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Directly cytoplasmic delivery and nuclear targeting delivery of HPMA-MT conjugates in a microtubules dependent fashion Jiaju Zhong, Xi Zhu, Lian Li, Manlin Tang, Yanxi Liu, Zhou Zhou, and Yuan Huang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00181 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 30, 2016

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Molecular Pharmaceutics

Directly cytoplasmic delivery and nuclear targeting delivery of HPMA-MT conjugates in a microtubules dependent fashion

Jiaju Zhong, Xi Zhu, Lian Li, Manlin Tang, Yanxi Liu, Zhou Zhou, Yuan Huang*. Key Laboratory of Drug Targeting and Drug Delivery System (Ministery of Education), West China School of Pharmacy. Sichuan University, NO. 17, Block 3, South Renmin Road, Chengdu 610041, P.R. China.

*Corresponding author: Prof. Yuan Huang, Tel.: +86-28-85501617, Fax: +86-28-85501617 Email: [email protected]

ACS Paragon Plus Environment


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Table of Contents Graphic

The nuclear delivery process of FITC labeled and MT peptide decorated HPMA copolymer conjugates.


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Abstract

As the hearts of tumor cells, the nucleus is the ultimate target of many chemotherapeutic agents and genes. However, the nuclear drug delivery is always hampered by multiple intracellular obstacles, such as low efficiency of lysosome escape and insufficient nuclear trafficking. Herein, an N-(2-hydroxypropyl) methacrylamide (HPMA) polymer-based drug delivery system was designed, which could achieve directly cytoplasmic delivery by non-endocytic pathway and transport into nucleus in a microtubules dependent fashion.

A special targeting

peptide (MT), derived from an endogenic parathyroid hormone-related protein, was conjugated to the polymer backbone, which could accumulate into nucleus by microtubule-mediated pathway. The in vitro studies found that low temperature and NaN3 could not influence the cell internalization of the conjugates. Besides, no obvious overlay of the conjugates with lysosome demonstrated that the polymer conjugates could enter the tumor cell cytoplasm by non-endocytic pathway, thus avoiding the drug degradation in lysosome. Furthermore, after suppression of the microtubule dynamics with microtubule stabilizing docetaxel (DTX) and destabilizing nocodazole (Noc), the nuclear accumulation of polymeric conjugates was significantly inhibited. Living cells fluorescence recovery after photobleaching study found that the nuclear import rate of conjugates was 2-fold faster compared with the



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DTX and Noc treated groups. These results demonstrated that the conjugates transported into nucleus in a microtubules dependent way. Therefore, in addition to directly cytoplasmic delivery, our peptide conjugated polymeric platform could simultaneously mediate nuclear drug accumulation, which may open a new path for further intracellular genes/peptides delivery.

Key words: Nuclear drug delivery, Cytoplasmic delivery, Microtubules,

HPMA copolymer, Cancer therapy


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1. Introduction

Recently, the interesting of sub-cellular compartment-specific antitumor drug delivery system of nanomaterials has skyrocketed with the development of molecular pharmaceutics.1-3

As the heart of cells, the

nucleus is the ultimate target of a large number of chemotherapeutic agents and genes, in which the genetic information exists and also a lot of anti-cancer drugs act efficiently.4 Therefore, quite a lot of nanoscale carriers such as polymers, micelles and inorganic nanoparticles were designed to achieve nuclear drug delivery.5-7

However, significant

limitations exist with those strategies, including insufficient cellular uptake, lyso/endosomal trap and nuclear barriers. Most nano-carriers developed for intracellular drug delivery take advantage of endocytic pathways to reach cell cytoplasm and intracellular compartment.8,

9

For enzyme-based or pH-sensitive therapies, these

pathways provide a possible target location, while in term of biological drugs, lysosomes are the degradation site.10 Therefore, to achieve cytoplasmic or organelles targeting, the drug carriers should escape from the harsh endo/lysosome before the therapeutics being degradation.11 Thus, quiet a lot of recent intracellular drug delivery platform were focused on the promoting of the endo/lysosomal escape, for example, by using endo/lysosomal membrane-destabilizing peptide HA2, GALA,



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INF7 and p5RHH,12, 13 Polymers incorporated of secondary or tertiary amine group (PEI, histidine) could rupture the endo/lysosomal membrane by "proton sponge effect".14, 15 Nanoparticles with positive charge could interact with negatively charged lysosomal membrane, which accounted for membrane flipping and subsequent destabilization.16 However, the highly acidic environment of lysosome with rich enzymes contributed to the degradation of carriers, drugs and especially for therapeutics peptide or gene inevitably. Thus, the inadequate lysosomal escape has become a development bottleneck for subcellular dug delivery. An alternative way to prevent nanoparticle/drug from degradation is to enable their directly cytoplasmic delivery by non-endocytic pathway. The study by Kostas found that functionalized multiwelled carbon nanotubes could penetrate into cytoplasm by energy-independent mechanisms.17 Also Jungmin Lee group developed a microfluidic device to deliver nanoparticles into cytoplasm by non-endocytic pathway.18

Both of these nanoparticle

systems exhibited great potential in cytoplasmic and subcellular organelles drug delivery. After escaping the lysosome, the next important issue for the nano vehicles is to overcome the degradation by cytoplasmic nucleases and gain entry into the nucleus. Double layer membrane limited the access to nucleus from cytoplasm. Therefore, macromolecules with molecular weight ~40 KDa or smaller could enter the nuclear region by nuclear


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pore.19,

20

Various nuclear targeting strategies were reported with

different mechanisms including nuclear location signal (NLS) and nuclear receptor (estrogen receptors, retinoic acid receptors and glucocorticoid receptors)-mediated active nuclear transport,21-25 positive nuclear delivery through microtubule cytoskeleton/motor/dynein mediation or cationic polymers mediated electrostatic interaction.26 Previous studies showed that some specific endogenic protein such as retinoblastoma (Rb), p53 and parathyroid hormone-related protein (PTHrP) could undergo fast nuclear import by MTs/dynein.27, 28 Detailed study finished by Daniela and coworkers found that PTHrP amino acids 82-108 (MT) are sufficient to confer MT-enhanced nuclear accumulation by microtubule-mediated pathway.29 However, the MT peptide so far were not adequately study in facilitating nuclear drug delivery and the applied prospects in drug delivery system need further research. Herein, an innovative nuclear drug delivery was designed, which entered cells by non-endocytic pathway and transported into nucleus in a microtubule dependent fashion (Scheme 1). N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer was served as drug carrier. By covalently conjugating to HPMA copolymers, therapeutic drugs could be preferentially delivered to tumor tissue by the “enhanced permeation and retention (EPR) effect”.30-32 Recently, several HPMA copolymer-drug conjugates have been evaluated in clinical trials.33, 34 MT peptide was



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conjugated to the HPMA backbone by maleimide-thiol coupling reaction. A c-Myc protein derived H1-S6A, F8A (H1) peptide served as therapeutic agent, which suppressed tumor cells growth by interference the nuclear interaction of c-Myc with MAX.35, 36

Our results showed that MT could

mediate HPMA polymer enter into HeLa cells cytoplasm by non-endocytic pathway and facilitate the fast nuclear delivery of drug in a microtubules dependent fashion. The unique mechanism accounted for higher cellular uptake and nuclear accumulation compared with non-MT modified polymer-drug conjugates.

2. Method and Materials 2.1 Materials

Microtubule-dependent

nuclear

targeting

PLKTPGKKKKGKPGKRKEQEKKKRRTR,

peptide

MW:3320

(MT:

Da)

the

scrambled MT (MT-m: CRPKLKTRKPKGKGKPKGKRKEKQKETR, MW:3320

Da)

peptide

and

H1-S6A,

F8A

peptide

(H1,N3-DNELKRAFAALRDQI, MW:1842 Da) were purchased from Chinese

Peptide

Co.Ltd

(Zhejiang,

China).

N-3-aminopropylmethacrylamide hydrochloride (APMA) was obtained from Poly Sciences (USA). Nuclear localization signal (NLS: CKRKKKP, MW: 886 Da) was bought from Kai jie Bioapharm Co. Ltd (Sichuan, China).

The

fluorescein

isothiocyanate

(FITC),


4,

4’-azobis

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(4-cyanovaleric acid) (V-501), Succinimidyl 3-maleimidopropanoate (SMP), 6-diamidino-2-phenylindole (DAPI), 4-cyanopentanoic acid dithiobenzoate

(CTA),

and

3-(4,

5-dimethyl-2-tetrazolyl)-2,

5-diphenyl-2H tetrazolium bromide (MTT) were from Sigma Aldrich (St. Louis, MO). 2.2 Preparation and characterization of HPMA-H1

peptide

conjugates 2.2.1 Preparation of N-methacryloyl-glycylglycyl-propargylamide (MA-GG-C≡CH)

Firstly,

N-methacryloyl-glycylglycyl-pnitrophenyl

ester

(MA-GG-ONp) was prepared according to previously reported method.37 MA-GG-C≡CH was then synthesized following established protocols.38 Briefly, 100 mg MA-GG-ONP (0.3112 mmol) was added into 3 ml N, N-dimethylformamide (DMF), then 30 µL propargylamine (0.4668 mmol) was added dropwise with N, N-diisopropylethylamine to control the basic reaction environment, agitated in room temperature. And the reactive progress was monitored using thin layer chromatography (ethyl acetate: methanol=10:1 as eluent). After the reaction finished, the mixture was separated with silica gel chromatography (ethyl acetate: methanol=10:1 as eluent). Yield: 63%. 2.2.2 Synthesis of HPMA copolymer precursors (P-APMA-C≡CH)

HPMA monomers was synthesized as reported.37 HPMA copolymer



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precursors

containing

MA-GG-C≡CH

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(10

mol%)

N-3-aminopropylmethacrylamide hydrochloride (APMA, 10

and mol%)

was synthesized by reversible addition-fragmentation chain transfer polymerization reaction (RAFT). Briefly, HPMA (1.14 g, 8 mmol), MA-GG-C≡CH (238 mg, 1 mmol), APMA (179 mg, 1 mmol) were dissolved in cold methanol/water (2:3, v/v, 7.8 ml) and initiator VA044 was added via syringe. Ar2 was used to protect the reaction and the reaction was agitated at 50℃ for 12 h. Acetone/diethyl ether (3:1, v/v, 150 ml) was used to precipitate the product, and acetone/diethyl ether (1:1 v/v, 150 ml) was applied to purify the polymer. Finally, we got a slightly pink powder. Yield: 55%. 2.2.3 Preparation of HPMA copolymer with alkynyl and maleimide group (P-AM)

The amino groups at side chain of HPMA precursor were converted to maleimide group by reaction with a heterobifunctional regent: succinimidyl

3-maleimidopropanoate

(SMP).

In

brief,

70.2

mg

P-APMA-C≡CH (0.0484 mmol -NH2) and 19.3 mg SMP (0.0726 mmol) was added into 3 ml DMF. The mixture was reacted for 24 h in the presence of trimethylamine to keep the pH near 8.5. The product was purified by dialysis (MWCO: 8000~14,000) and dried under vacuum. Yield: 75%. 2.2.4 Preparation of HPMA-H1 peptide conjugates


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Azide-modified H1 peptide was conjugated to HPMA copolymer by click reaction (azide-alkyne cycloaddition). 200 mg P-AM (0.007 mmol), 30 mg H1 peptide (0.016 mmol), 2.5 mg CuSO4.5H2O (0.01 mmol) and 20 mg sodium ascorbate (0.1 mmol) were added into water/tertbutyl alcohol (1:1, v/v, 5 ml) stirring 24 h in dark environment and room temperature. The mixture was dialyzed to move non-reacted materials, sodium ascorbate and CuSO4.5H2O.Yield:81%. 5-{(3-azidopropyl)

thioureido}-fluorescein

(N3-FITC)

were

synthesized following the previous method.5 N3-FITC was conjugated to HPMA copolymer conjugates following the same method. 2.2.5 Synthesis of MT peptide decorated HPMA-H1 peptide conjugates.

MT modified HPMA copolymer H1 peptide conjugates (P-H1-MT) was obtained by the reaction of 100 mg P-H1 (0.05 mmol) in 3 ml PBS buffer with 15 mg MT peptide (0.005 mmol), and agitated overnight (under nitrogen). And the polymer was isolated by dialysis (MWCO: 8000~14,000) against distilled water and freezing dried. Yield: 76%. Other peptide modified HPMA polymer was synthesized in the same method. 2.2.6 Characterization of HPMA copolymer conjugates.

The NH2, FITC and peptides content, molecular weight and polydispersity index of HPMA polymers were evaluated. The methods



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were added in Supporting Information. 2.3 The cell internalization and intracellular distribution

The cellular uptake of different FITC-labeled polymers was quantitatively study by flow cytometry. HeLa cells (China Center for Type Culture Collection) were seeded in 12-well plates (1 x104 cells/well). After 24 h culture, different polymers (0.2 mg/mL) were incubated with cells at 37 ℃ for 4/18 h. After removing the the medium, cells were harvested and washed with cold PBS. Flow cytometry was applied to determine the samples immediately. As confocal microscopy analyses, HeLa cells were seeded into coverslips (1 x 104 cells/well). After incubation at 37 ℃ for 24 h, cells then cultured

with FITC-labeled

polymers (0.2 mg/mL) and H1-FITC, P-H1-FITC, P-H1-MT-FITC (0.1 mg/mL)

at 37 ℃ for 18h. In some experiments, cells were exposed to

6 nM nocodazole (Noc) or 2 nM docetaxel (DTX) in growth media 30 min prior to conjugates addition. The nucleus were stained with DAPI and analyzed by confocal laser scanning microscopy (CLSM, BD). 2.4 In vitro cellular uptake mechanism

In order to identify possible mechanisms of FITC-labeled polymers, HeLa cells were incubated with polymers 30 min at 37 ℃ in the absence or presence of 100 mM sodium azide (NaN3).39, 40 After that, cells were cultured at 37 ℃ or 4 ℃ for 4 h. Fluorescence intensity was analyzed by Varioskan Flash reader and the protein content was measured using


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bicinchoninic acid BCA assay kit. To further determine the intracellular trafficking mechanism, FITC-labeled polymers (0.2 mg/mL) were cultured with HeLa cells at 37 ℃ for 18 h. The living cells were marked by lysosome red tracker, and imaged by CLSM. 2.5 The nuclear trafficking mechanism

The cells were incubated with 6 nM Noc or 2 nM DTX and then incubated with alpha-tubulin polyclonal antibody (1:100, Biotechnology Co. Ltd) in PBS with 1% BSA. Cy5-labeled anti-mouse secondary antibody (1:200, BD Bioscience) was used to mark microtubule (red) and observed by CLSM. 2.6 Quantitative analysis of FITC-labeled polymers in nuclear region

HeLa cells were seeded in 6-cm petri dish (1.5 x 106 cells). After 24 h culture, cells were incubated with 0.2 mg/mL FITC-labeled polymers for 18 h. In some experiments, cells were exposed to 6 nM Noc or 2 nM DTX in growth media 30 min prior to conjugates addition. Quantitative analysis of polymer in nuclear region was performed as described.6, 41 Free FITC was determined following the same process to get a standard curve. 2.7 Living cell nuclear fluorescence return after photobleaching (FRAP)

FRAP was performed to evaluate the microtubules functions.29



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HeLa cells were exposed to 6 nM Noc or 2 nM DTX in growth media 30 min prior to conjugates addition (18h co-incubation). Living cells were imaged using CLSM. Before bleaching, three images were collected as control, and then the total laser power in 488 nm was used to bleach the nuclear fluorescence. The fluorescence return was measured by scanning the images at 8-s /pixel over a period of 400 s. The Fn/c ratio (the ratio of fluorescence in nucleus/ cytosol) was calculated by Image-J software to evaluate nuclear polymer delivery from cytoplasm. The average rate of nuclear import (Fn/c s-1) was calculated. 2.8 In vitro cytotoxicity and apoptosis analysis

The in vitro antitumor efficiency of free H1 peptide and H1-loaded conjugates (P-H1, P-H1-MT) was assessed by MTT assay with no drug conjugated polymer (P, P-MT) as control. HeLa cells were seeded into 96-well plates (3 × 103 cells/well). After 24 h culture, cells were pre-incubated 18 h with polymer conjugates (P-H1 and P-H1-MT, 0.1-1.6 mg/mL) or H1 peptide (5-84 µg/mL), then the cell viability was calculated. And apoptosis analysis was following the previous method.5

3 Results and discussion 3.1 Preparation and characterization of polymers conjugates

The polymer precursor (P-APMA-C≡CH) was synthesized through RAFT reaction shown in Figure 1. Succinimidyl 3-maleimidopropanoate


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(SMP) was conjugated to polymer backbone to link targeting peptide (NLS, MT-s and MT) by maleimide-thiol coupling reaction. And N3-H1/N3-FITC was conjugated to the polymer by azide-alkyne cycloaddition reaction. These two highly specific click reactions were used to keep the bioactivity of the peptides42, 43.

The characteristics

including molecular weight (Mw), poly dispersity index (PDI), -NH2, acetylene, peptides and FITC content were shown in Table.1. Because of the use of RAFT method, the resulting copolymer showed narrow and unimodal distribution with PDI below 1.4. The -NH2 content was among 11.9%-13.2%. Acetylene (MA-GG-C≡CH) content was determined from the amount of GG sequences and the content was 4.5%-4.7，the FITC content was 3.2%-5.5%, and the peptide content was between 4.2% and 6.6%.

3.2 Cellular uptake mechanism

In the area of organelles targeting drug delivery, endo/lysosome was one of the most difficult barriers, it’s assumed that less than 10% of the active drug could successfully escape endo/lysosome.26, 44 However, some endogenous protein such as p53 tumor suppressors and parathyroid hormone-related protein (PTHrP) could achieve efficient nuclear trafficking by microtubule cytoskeleton-mediated pathway.27, 28 Further study demonstrated that PTHrP amino acids 82-108 (MT) are sufficient to



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confer MT-enhanced nuclear accumulation, which contains the two basic regions (KKKK91 and KKRR106) facilitated efficient MT-dependent nuclear accumulation.29 signals

(NLS,

In our study, a traditional nuclear location

CKRKKKP)

and

the

MT

peptide

(PLKTPGKKKKGKPGKRKEQEKKKRRTR) were designed to evaluate their nuclear targeting ability and subcellular trafficking mechanisms. Also,

a

scrambled

MT

peptide

(MT-m,

CRPKLKTRKPKGKGKPKGKRKEKQKETR) was used as a control, which disorganized the basic regions of MT peptide (KKKK91 and KKRR106). Firstly, flow cytometry and CLSM were used to measure the cellular uptake of different fluorescence labeled polymer conjugates (P-FITC, P-FITC-NLS, P-FITC-MT-m and P-FITC-MT). As shown in Figure 2A&B, peptide conjugated polymer could significant enhance the

cellular uptake (P

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