Rational Design of a Polymer with Robust Efficacy for Intracellular

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Rational Design of a Polymer with Robust Efficacy for Intracellular Protein and Peptide Delivery Hong Chang, Jia Lv, Xin Gao, Xing Wang, Hui Wang, Hui Chen, Xu He, Lei Li, and Yiyun Cheng Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.6b04955 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 17, 2017

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Rational Design of a Polymer with Robust Efficacy for Intracellular Protein and Peptide Delivery Hong Chang1,†, Jia Lv1,†, Xin Gao2, Xing Wang1, Hui Wang1, Hui Chen1, Xu He1, Lei Li1, Yiyun Cheng1,* 1

Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East

China Normal University, Shanghai 200241, P. R. China. 2

Department of Orthopedic Oncology, Changzheng Hospital, the Second Military

Medical University, Shanghai, 200003, P.R. China. ABSTRACT: The efficient delivery of biopharmaceutical drugs such as proteins and peptides into the cytosol of target cells poses substantial challenges owing to their large size and susceptibility to degradation. Current protein delivery vehicles have limitations such as the need for protein modification, insufficient delivery of large-size proteins or small peptides, and loss of protein function after the delivery. Here, we adopted a rational approach to design a polymer with robust efficacy for intracellular protein and peptide delivery. The polymer is composed of a dendrimer scaffold, a hydrophobic membrane-disruptive region, and a multivalent protein binding surface. It allows efficient protein/peptide binding, endocytosis and endosomal

disruption,

and

is

capable

of

efficiently

delivering

various

biomacromolecules including bovine serum albumin, R-phycoerythrin, p53, saporin, β-galactosidase and peptides into the cytosol of living cells. Transduction of apoptotic proteins and peptides successfully induces apoptosis in cancer cells, suggesting that the activities of proteins and peptides are maintained during the delivery. This technology represents an efficient and useful tool for intracellular protein and peptide delivery and has broad applicability for basic research and clinical applications.

KEYWORDS: Rational design, polymer, robust efficacy, protein delivery, peptide delivery

ACS Paragon Plus Environment

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Since the launch of recombinant human insulin, therapeutic proteins including cytokines, transcription factors, enzymes, peptide hormones and monoclonal antibodies have revolutionized the pharmaceutical industry.1,2 A large number of protein-based therapeutics have been discovered or engineered for treatment of cancer, diabetes, bacterial infection, and many other disorders.3,4 Protein therapeutics has exhibited the advantages of high specificity and activity even at an extremely low concentration compared to small molecule drugs.1 However, they are susceptible to enzymatic degradation and generally membrane-impermeable owing to the macromolecular nature,4 and thus the clinical use of protein drugs has been hindered by inefficient delivery of proteins into the cytosol of target cells.5 For nucleic acids such as DNA and siRNA, cationic polymers and liposomes were usually used to complex the negatively charged biomacromolecules via electrostatic interactions.6-8 However, this approach is infeasible for intracellular protein delivery because the protein might be positively, negatively or even neutrally charged at physiological condition depending on its isoelectric point.4 One solution for reducing the uncertainty of protein charge is to conjugate the protein with polyanionic molecules such as aconitic acid,9,10 green fluorescent protein,3 anionic polymer,4 and oligonucleotide.11 However, this approach is usually involved with sophisticated syntheses and the protein structure might be irreversibly changed after chemical modification. Besides protein binding, the vehicle/protein complex should be efficiently internalized by target cells and then escaped from the acidic endosomes to the cytosol. During the past decade, strategies including using nanoparticles,12-14 cationic lipids,3,15,16 polymers,9,10,17,18 and cell-penetrating peptides19,20 were developed to achieve efficient intracellular delivery of proteins. Despite significant advances, the current protein vehicles have limitations including the need for protein modification, insufficient delivery of large proteins or small peptides, ineffective endosomal escape, loss of protein function, and severe cytotoxicity.17 An efficient and biocompatible vehicle available for the delivery of a variety of proteins and peptides is highly desirable to advance protein-based basic research and therapeutics.15 In this study, we adopted a rational approach to design an efficient polymer for the


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delivery of a variety of proteins and peptides without chemical modification. The chemical structure of the polymer is shown in Figure 1A. It is consisted of three parts, a multivalent protein binding surface, a hydrophobic membrane-disruptive region, and a dendrimer scaffold. There are abundant hydrogen-bonding donors and receptors in a protein structure, and thus we hypothesize that these donors/receptors can serve as binding sites to facilitate protein binding by the vehicle with hydrogen bonding motifs. Guanidyl ligand is reported to form strong hydrogen bonds and salt bridges with amides and oxyanions in proteins,21,22 and the multivalent display of guanidyl groups on a vehicle surface can significantly strengthen the affinity between protein and the vehicle. The high density of guanidyl groups on the polymer is also beneficial for efficient endocytosis by a similar mechanism to guanidine-rich peptides, such as oligoarginines

and

trans-activating

transcriptional

activator

peptide.19,23-25

Considering the intrinsic hydrophobic property of lipid bilayers in cell membrane, we integrate a hydrophobic phenyl ligand adjacent to the guanidyl group to promote the endocytosis process.26 In addition, the integrated phenyl groups are able to disrupt the endosomal membrane, which facilitates the endosomal escape of vehicle/protein complexes.27,28 The dendrimer was used as the polymer scaffold because it allows multivalent display of guanidyl and phenyl groups and is well-established for intracellular nucleic acid delivery.8,29-31 The synthesized polymer shows several promising features for intracellular protein delivery when compared to most existing protein vehicles: (1) no need for protein modification; (2) superior delivery efficacy; (3) enabling the delivery of various proteins; (4) applicable for large-size proteins and small peptides; (5) retention of protein activity; (6) minimal cytotoxicity (Figure 1A). Both the guanidyl and phenyl groups on the dendrimer are essential for efficient intracellular protein delivery. The dendrimer with only phenyl ligands failed to complex the proteins, while the one with only guanidyl moieties failed to effectively escape from the endosomal membranes (Figure 1B).


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Figure 1. Structure of the designed polymer and its features in intracellular protein delivery (A). Proposed mechanism of the designed polymer and control materials in intracellular protein delivery (B).

Result and Disussion. An amine-terminated generation 5 polyamidoamine dendrimer was conjugated with guanidinobenzoic acid (GBA) by a facile chemistry to synthesize the protein vehicle (Figure S1). According to the 1H NMR in Figure S2, an average number of 60 GBA molecules were modified on each dendrimer (DGBA60). Similarly, the dendrimers modified with 60 benzoic acid (BA) or guanidyl groups were synthesized as control materials (DBA60 and DG60, Figure S2). As expected, the guanidyl groups on dendrimer play a critical role in protein binding (Figure 2A and 2B, Figure S3), and hydrogen bond interactions between guanidyl groups and the amino acids contribute to complex formation (Figure S4). DGBA60 and DG60 successfully formed nanoparticles around 200 nm with bovine serum albumin (BSA), while DBA60 with phenyl groups showed inefficient protein complexation (Figure S3). The formed DGBA60/BSA nanoparticles are positively charged, which is benefical for efficient cellular uptake (Figure S5). The endocytosis and intracellular trafficking of the protein/vehicle complexes were further investigated by confocal microscopy. BSA labeled with fluorescein isothiocyanate (BSA-FITC) was efficiently internalized into


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HeLa cells mediated by DGBA60 and the protein nanoparticles successfully escaped from the endosomal membrane within 2 h (Figure 2C and Figure S6). In comparison, low BSA-FITC endocytosis was obtained for DBA60, and entrapment of BSA-FITC within acidic compartments labeled with LysoTracker Red was observed for DG60 after 4 h incubation (Figure S6). The weak fluorescence observed for cells treated with DG60/BSA-FITC in Figure 2C is probably due to the fluorescence quenching of FITC in the complex, which failed to escape from the acidic compartments (Figure S7). DGBA60 exhibited dramatically higher efficacy than unmodified dendrimer, DBA60 and DG60, and was much more efficient than a commercial protein delivery reagent PULSinTM when delivering BSA-FITC into HeLa cells (Figure 2D, 2E, S8 and S9). HeLa cells incubated with DGBA60/BSA-FITC complexes for 8 h exhibited the highest fluoresence intensity (Figure S10). When the guanidinium groups on DGBA60 were replaced by amine groups, efficacy of the yielding polymer in the delivery of BSA-FITC was significantly decreased (Figure S11). Besides high efficacy, both DGBA60 and its complex with BSA caused negligible toxicity on the transfected cells (Figure 2F). These results suggest that both guanidyl and phenyl groups are essential for the intracellular protein delivery mediated by the dendrimer.


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Figure 2. Intracellular delivery of BSA into HeLa cells. Characterization of polymer/BSA complexes by dynamic light scattering (A) and transmission electron microscopy (B). Confocal images of HeLa cells treated with polymer/BSA-FITC complexes (green) for 4 h (C). The nuclei were stained with Hoechst (blue) and the acidic compartments were stained with LysoTracker (red). Fluorescence images (D) and

the

mean

fluorescence

intensity

(E)

of

HeLa

cells

treated

with

polymer/BSA-FITC or BSA-FITC only for 4 h. PULSinTM was tested as a positive control. The doses of polymer and protein in each well were 8 µg and 2 µg, respectively. Viability of HeLa cells treated with DGBA60 and DGBA60/BSA complexes at optimal transfection conditions (F).

It is reported that the delivery of large-size proteins is challenging due to the difficulties in effective protein complexation and membrane penetration.1 Here, we investigated the ability of DGBA60 to deliver relatively large-size proteins such as R-phycoerythrin (R-PE, 240 kDa) and β-galactosidase (β-Gal, 430 kDa) into different cell lines. As shown in Figure 3, S12 and S13, nearly 100% of the HeLa, NIH3T3 and Raw264.7 cells were successfully transfected with the red-fluorescent protein R-PE by the designed vehicle DGBA60, while the cells treated with DBA60/R-PE or DG60/R-PE complexes show extremely weak fluorescence. The efficacy of DGBA60 is also superior to PULSinTM. The cells treated with DGBA60/R-PE complexes for 8 h exhibited the highest efficacy (Figure S14). Similarly, DGBA60 showed high efficacy in intracellular β-Gal delivery. β-Gal is an enzyme that hydrolyzes 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) into a colorless galactose and an insoluble blue pigment. As shown in Figure 4 and Figure S15, nearly all the cells transfected with DGBA60/β-gal showed accumulation of the blue pigments after X-Gal staining, demonstrating that β-Gal was efficiently delivered into the cells. The transfected β-Gal mediated by DGBA60 remained active intracellularly (Figure 4B). Though β-Gal transfected by DG60 showed relatively high activity after 4 h incubation, the enzymatic activity was almost lost at 24 h, owing to ineffective endosomal escape mediated by DG60 and protein degradation in the acidic


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compartments (Figure 4C and 4D). The decrease of β-Gal activity delivered by DGBA60 at 24 h compared to the value at 4 h was also observed, which could be explained by the autophagic protein degradation by the transfected cells.32,33 These results demonstrate that DGBA60 is capable of delivering relatively large-size proteins into the cytosol of living cells with the retention of protein activity.

Figure 3. Intracellular delivery of R-PE into HeLa (A, B) and NIH3T3 (C, D) cells for 4 h. The doses of polymers and R-PE in each well were 8 µg and 1 µg, respectively. PULSinTM was used as a positive control.


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Figure 4. Intracellular delivery of β-Gal into HeLa cells. X-Gal staining of the cells transfected by β-Gal after 24 h (A). Relative β-Gal activity after intracellular delivery for 4 h (B) and 24 h (C). The doses of polymers and β-Gal in each well were 8 µg and 5 µg, respectively. Proposed mechanism of intracellular β-Gal delivery mediated by DGBA60 and DG60, respectively (D). PULSinTM was used as a positive control.

We further investigated the capability of DGBA60 in the delivery of apoptotic peptides and proteins into cancer cells. A cyclic hendecapeptide (CWMSPRHLGTC, P1) and a heptapeptide (AVPIAQK, P2) were used as model peptides. These peptides were labeled with FITC during their synthesis to visualize the intracellular peptide delivery. As shown in Figure 5A, 5B and S16, both P1 and P2 were efficiently delivered into HeLa cells mediatd by DGBA60 at 4 h and 24 h, while extremely weak green fluorescence was observed in the cells treated with the peptides only. The peptide P1 was reported as ligand to block the protein kinase CK2 phosphorylation34, and P2 is a pro-apoptotic peptide35. Internalization of these peptides by cancer cells will result in cell apoptosis. As shown in Figure 5C, the cells showed significant inhibition after treatment with DGBA60/P1 or DGBA60/P2 complexes. If we alter the sequence of P1 to a random one (P1-Scr, CRWSPLGMTHC), the DGBA60/P1-Scr complex caused minimal toxicity to the HeLa cells, suggesting that the cell death was


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caused by the specific delivery of P1 into cytosol (Figure S17). Similarly, DGBA60 efficiently transported an apoptotic protein p53 and a cytotoxic protein saporin into HeLa cells and PC-9 cells, respectively (Figure S18 and Figure 6). p53 is a protein that promotes the apoptosis of aberrant cells through both transcription-dependent and -independent mechanisms.36 Restoring p53 function in cancer cells is considered as a promising option for cancer treatment.37 The induction of p53 into HeLa cells by DGBA60 successfully activates the expression of p21, a downstream gene of p53, and induces the apoptosis of cancer cells (Figure S18). Saporin is a 30 kDa protein with

N-glycosidase activity, which could irreversibly blocking protein synthesis in eukaryotic cells by depurinating a specific nucleotide in 28S subunit of ribosomes.15 Saporin has been used in clinical trials to treat cancer patients that are refractory to traditional chemotherapy. Since saporin is a membrane-impermeable protein, it causes minimal toxicity to PC-9 cancer cells even at a high protein dose of 16 nM. After complexation with DGBA60, the protein can significantly inhibit the cell proliferation with an IC50 value of 2.2 nM on PC-9 cells (Figure 6A). The mice bearing PC-9 tumors were administrated with PBS, saporin, DGBA60, and DGBA60/saporin complex twice via intratumoral injection. After treatment, the tumor growth in the DGBA60/saporin complex group was notably reduced compared with those of tumors in the control groups (Figure 6B). There were no obvious body weight changes during the therapeutic period, indicating that DGBA60 and its complex with saporin had minimal systematic toxicity (Figure 6C). In addition, the polymer DGBA60 and its complex with saporin showed minimal hemolytic activity (Figure 6D) and negligible hematotoxicity in vivo (Figure S19). These results suggest that DGBA60 can be used as an efficient and non-toxic vector for intracellular protein and peptide delivery.


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Figure 5. Intracellular peptide delivery mediated by DGBA60. Fluorescence images of HeLa cells treated with DGBA60/P1-FITC or DGBA60/P2-FITC for 4 h (A). The polymer dose is 8 µg, and the peptide dose is 1 µg. Fluorescence intensity of the transfected cells (B). Viability of cells treated with DGBA60/P1-FITC or DGBA60/P2-FITC complexes for 24 h (C). P1-FITC and P2-FITC were tested as negative controls.

Figure 6. Concentration-dependent cytotoxicity of saporin and DGBA60/saporin


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complex on PC-9 cells (A). Evolution of tumor volumes in mice bearing PC-9 tumors treated with PBS, DGBA60, saporin, and DGBA60/saporin complex, respectively (B). Body weight changes during the therapeutic period (C). The black arrows indicate the injections. Data are presented as mean ± SD (n=5). **p < 0.01 analyzed by student’s t-test. Hemolytic activity of DGBA60 and DGBA60/Saporin complex at concentrations ranging from 20 to 100 µg/mL (D). PBS and Triton X-100 (0.5%) were used as the negative and positive control, respectively.

Conclusions. In summary, we designed a polymeric protein vehicle with robust efficacy in the delivery of proteins into the cytosol of living cells. Both the guanidyl and phenyl groups on the polymer contribute to the efficient intracellular protein delivery. The designed polymer efficiently delivered various proteins and peptides into cells without the need for protein modification, while maintaining high protein activity and cell viability. This study provides a facile and promising strategy for the rational design of polymeric vehicles for intracellular protein and peptide delivery. To transfer the polymer DGBA60 for in vivo protein delivery, further modifications on the polymer or polymer/protein complex are needed to shield the positive charges on the complex.38,39 We are now trying to attach the DGBA60/saporin complex with a polyethylene glycol (PEG) shell or encapsulate the complex within a biocompatible nanocapsule such as PLGA to improve its blood circulating time.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available via the Internet at http://pubs.acs.org. Additional details on experimental methods. Figures showing synthesis and characterization of DGBA60, DBA60 and DG60; optimal condition for the delivery of BSA-FITC into HeLa cells mediated by DGBA60, DBA60, DG60 and unmodified dendrimer; intracellular delivery of R-PE into Raw264.7 cells; p53 delivery efficacy. (PDF)


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AUTHOR INFORMATION Corresponding Author *E-mail (Yiyun Cheng): [email protected]

†These authors contributed equally on this manuscript. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS We thank financial supports from the National Natural Science Foundation of China (No. 21474030 and No. 21322405) and the Shanghai Municipal Science and Technology Commission (17XD1401600 and 148014518) on this work.

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Nat. Mater. 2012, 11, 82-90. (39) Yang, J.; Hendricks, W.; Liu, G.; McCaffery, J. M.; Kinzler, K. W.; Huso, D. L.; Vogelstein, B.; Zhou, S. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 14717-14722.

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Figure 1. Structure of the designed polymer and its features in intracellular protein delivery (A). Proposed mechanism of the designed polymer and control materials in intracellular protein delivery (B). 177x112mm (300 x 300 DPI)


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Figure 2. Intracellular delivery of BSA into HeLa cells. Characterization of polymer/BSA complexes by dynamic light scattering (A) and transmission electron microscopy (B). Confocal images of HeLa cells treated with polymer/BSA-FITC complexes (green) for 4 h (C). The nuclei were stained with Hoechst (blue) and the acidic compartments were stained with LysoTracker (red). Fluorescence images (D) and the mean fluorescence intensity (E) of HeLa cells treated with polymer/BSA-FITC or BSA-FITC only for 4 h. PULSinTM was tested as a positive control. The doses of polymer and protein in each well were 8 µg and 2 µg, respectively. Viability of HeLa cells treated with DGBA60 and DGBA60/BSA complexes at optimal transfection conditions (F). 177x140mm (300 x 300 DPI)


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Figure 3. Intracellular delivery of R-PE into HeLa (A, B) and NIH3T3 (C, D) cells for 4 h. The doses of polymers and R-PE in each well were 8 µg and 1 µg, respectively. PULSinTM was used as a positive control. 177x115mm (300 x 300 DPI)


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Figure 4. Intracellular delivery of β-Gal into HeLa cells. X-Gal staining of the cells transfected by β-Gal after 24 h (A). Relative β-Gal activity after intracellular delivery for 4 h (B) and 24 h (C). The doses of polymers and β-Gal in each well were 8 µg and 5 µg, respectively. Proposed mechanism of intracellular β-Gal delivery mediated by DGBA60 and DG60, respectively (D). PULSinTM was used as a positive control. 177x125mm (300 x 300 DPI)


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Intracellular peptide delivery mediated by DGBA60. Fluorescence images of HeLa cells treated with DGBA60/P1-FITC or DGBA60/P2-FITC for 4 h (A). The polymer dose is 8 µg, and the peptide dose is 1 µg. Fluorescence intensity of the transfected cells (B). Viability of cells treated with DGBA60/P1-FITC or DGBA60/P2-FITC complexes for 24 h (C). P1-FITC and P2-FITC were tested as negative controls. 177x97mm (300 x 300 DPI)


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Figure 6. Concentration-dependent cytotoxicity of saporin and DGBA60/saporin complex on PC-9 cells (A). Evolution of tumor volumes in mice bearing PC-9 tumors treated with PBS, DGBA60, saporin, and DGBA60/saporin complex, respectively (B). Body weight changes during the therapeutic period (C). The black arrows indicate the injections. Data are presented as mean ± SD (n=5). **p < 0.01 analyzed by student’s t-test. Hemolytic activity of DGBA60 and DGBA60/Saporin complex at concentrations ranging from 20 to 100 µg/mL (D). PBS and Triton X-100 (0.5%) were used as the negative and positive control, respectively. 177x132mm (300 x 300 DPI)


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80x39mm (300 x 300 DPI)


Rational Design of a Polymer with Robust Efficacy for Intracellular

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