Polymeric Drug Delivery System with Actively Targeted Cell

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Polymeric drug delivery system with actively-targeted cell penetration and nuclear targeting for cancer therapy Qiaochu Hua, Zhe Qiang, Maoquan Chu, Donglu Shi, and Jie Ren ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00097 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 15, 2019

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ACS Applied Bio Materials

Polymeric drug delivery system with activelytargeted cell penetration and nuclear targeting for cancer therapy Qiaochu Hua1, Zhe Qiang2, Maoquan Chu3 , Donglu Shi4,5 , Jie Ren1* 1. Institute of Nano and Biopolymeric Materials, School of Materials Science and Engineering, Tongji University, Shanghai 201804, China 2. Department of Chemical and Biological Engineering. Northwestern University, Evanston, IL 60208, USA 3. Biomedical Multidisciplinary Innovation Research Institute and Research Center for Translational Medicine at Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China 4. Materials Science and Engineering Program, Department of Mechanical and Materials Engineering, College of Engineering and Applied Science, University of Cincinnati, Cincinnati, OH 45221-0072, USA 5. Key Laboratory of Basic Research in Cardiology, Ministry of Education, Shanghai East Hospital, Institute for Biomedical Engineering and Nano Science, School of Medicine, Tongji University, Shanghai 200120, China

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ABSTRACT: Targeted tumor cell killing using polymeric micelles with active targeting strategies has been demonstrated to be effectively therapeutic for liver cancers. To implement this strategy, enhancing the cellular uptake of the drug delivery system with targeted anti-cancer drugs, such as doxorubicin towards nuclear, is of vital importance for increasing drug efficiency and reducing the systemic side effects of encapsulated drugs. In this study, a multifunctional polymeric drug delivery system was designed with actively targeted cell penetration and nuclear targeting for efficient cancer therapy. The nanocarriers were self-assembled from poly (ethylene glycol)‐block-poly(ε‐caprolactone), decorated with folic acid (FA-PECL) for active targeting via amide reaction for selective delivery of drugs to tumors. Cell penetration peptide (CPP) was decorated with doxorubicin (DOX), and the conjugate (CPP-DOX) was encapsulated in the carrier system for efficient cell-penetrating and nuclear targeting of drugs. In vitro study showed enhanced in vitro cytotoxicity and tumor volume decreased more than 5 times compared with the nontargeted system, by utilizing the drug-loaded system (FA-PECL/CPP-DOX) with active tumor cell targeting and subsequent nuclear targeting. The FA-PECL/CPP-DOX drug loading system was well-targeted and enriched on tumor sites, resulting in significant suppression of liver tumor growth.

KEYWORDS: active targeting, drug-loaded micelle, cell penetrating peptide, tumor inhibition


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1.

Introduction Cancer has been the leading disease of mortality that threatens human life. Traditional

chemotherapy has been effective for treating tumors to certain extent, but severe toxic side effects and low therapeutic efficiency are major concerns1-5. In recent years, many targeted drug delivery systems, including micelles, vesicles, nanomagnets and nanogels, demonstrate great promise in cancer therapeutics due to the advantage of significantly reduced side effects6-8. Micelles with the core-shell structure are capable to spontaneously accumulate in tumors through EPR (enhanced permeability and retention) effect9-10. To further enhance cellular internalization of micelles with active targeting strategy has been implemented, utilizing ligands such as anti-bodies, small molecules and affibodies to enable specific binding between the drug delivery systems and molecular biomarkers that are over-expressed on tumor cell membranes. These results lead to effective tumor cell internalization via ligand/ receptor-mediated endocytosis11-16. Since folic receptor protein (FR) is highly expressed in many kinds of cancer cells but not in normal cells, folic acid (FA) is frequently applied to functionalize various of nanoparticles and molecules including polymeric micelles for active targeting towards tumor cell membrane and subsequently internalize the system, which has shown enhanced therapeutic efficacy and reduced systemic side effects17-20.



Furthermore, anticancer drugs need to enter nucleus for high efficacy. For instance, doxorubicin (DOX) induces tumor cells apoptosis mainly through oxidative DNA damage and topoisomerase II inhibition in tumor cell nucleus. However, most of targeted drug delivery micelles can only transport the encapsulated drugs or agents in the cytoplasm but rarely into nucleus. Therefore, it is very important to render the nano-sized drug delivery systems with nuclear-targeted for further improving the anticancer efficiency of drugs presently available21. Cell penetrating peptides (CPPs), capable to penetrate into many different kinds of cells with a nuclear localization signal (NLS), have provided new strategy for intranuclear transport of drug delivery. Recent studies show that CPPs have been effective in delivering proteins, nucleic acids, quantum dots and small molecule therapeutics to cancer cells and tumor sites, therefore serving as the important conjugates for improved efficacy of anticancer drugs22-26. However, this approach has been hindered by CPPs not having specific recognition of cells or tissues, which is a major disadvantage for being utilized as the major drug delivery systems. One solution for potentially addressing this critical issue is via steric protection by long polymeric chains and the stimuli-responsive activation delivery systems27-32. In this study, we designed and fabricated a multifunctional polymeric drug delivery system (named as FA-PECL/CPP-DOX) with the capabilities of cell penetration and nucleus targeting (Scheme. 1). As shown in the Scheme 1, the poly (ethylene glycol)-blockpoly(ε‐caprolactone) are self-assembled into active targeting micelles with decorated by folic acid for specific targeting the tumor cells. CPPs (CKRRMKWKK), which have high


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membrane translocation and nuclear localization efficiency24, are conjugated with DOX to form the CPP-DOX. The conjugation is then encapsulated into the FA-PECL micelle for delivery. This unique drug delivery system decorated with FA is initially combined with the folic receptor (FR) over-expressed on tumor cells, enabling accumulation and endocytosis, and consequently releasing the conjugation from the micelles. In this study, CPPs was used to target nucleus for delivering DOX, which significantly enhanced intracellular efficiency of anticancer drugs. Moreover, we also evaluated anti-tumor efficiency of the drug delivery system in vivo and in vitro. This work provides a novel methodology platform to successfully combine both active targeting and nuclear targeting in the drug delivery system.

Scheme 1. Schematic diagram of the FA-PECL/CPP-DOX drug delivery system.



2.

Experimental section

2.1 Sample preparation All the synthetic details can be found in the Supporting Information (SI). SMP-DOX was synthesized via previous method with changes[24]. Briefly, the carboxyl group of folic acid was activated and conjugated with NH2-PEG2000-PCL3400 via carboxylate/amine condensation (Fig.1a). Through N-Succinimidyl 3- maleimidopropionate (SMP) as linker, CPP and DOX was coupled as CPP-DOX conjugate (Fig.1b). CPP-DOX was prepared by Michael addition, which covalently attached DOX-SMP to a chemically reactive thiol group for drug conjugation with maleimide on the cysteine sulfur of CPP. FA-PECL or NH2-PECL polymer were dissolved in DMSO (20mg/mL) and added into 15 mL of deionized water (or PBS for biological tests) dropwise under ultrasonication. The resulting mixture was dialyzed against deionized water (or PBS) at 25℃ for 48 h, in between DMSO was removed by refreshing the medium for every 6 hours. DOX·HCL, CPP-DOX and TEA were dissolved in DMSO in dark at 25℃ for overnight. The polymer solution was then added into drug solution, followed by stirring for 30 min. Subsequently, the resulting solution was added into 15 mL of deionized water (or PBS for biological tests) dropwise under ultrasonication, then dialyzed against PBS at 25℃ for 24 h in dark, with the medium refreshed every 4 h to completely remove excess DMSO.


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(a)

(b) Figure 1. Synthesis of (a) FA-PECL copolymer and (b) CPP-DOX conjugate. 2.2 Sample characterization



1H

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NMR spectra of samples were obtained on a Bruker DMX 500 NMR spectrometer

operating at 400MHz, using deuterated dimethyl sulfoxide (DMSO-d6) as solvents. The chemical shifts were relative to TMS. Attenuated total reflection Fourier transform infrared (ATR FT-IR) spectra of samples were recorded on an Equinoxes/Hyperion2000 FT-IR spectrometer (Bruker, Germany). MALDI-TOF Mass spectra of CPP-DOX was obtained on AB SCIEX 4800 Plus MALDI-TOF-MS and samples were dissolved in DMSO. The morphology of blank and drug-loaded micelles was observed using a JEOL JEM-2010F transmission electron microscope (TEM), at the accelerating voltage of 120 kV. TEM samples were prepared by dropping 10 μL of 1.0 mg/mL micellar solution on a Formvarcoated copper grid, and then air-dried at room temperature. The hydrodynamic diameter (Dh) of each micelle was measured on a Malvern Zetasizer Nano-ZS90 dynamic light scattering (DLS) spectrometer at 25°C. DLS was performed at a scattering angle of 90°. Dh was determined by a cumulant analysis. Drug-loading content (DLC) of each micelle was measured by UV-vis on UV-vis Spectrophotometer (U-1800, Hitachi, Japan) at 480 nm. Drug loading content (DLC) is determined by formula (1): DLC (%)= (mdrug loaded in micelles / mmicelles) x 100%

(1)

2.3 In vitro characterization The in vitro release study of DOX and CPP-DOX from each micelle was conducted. Briefly, 2.0 mL of the FA-PECL/DOX or FA-PECL/CPP-DOX micelles with the concentration of 1.0 mg/mL was transferred to a dialysis bag and then soaked with 25 mL


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of PBS. The release experiments were performed at 37℃, with continuous shaking at 80 rpm in darkness. At predetermined intervals, 2.0 mL of release medium was collected for UV-Vis measurements, and then 2.0 mL of PBS was replenished. The amount of DOX was determined by spectrophotometry at 480 nm using the standard curve (DOX/PBS solutions) and the cumulative release was calculated according to the previous article.7 All measurements were performed in triplicate. The immortalized human hepatocellular carcinoma cell line (Huh-7 cells) was used to evaluate the targeting effect and cytotoxicity. The cells were cultured in DMEM supplement with 10% fetal bovine serum (FBS), 100 unit per mL penicillin and 100 μg/mL streptomycin, maintained in a 37℃, humidified incubator with a 5% CO2 atmosphere. The cellular uptake experiments were performed by using the confocal laser scanning microscopy (CLSM) as previous study15. The detailed procedure could be found in the SI. In vitro cytotoxicity assays of FA-PECL blank micelle and FA-PECL/CPP-DOX, FAPECL/DOX, PECL/CPP-DOX, PECL/DOX drug-loaded micelle were evaluated by MTT method in Huh-7 cell. Experiments of each micelle were conducted in quintuplicate. The antitumor efficacy of drug loaded micelles was evaluated using the xenograft model developed by injecting human hepatocellular carcinoma Huh-7 cells on the back of 4-5 weeks old male nude mice. The treatment started after tumor models had grown to 50 mm3. Consequently, the mice were divided randomly into 5 groups with numbered. Each group contains 5 mice and the weight (W0) and the initial tumor volume (V0) were measured and recorded before treatment. Tumor volume of mice was calculated using equation (2).



V(mm3) =L(mm)×W2 (mm2)/2

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(2)

where a represented the longest width and b represents the shortest width of tumors. The mice received intravenous injections of each formula (0.1 ml, 10 mg/kg body weight) with an interval of 2 days via tail vein. The control group was injected with PBS. During the treatment period of 18 days, the body weight and tumor volume were recorded before each injection. All measures were performed in triplicate. PECL and FA-PECL blank micelles was conjugated with fluorescent dye, cy-5 (5 mg/kg body weight) and intravenously injected through tail vein into test mice . The image of the mice were obtained via in-vivo imaging system (Night Owl LB983, Berthold, Germany) at 1h, 6h and 24h. The emission and excitation fluorescence were set as 662 nm and 642 nm, separately. All experiments were performed at least three different times (n≥3). All data were expressed as the mean ± the standard error of the mean. Statistical differences were assessed by one-way analysis of variance test in Excel 2017. P 

Polymeric Drug Delivery System with Actively Targeted Cell

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