,e Xi Yang,f Qixian Chene,* a

School of Public Health, Dalian Medical University, No. 9 West Section Lvshun South Road,

Dalian 116044, China b

c

College of Science, Dalian Ocean University, No. 52 Heishijiao Street, Dalian 116023, China

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

Xiqing District, Tianjing 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. 1177 Lingyun Road, High-Tech Zone,

Ningbo 315040, China f

Department of Neurosurgery, Renji Hospital, Shanghai Jiao Tong University School of

Medicine, Shanghai 200127, China Corresponding Authors *

Address correspondence to [email protected] (X. Chen) and [email protected] (Q. Chen).

KEYWORDS: polymeric micelle; PEGylation, cyclic RGD, mRNA delivery, endosome escape

ACS Paragon Plus Environment

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Abstract A facile poly (ethylene glycol) (PEG) detachment scheme was utilized to control the PEGylation degree of the polymeric micelles. The performance of cyclic Arg-Gly-Asp (cRGD) as targeted moiety was studied on a class of polymeric micelles with varied PEGylation degree, revealing that the specific cRGD-mediated cell affinity, thus cellular uptake and implicated privileges including the ligand-specified favorable intracellular trafficking and consequent favorable biofunctions, was prominent for the polymeric micelles with high PEGylation degree. These results endow important information and implications for design and development of targetednanomedicine, particularly delivery of vulnerable biological compounds.


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The remarkable advance in nanotechnology and synthetic chemistry has spurred the development of nanoscaled architectures to accommodate a variety of bioactive compounds as a reservoir for biomedical applications, e.g. delivery of the pharmaceutics substances to the pathological cells for therapeutics.1−7 Aiming for selective delivery of the bioactive payloads to the targeted cells, targeting moieties attached onto poly(ethylene glycol) surface modified (PEGylated) polymeric delivery vehicles were extensively used strategies,8,9 where the biocompatible and stealth characters of surface PEG have been validated to diminish non-specific interactions in the biological milieu while the target ligands (such as peptides, antibodies), capable of specifically recognizing the receptors overexpressed at the surface of the targeted cells, thus could prompt preferential accumulation and internalization into the targeted cells.10 Nonetheless, high PEG surface modification (PEGylation) degree is postulated to interfere the affinity between the targeting moieties and the receptors,11 moreover, question may be also raised: the involved potential non-specific interactions of the polymeric micelles with the targeted cells whether or not will subsequently end up with targeting moieties/receptors-mediated association and consequently specific cell internalization pathway?

To answer this critical question, we attempted to develop a polymeric micelle platform, possessing a constant number of ligand moieties on the surface of the polymeric micelles but varied PEGylation degree. A systematic study was carried out to explore the impact of PEGylation degree on the targeting moieties-mediated cellular uptake enhancement and also targeting moieties-implicated benefits post cellular uptake. Herein, a template polymeric micelle was manufactured based on electrostatic self-assembly of polycationic PEG-poly{N'-[N-(2aminoethyl)-2-aminoehtyl] aspartamide}PAsp(DET) [(PEG-PAsp(DET)] and polyanionic


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poly(aspartic acids) (PAsp) (chemical descriptions were summarized in Table S1). Aiming to modulate the PEGylation degrees of the polymeric micelles, PEG-SS-PAsp(DET), characterized by its facile cleavage into individual PEG block and PAsp(DET) block through disulfide breakage in the reducing milieu, was proposed to substitute varied polycationic composition of PEG-PAsp(DET) in the original template polymeric micelle.12 Accordingly, the polymeric micelles with varied PEGylation degree could be obtained by post-treatment of a class of polymeric micelles [composed of varying compositions of polycationic PEG-PAsp(DET)&PEGSS-PAsp(DET) hybrid and polyanionic PAsp] with the reducing agent. Moreover, cyclic ArgGly-Asp (cRGD)-PEG-PAsp(DET) [cRGD-PEG-PAsp(DET)] was proposed to substitute a constant polycationic composition (10%) of PEG-PAsp(DET) to functionalize the polymeric micelles aiming for targeted affinities to the pathologic cells (here human U87 astrocytoma cells was used as an example).13 Note that cRGD was determined to have selective affinities to the αVβ3 and αVβ5 integrins, which are overexpressed on the surface of a variety of cancerous cell lines,14 including U87 cells. In this study, cRGD as the targeting moiety was utilized to study the impact of PEGylation degree on the targeting moieties-mediated cellular uptake enhancement and also consequent benefits in U87 cells. Prior to the polymeric complexation, it is important to confirm the facile cleavage activity of disulfide linkage in the diblock copolymer [PEG-SSPAsp(DET)] into PEG and PAsp(DET) segments responsive to a reducing milieu. To serve this purpose, the PEG-SS-PAsp(DET) was incubated in a dithiothreitol (DTT)-containing PBS solution (50 mM DTT, pH 7.4). At 1 h post incubation, GPC measurement was performed for the aforementioned reaction solution. Meanwhile, the solution of original PEG-SS-PAsp(DET) solution without DTT was included as a control. Complete cleavage of PEG-SS-PAsp(DET) (high Mw) was observed (Fig. 1a) to covert into two fractions of lower Mw products, which could


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be assigned as the individual PEG block and PAsp(DET) block, respectively. This result affirmed the possibility of utilizing the facile redox-responsive of disulfide linkage in controlling PEG shedding from PEG-SS-PAsp(DET) formulated polymeric micelles and consequently precisely modulating the PEGylation density for a given polymeric micelle.

The polymeric micelles were prepared by electrostatic complexation between opposite-charged cRGD-PEG-PAsp(DET) [together with PEG-PAsp(DET) and PEG-SS-PAsp(DET)] and PAsp at equal charge (+/- = 1). Note that cRGD-PEG-PAsp(DET), PEG-PAsp(DET) and PEG-SSPAsp(DET) was mixed at varying molar ratios with the aim of controlling the ultimate PEGylation density. All the polymeric micelles contains a constant composition of cRGD-PEGPAsp(DET) (10%). The total composition of PEG-PAsp(DET) and cRGD-PEG-PAsp(DET) was referred as the PEGylation degree. Given that DTT could selectively cleave the disulfide bond in PEG-SS-PAsp(DET), the facile control of PEG-PAsp(DET) and PEG-SS-PAsp(DET) compositions would allow the precise control of PEGylation degree for the polymeric micelles with DTT treatment post polymeric complexation. On the other hand, the cRGD-negative control was also prepared for comparison purpose, where the composition cRGD-PEG-PAsp(DET) (10%) was replaced by PEG-PAsp(DET). To avoid the potential structural transformation, we conducted crosslinking treatment for the polymeric complex core of PAsp(DET)/PAsp by an [1ethyl-3-(3-dimethylaminopropyl)-carbodiimide] (EDC) coupling reaction.15 The formation of the complexed structures were characterized by Dynamic Light Scattering (DLS) (Table S2), which verified a uniform spherical structures with uniform size distribution [polydispersity Index (PDI) ranging from 0.06 - 0.10], and approximate 50 nm in diameter. To verify the feasibility of PEG being fully detached from the polymeric micelles under DTT treatment (10 mM), we prepared


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the polymeric micelles with only PEG-SS-PAsp(DET) and PAsp. DLS observation determined its possessing comparable size (51.2 nm in diameter). At 1 h post incubation with DTT, a markedly reduced DLS size was observed from approximate 51.2 nm to 30.1 nm (Fig. 1b). Given that 12 kDa PEG has a Rg of 4.7 nm in an unperturbed state,16 the obtained 30.1 nm (approximate size reduced by 4Rg) should represent the complex structure of PAsp(DET)/PAsp. In consistency, zeta-potential measurement for the DTT-treated sample captured a pronounced jump from + 3.1 mV (Table S2) to 21 mV, again confirming the complete dePEGylation so as for disappearance of charge-shielding activity. To verify the impact of PEGylation on the cell uptake efficiency, a class of polymeric micelles with a same number of cRGD moieties but varied PEGylation degree was incubated with U87 cells (cancerous cells characterized with overexpression of cRGD specified receptor integrins), meanwhile another class of polymeric micelles without cRGD moieties was also included as cRGD-negative control in the experiment. Overall, cRGD-functionalized samples mediated markedly higher cellular uptake activity than those without cRGD moieties (Fig. 2b). This result approved the utility of cRGD for introducing specific cell affinity and promoting cellular uptake activities.

For non-cRGD samples, the cellular uptake of the polymeric micelles appeared to follow a clear PEGylation degree-dependent manner that the polymeric micelles with relatively lower PEGylation degree tend to induce a higher mediated cellular uptake activity (Fig. 2a). This PEGmediated reduced cellular uptake activity was actually documented repeatedly that the intrinsic passivation character of PEG has propensity of reducing non-specific interactions with the biological species or biological structures. In this regards, it is not surprising that cRGDfunctionalized samples also exhibit PEGylation degree-dependent cellular uptake activities (Fig.


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2a). Higher cellular uptake activities tend to be obtained for the polymeric micelles with lower PEGylation degree. Nevertheless, close observation could discern that the tendency of the class of cRGD-functional samples was not as evident as the cRGD-negative samples. Here, a promotion index by cRGD was calculated based on the following formula:

α (promotion index) = [cellular uptake of cRGD(+)] / [cellular uptake of cRGD(-)]

higher promotion index stands for a larger contribution of cRGD-mediated cell affinity to the ultimate cellular uptake. Apparently, the promotion index appeared to consistently rise along a rising PEGylation degree (Fig. 2b), which suggest cRGD effect was more prominent at higher PEGylation degree. On the other word, the non-specific interaction of polymeric micelles with the cells (and subsequent cellular uptake) was largely involved for the polymeric micelles with low PEGylation degree,17 and the cRGD-mediated cellular uptake could be maximized by strategically elevating PEGylation degree. Subsequent investigations on the protein adsorption activity of a class of polymeric micelles with varying PEGylation degrees revealed distinctive PEGylation-degree-dependent protein adsorption behaviors, where polymeric micelles with lower PEGylation degree is markedly more susceptible to protein adsorption [Fig. 3], possible inducing formation of an external protein corona,18,19 consequently reducing the possibility of presentation of ligand moiety and recognition by the cell surface receptors. Eventually, the polymeric micelles with high PEGylation degrees could manage to resist the potential surface interactions with the biological compounds in the complex biological milieu and facilitating ligand-receptor recognition.


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As suggested by the pioneering researches, cRGD-mediated cell internalization could possible detour the endosome entrapment and steer an alternative tempting pathway to enter the cells,20 note that most of the nanoparticles was subjected to non-specific interactions with cellular membrane, and end up with entrapment and degradation in endosomes and lysosomes. Therefore, we could speculate that our proposed system could serve as a rationale template to confirm the relevance of cRGD-mediated cellular uptake in pursuit of the subsequent cRGDentitled benefits. Herein, we utilized mRNA as the testing polyanionic bioactive molecules, with respect to its vulnerability in the digestive endosomes and lysosomes,21 for complexation with cRGD-PEG-PAsp(DET) together with PEG-SS-PAsp(DET) and PEG-PAsp(DET) according to the same way. The polymeric structure was crosslinked by 3,3'-Dithiodipropionic acid di(Nhydroxysuccinimide ester) (NHS-SS-NHS), followed by dialysis for purification. A class of mRNA polymeric micelles (with a same number of cRGD moieties but varied PEGylation degrees) was transfected in U87 cells. The cellular uptake efficiencies (mRNA-labeled by Cy5 for Flow Cytometry) and gene expression efficiencies (LUC as reporter sequence) were evaluated. Similar to the model polymeric micelles, the promotion index (α) of the cellular uptake was also observed to follow consistent upward trend along a rising PEGylation degree (Fig. 4a). Meanwhile, higher promotion index (β) of the gene expression efficiency appeared to obtain at a higher PEGylation degree (Fig. 4b). The normalized expression efficiency (promotion index γ = β/α) by normalization of gene expression efficiency with cellular uptake efficiency was reconstructed and summarized in Fig. 4c, which confirmed remarkably higher expression efficiencies at higher PEGylation degrees. This indicates that cRGD-mediated cellular uptake (high PEGylation degrees) has to facilitate the intracellular trafficking of the vulnerable payload which is consistent with the claim that cRGD is capable of inducing an alternative pathway for


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intracellular trafficking of the nanoparticles rather than entrapment in endosomes and lysosomes. Indeed, our subsequent results verified PEGylation-dependent endosome entrapment activities, where polymeric micelles with lower PEGylation degree tend to entrap in the endosomes, e.g. the polymeric micelle with 10% PEGylation has approximate 89% colocalization degree with endosomes (Fig. 5), indicating involving non-specific cell interactions and endocytosis. This is in start contrast to the polymeric micelles with higher PEGylation degrees (e.g. 100%) have merely 23% entrapment in the endosomes. Hence, these results encourage us to pursue high PEGylation in development of active-targeted PEGylated polymeric vehicles for delivery of bioactive vulnerable substances. In conclusion, we have devised a nanoscaled cRGD-installed polymeric platform to study the relevance of PEGylation degree and ligand-mediated cellular uptake behaviors. The subsequent results revealed that ligand-mediated cellular uptake is prominent for the polymeric micelles with higher PEGylation degree, which was consequently entitled the ligand-specified benefits to the payloads by steering an appreciable intracellular trafficking pathway for the payloads and accomplish their bio-functions. These results should endow important information and implications for design and development of targeted-nanomedicine. Supporting Information Materials and equipment, polymer characterizations, methodologies for preparation and characterizations

of

polymeric

micelles

and

polyplex

micelles

containing

mRNA,

electrophorogram of polyplex micelles containing mRNA, snapshot of GFP expression of polyplex micelles containing GFP mRNA by INCELL Aanalyzer

Notes Conflict of Interest: The authors declare no competing financial interest.


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Acknowledgement This research was funded by National Natural Science Foundation of China (No. 21304070), Natural Science Foundation of Tianjin City (No. 15JCYBJC47300).

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Fig. 1 Investigation of DTT-responsive PEG cleavage for PEG-SS-PAsp(DET). a): GPC measurement for the reaction solution of PEG-SS-PAsp(DET) under incubation of DTT (50 mM) for 1 h, where green dash line represents the sample of the reaction solution and solid line represents the control polymer solution of PEG-SS-PAsp(DET) without DTT treatment; b): DLS measurement for the polymeric micelle from PEGSS-PAsp(DET) and PAsp with DTT (50 mM) treatment (dash line) and without DTT treatment (solid line).


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Fig. 2 The impact of PEGylation degree and cRGD motif on the cellular uptake activity of polymeric micelles. a): Cellular uptake activity of two classes of polymeric micelles at varying PEGylation degree, cRGD (+) and cRGD (-); b): the impact of PEGylation degree on the cRGD-mediated enhanced cellular uptake.


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Fig. 3 Quantification of the adsorbed BAS on a class of polymeric micelles with varying PEGylation degrees. The protein amount is expressed as mg BSA/m2 of polymeric micelle surface area. (*p < 0.05, **p < 0.01, Student T. Test.)

Fig. 4 Demonstration of cRGD-mediated benefits accomplished for the polymeric micelles with higher PEGylation degree by using mRNA as payload. a): the impact of PEGylation degree on the cRGD-mediated enhanced cellular uptake; b): the impact of PEGylation degree on the cRGD-mediated enhanced gene


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expression; c): normalized expression efficiency to demonstrate cRGD-mediated benefits other than enhanced cellular uptake.

Fig. 5 Entrapment activities of cRGD-functionalized polymeric micelles in endosomes as a function of PEGylation degree. a): Representative intracellular distribution of cRGD-functionalized polymeric micelle with varying PEGylation degree. Blue: nuclei; Green: endosomes/lysosomes; Red: polymeric micelle; Scale bar: 20 µm. b): The quantified colocalization degree of polymeric micelles and endosomes/lysosomes. The colocalization degree was calculated according to the formula: Colocalization degree = the number of yellow pixels/the total number of yellow and red pixels, where yellow indicates the mRNA that is present in the compartment of endosome/lysosome, while red indicates the mRNA that is absent in the compartment of endosome/lysosome. (*p < 0.05, **p < 0.01, Student T. Test.)


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Graphic Abstract (Table of Contents)


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Controlled PEGylation Crowdedness for Polymeric Micelles To Pursue

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