Polarity Conversion of Conjugated Polymer for Lysosome Escaping

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Polarity Conversion of Conjugated Polymer for Lysosome Escaping Lingyun Zhou, Fengting Lv, Libing Liu, and Shu Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10105 • Publication Date (Web): 08 Aug 2017 Downloaded from http://pubs.acs.org on August 11, 2017

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ACS Applied Materials & Interfaces

Polarity Conversion of Conjugated Polymer for Lysosome Escaping Lingyun Zhou,a,b Fengting Lv,a Libing Liu,a Shu Wanga* a

Beijing National Laboratory of Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100910, P. R. China b

University of Chinese Academy of Sciences,Beijing 100049, P.R.China E-mail: [email protected]

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ABSTRACT: Polymers are mostly trapped in lysosomes when they enter cells and then expelled out, otherwise they were designed to be degradable to small molecules or to sabotage lysosomes. Therefore they have reached the limit of the unique functionalities as a whole. Different from other escaping strategy, we introduced the polarity exchanging approach to rigid-backboned conjugated polymer for controlled penetrating through endosome or lysosome membranes. With the aid of pH sensitive cleavage of water-soluble side chain, the rigid conjugated polymer turns to be highly hydrophobic after internalized into lysosomes then accomplishes escaping. Thus, polarity exchange of CPs could become a new strategy for their application on chemotherapeutics. KEYWORDS: lysosome escaping, conjugated polymers, polarity conversion, fluorescent tracer, pH response

Functional polymer-based materials have been applied in increasing extents of biological applications, such as drug delivery systems (DDS), protein or RNA carriers, chemotherapeutics, tissue engineering and so on1-7. Polymers entering cells could be through different pathways. Targeting ligand modified polymers could bind with the receptor anchored on cell membrane, which could be further transported into cell by receptor-mediated endocytosis and other endocytosis pathway. Macromolecules without targeting moieties mostly by forming water-dispersible micelle or particle, could contact with cell outer membrane through electrostatic interactions with the aid of cationic moieties on side chains and hydrophobic interactions from polymer backbones8-11. Following with continuous incubation, they could be internalized by clathrin-dependent endocytosis, caveolae-dependent endocytosis and micropinocytosis12,

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. Internalized polymers are enclosed by plasma

membrane and form early endosomes, which subsequently transform into late endosomes and finally merge with perinuclear lysosomes14. 2


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Although high molecular weight molecules could enter cells, they have reached the limits of the functionalities because halt before the barrier of endosome and lysosome membrane. After being up-taken through either endocytosis or pinocytosis, polymers were captured in endosomes or later in lysosomes mainly because of inability to escape15. Nature has developed a mature system for species to evade the arrest by lysosome. Viral and bacterial escape from endosomes using some peptides such as cationic amphiphilic peptides (AMP)16, which can generate and stabilize pores in the lipid membrane17. Another way for viruses is producing peptides, which can undergo conformational changes at low pH and fuse in the lipid bilayer18-21. Haemagglutinin, an influenza virus coat could convert from anionic hydrophilic coil to a hydrophobic α-helical structure at acidic environment leading to its fusion into endosome membrane22, 23. Similar strategies were also adopted by bacteria like Corynebacterium diphtheria, whose secreta, diphtheria toxin could undergo conformational changes and insert into membrane of endosomes, before bacteria were trapped and digested in lysosomes24,

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. Proton sponge effect (or pH-buffering effect) was utilized for synthetic

polymer such as widely used polyethylenimine (PEI) and histidine-rich molecules, to get rid of trapping in lysosomes. These molecules were capable of absorbing protons to become detergents in order to unbalance inner environment then disrupt the membrane of endosomes or lysosomes. Photochemical disruption of the endosomal membrane is also adopted, where sensitizer produces reactive oxygen species (ROS) under light irradiation to break membranes for escaping from endosomes or lysosomes26, 27. Conjugated polymers (CPs) as unique species among other polymers possess good optoelectronic properties, such as high bright fluorescence and large extinction coefficients. Modified with water-soluble moieties (such as oligo ethylene glycol, ammonium groups) or other targeting ligands, they can be applied in the fields of optical sensors, fluorescent imaging, drug screening, disease diagnosis and photodynamic therapy14,

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. However,


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particular method for CPs escaping endosome or lysosome is absent, so that to some degree the extent of application of CPs in biological area is restricted. Herein, inspired by the environmental responsive peptide that could change polarity and hydrophilic/hydrophobic property, we demonstrated a new strategy for CPs to evade from being captured. CPs mainly own hydrophilic substitutes to ensure good aqueous disperse for biological application, while they also possess highly hydrophobic and rigid backbones, as similar with pH sensitive amphiphilic peptides. Meanwhile, the low polarity of carbon-hydrogen bond endows polymer only weak interactions with phospholipids, and it is difficult for rigid molecules to disperse in the matrix of flexible molecules29, while with the aids of π-π interaction accorded by conjugated backbone, they could easily assemble with each other. Hence, we designed a poly(phenylene ethynelene) (PPE) with cleavable hydrophilic sidechains, which help polymer to contact cell membrane and enter cells, and then could be hydrolyzed under acid environment inside cells, precisely endosome or lysosome. After the side-chains of PPE molecule fully detached then hydrophobic main-chain exposed, the polymer would lose its water dispersity and form rigid and hydrophobic linear structure. The PPE polymer will be extruded out of the cytosol due to their weak interactions with phospholipids, to accomplish the escaping rather than remained in membrane structure (Scheme 1).

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Scheme 1. The illustration of polarity conversion of PPE molecules to escape from lysosomes.

PPE-A was water dispersible polymer with amide and oligo ethylene glycol (OEG) groups on the side chain with a molecular weight of 12500 approximately. The PPE was adopted as backbone of CPs here because it is more rigid compared with other CPs. Therefore, it was more mimic like amphiphilic peptides, which were able to penetrate the membrane. The linkage between amide group and polymer backbone was designed to be easily hydrolysable in acid environment and relatively stable in neutral environment, by inducing the beta-thiol substituted ester through Michael addition (Scheme 2) 30. After hydrolysis, water-soluble moieties would be separated apart and hydrophobic backbone would be

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exposed. PPE-B with less length of side chain compared with PPE-A (Scheme 2) and PPE-C without cleavable hydrophilic side-chains (Scheme S1) were also prepared for getting more insight into the structure-property relationship.

Scheme 2. Synthesis route of PPE-A and PPE-B.

To verify the occurrence of hydrolysis, PPE-A was respectively incubated for 1, 4, 24 hours in HCl aqueous solution (pH 4.0) simulating lysosome environment, and in phosphate buffer saline (pH 7.2) simulating cytosol or cell culture medium environment. The hydrolyzed products were separated through centrifugal ultrafiltration and small molecular products were collected for NMR tests. As illustrated in Figure 1, in pH 4.0 HCl aqueous solution, the 1HNMR signal of hydrolyzed product 2-(2-(2-((2-carboxyethyl)thio)ethoxy)ethoxy)-N,N,Ntrimethylethan-1-aminium bromide enhanced along with reaction time. The longer time sustained, the stronger signal of each peak was observed, which indicated that the gradual hydrolysis of PPE. Noted that the peak heights exhibit little diversity after 4-hour hydrolysis,

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indicating the completion of hydrolysis process. But in neutral solution (PBS), the unconspicuous signal indicated that very less hydrolysis even after 24 hours.

Figure

1.

1

H-NMR

and

the

chemical

structure

of

hydrolysate,

2-(2-(2-((2-

carboxyethyl)thio)ethoxy)ethoxy)-N,N,N-trimethylethan-1-aminium bromide. a) Zoomed 1HNMR spectra of the hydrolysate under HCl aqueous solution (pH 4.0). b) Zoomed 1H-NMR spectra of the hydrolysate under PBS (pH 7.2). c) Stacked all 1H-NMR spectra, the water peak intensity was unified. Dash-line frame is the zoomed part in a) and b). d) Hydrolysis of PPEs and the chemical structure of hydrolysate.

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PPE-A exhibits a maximum absorption at 414 nm, and a maximum emission at 498 nm with an absolute fluorescence quantum yield of 1% in water (Figure S1). The fluorescent intensity varies with the hydrolysis process of PPE-A in acid environment. Figure 2a (Figure S2) showed the descent of fluorescent intensity of PPE-A at maximum emission wavelength as the hydrolysis time increased. As detaching of water soluble side-chains, PPE-A became hydrophobic in water and aggregated with each other inducing the quenching of fluorescence. Comparatively, fluorescent intensity showed little variation in PBS, which further indicates that hydrolysis process was much more difficult in neutral environment and PPE remained dispersion in solution. It is noteworthy that the precipitates were imperceptible with the naked eyes because of low concentration of 2.5 µM and continuously stirring during the fluorescent intensity measurements The hydrolyzed particle was still small in diameter (around 300 nm), which was further verified by dynamic light scattering (DLS) data. DLS measurement and transmission electron microscope (TEM) imaging were conducted to testify the aggregation changes upon hydrolysis. As shown in Figure 2b (Figure S3), the size of PPE-A measured by DLS enlarged along with the extension of reaction time in HAc-NaAc buffer (pH 4.0). TEM images (Figure 2c, d) also showed amorphous macule without hydrolysis, while nonuniform triangle shaped particles with a size of around 100 nm emerged with hydrolysis. With the aid of OEG and amide groups on cleavable side-chain, PPE-A molecules were separately dispersed in solution. After hydrolysis, the molecules stack with each other through π-π interactions and hydrophobic interactions, resulting in bigger particle sizes.

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Figure 2. a) The variation of fluorescent intensity of PPE-A in maximum emission wavelength during hydrolysis. b) The variation of diameter (average size) of PPE-A molecules during hydrolysis. c) TEM image of PPE-A without hydrolysis. The scale bar is 1 µm. d) TEM image of PPE-A with 24 hours hydrolysis in pH 4.0 HCl aqueous solutions. The scale bar is 1 µm.

The cytotoxicity of PPE-A on A549 cell line was confirmed to be nontoxic at the concentration lower than 2.5 µM upon incubation for 24 hours (Figure S1), and only 20% inhibition rate of cell growth was observed when the concentration of PPE-A was up to 16 µM. Colocalization experiment was conducted to testify the escaping capacity of PPE-A. Figure 3 shows that after 10 minutes incubation, PPE-A successfully contacted with cell membrane mainly through electrostatic interactions between positive charged ammonium

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groups and negative charged cell outer membrane. Further endocytosis procedure of cell membrane started to actively internalize the molecules in 20 minutes, as some green fluorescent macule generated by PPE-A overlapped with red fluorescence generated by lysotracker DND 99 inside and near the cell membrane. Later the overlapping of lyso-tracker and PPE-A signal became more obvious after 1 hour, at this case, most PPE-A’s green fluorescence were colocalized with red ones of lyso-tracker DND 99, which indicated that PPE-A molecules were endocytosed and transferred to lysosomes, yet without escaping. Three hours later, PPE-A penetrated out of lysosome membrane and was observed in cytosol, which confirmed by the distinct non-overlapping green and red fluorescence. Not pervading every part of cytosol but presenting as aggregated dots resulted from the hydrolyzed polymers remaining hydrophobic backbone that prefer gathering mutually.

Figure 3. Confocal laser scanning microscope images of A549 cells after incubated with 2.5 µM PPE-A for different length of time and labeled with lysotracker DND-99.

We further investigated the influence of the length of spacer that connects hydrophobic backbone and hydrophilic side chain on lysosome escaping capacity. As comparison, PPE-B was synthesized with a spacer of two carbons length rather than six carbons length in PPE-A.

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Similar escaping capacity was illustrated in Figure 4. Differently, the fluorescent intensity of PPE-B in cytosol was revealed a little bit weaker and the distribution was relatively less than that of PPE-A. Furthermore, PPE-C was synthesized without cleavable amide group on side chain, where ester bond was used instead. It showed almost nontoxicity and was incapable of escaping from lysosome.

Figure 4. CLSM images of A549 cells after incubated with 2.5 µM PPE-B or PPE-C for different length of time and labeled with lyso-tracker DND-99. Images of cells incubated with PPE-C are red framed.

In conclusion, different from other escaping strategy for CPs, we introduced the polarity exchanging approach for controlled penetrating through endosome or lysosome membranes. After water-soluble side chain of CPs undergoes cleavage, they successfully escape from acid environments in endosomes or lysosomes to cytosol and form self-assembled particles. Speculatively unlike the virus, bacteria or synthetic fusogenic peptide remaining in membrane structure and disrupting membrane stability, retention in phospholipid is not easy for CPs. Therefore, polarity exchange of CPs could become a new strategy for their application on chemotherapeutics. 11



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ASSOCIATED CONTENT Supporting Information. Experimental procedures, additional Scheme S1 and Figures S1S3. This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] (S.W.)

ACKNOWLEDGEMENTS The authors are grateful to the National Natural Science Foundation of China (Nos. 21373243, 91527306, 21661132006).

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Polarity Conversion of Conjugated Polymer for Lysosome Escaping

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