Targeted Biomimetic Nanoparticles for Synergistic Combination

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Targeted Biomimetic Nanoparticles for Synergistic Combination Chemotherapy of Paclitaxel and Doxorubicin Mengjie Rui, Yuanrong Xin, Ran Li, Yanru Ge, Chunlai Feng, and Ximing Xu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00732 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 11, 2016

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Molecular Pharmaceutics

Targeted Biomimetic Nanoparticles for Synergistic Combination Chemotherapy of Paclitaxel and Doxorubicin Mengjie Rui, Yuanrong Xin, Ran Li, Yanru Ge, Chunlai Feng**, Ximing Xu* Department of Pharmaceutics, School of Pharmacy, Jiangsu University, Zhenjiang 212013, PR China

KEYWORDS Combination antitumor chemotherapy, Co-delivery high density lipoprotein, Synergistic effect, Ratio dependent synergy, Paclitaxel; Doxorubicin

ABSTRACT

Co-delivery of multiple chemotherapeutics has become a versatile strategy in recent cancer treatment, but antagonistic behavior of combined drugs limited their application. We developed a recombinant high-density lipoprotein (rHDL) nanoparticle for the precise co-encapsulation and co-delivery of two established drugs and hypothesized that they could act synergistically to

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improve anticancer efficacy. The co-loaded rHDL was formulated by passively incorporating hydrophobic paclitaxel (PTX), and subsequently remotely loading hydrophilic doxorubicin (Dox) into the same nanoparticles. The resultant rHDL system restored targeted delivery function toward cancer cells via scavenger receptor class B (SR-BI), as confirmed by in vitro confocal imaging and flow cytometry. These co-loaded rHDL nanoparticles were remarkably effective in increasing the ratiometric accumulation of drugs in cancer cells and enhancing antitumor response at synergistic drug ratios. In particular, they exhibited more efficacious anticancer effects in an in vitro cytotoxicity evaluation and in a xenograft tumor model of hepatoma compared with free drug cocktail solutions. These results confirm that the co-loaded rHDL nanoparticles are promising candidates for the synergistic delivery of drugs with diverse physicochemical properties in cancer treatment integrating efficiency and safety considerations.

INTRODUCTION Chemotherapy is an effective strategy in clinical cancer treatment, however, the application is intensively limited by the transient therapeutic response. Previous studies identified that these failures were partially attributed to intrinsic compensatory mechanisms and the high degree of heterogeneity of cancer cells, which could also lead to drug resistance1, 2. Currently, this issue might be addressed by combining multiple chemotherapeutics to achieve an efficacious inhibition of tumor cell proliferation, invasion and metastasis3, 4. Numerous anticancer agents generally do not reach their full potential unless they are given simultaneously with other drugs designed against alternative targets5. For example, the clinical benefits of a combination of paclitaxel (PTX) and doxorubicin (Dox), which has been applied for the treatment of metastatic


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breast cancer, further confirmed their therapeutic efficacy6, 7. Nevertheless, traditional combination therapy is far from perfect8. It’s previously revealed that the non-coordinated pharmacokinetics, biodistribution and diverse physicochemical properties of individual drugs within conventional "cocktail" contributed to unsatisfactory therapeutic outcomes during systemic delivery9, 10. Moreover, the inconsistent pharmacokinetic profiles present a real clinical challenge for predicting antitumor efficacy under in vivo conditions from in vitro synergistic cytotoxicity. Therefore, it is desirable to develop a new approach to improve the combinatorial effects without significant systemic toxicity.

To overcome these limitations, we sought to develop a biocompatible and biodegradable drug system to load drugs with varying pharmacological action mechanisms and to unify combined drug pharmacokinetics. A variety of nanoparticle delivery systems, including liposomes10, 11, micelles12, 13, polymeric nanoparticles14, 15, and emulsions16, have been investigated for their potential in chemotherapy due to the promising ability to increase drug solubility, prolong circulation time, alleviate systemic toxicity, and improve tumor accumulation17. Additionally, the effective tumor penetration behavior of nanoparticles could potentially coordinate the pharmacokinetic properties of individual drugs in combination while maximizing the antitumor efficiency and reducing side cytotoxicity. Nevertheless, as synthetic delivery systems are required to meet clinical expectations, endogenous nanoparticles composed of natural biomaterials have captured increasing attention, due to their prominent characteristics, including nano-scaled dimensions, low immunogenicity, and superior binding ability to diverse targets. Human endogenous lipoproteins, such as high-density lipoproteins (HDL), are considered as ideal carriers that consist of phospholipids, apolipoproteins and cholesterol18,

19

. These


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components can self-assemble in aqueous environments to form core-shell spherical or discoidal nanoparticles, which can deliver hydrophobic compounds (such as cholesterol), or hydrophilic RNA20 to specific tissues. With regard to the detailed structure of HDL nanoparticles, the core, acting as a reservoir for multiple agents, is enclosed by amphiphilic lipids, meanwhile, the wrapped apolipoproteins stabilize the shell, enhance loading capacity and effectively protect the encapsulated drugs from degradation. Previous studies showed that synergistic combination effect is dependent on combined drug ratios, reflecting the significance of precise loading and ratiometric delivery of drugs5, 21. In this regard, the specific structural properties of lipoproteininspired nanoparticles enabled them to facilitate high loading of drugs and fine-tuning of the combinatorial ratio, which were potentially suitable for synergistic delivery22, 23.

For an advanced drug vehicle, tumor-targeting delivery is crucial for efficient oncotherapy and minimizing drug adverse toxicity. Therefore, selecting an appropriate targeting moiety is critical in the design of drug carriers for synergistic combination chemotherapy. So far, most strategies based on nanoparticles have focused on encapsulating two different drugs and modification with a variety of targeting moieties into one single delivery system24-27. Nevertheless, this sort of pharmaceutical design is complex and difficult to produce. There is a strong need to develop a simple but versatile approach that can be applied to targeted combinatorial therapy. In recent decades, it has been revealed that apolipoprotein A-I (apo A-I), the major functional protein in natural HDL, possessed specific affinity toward its endogenous receptors and confered superb ability for targeted transportation. In particular, previous studies demonstrated increased levels of expression of HDL receptors, such as scavenger receptor class B type I (SR-BI), in malignant cells, compared with normal cells28-30. In addition, an alternative pathway, comprising


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endocytosis of HDL nanoparticles followed by re-secretion, was discovered to regulate lipid exchange between HDL and target cells31. These HDL receptors, which mediate cellular uptake of cholesterols, are significantly involved in various cancer processes, i.e., cell survival, migration, and angiogenesis, indicating the potential for tumor-targeted delivery. Moreover, the non-endocytic transmembrane pathway mediated by SR-BI could transfer cargo to the cytoplasm directly without destruction of whole particles, while evading the endosome/lysosome pathways employed by commonly reported nanoparticle systems32,

33

. Recombinant HDL (rHDL)

nanoparticles have been verified to mimic the function of targeting SR-BI and other HDL receptors and to specifically deliver their cargo into cancer cells 34-37. Inspired by this knowledge, an rHDL formulation co-loaded of two chemotherapeutic agents might provide an avenue for combinatorial delivery, particularly, to address the fast plasma elimination and diverse biodistribution of drugs in conventional cocktail therapy. . Based on this rational, we designed an rHDL delivery system with the aim to achieve precise ratiometric co-loading of drugs and synergistic inhibition of tumor proliferation. In the present work, the combination of paclitaxel (PTX) and doxorubicin (Dox) was selected as the model due to their different physicochemical properties and varied targets of action. PTX, a typical hydrophobic drug, causes depolymerization of microtubules, leading to mitotic arrest, whereas the hydrophilic Dox intercalates into the DNA duplex and prevents biosynthesis of nucleic acids, leading to cell apoptosis. As shown in Scheme 1A, hydrophobic PTX was passively encapsulated into liposomal nanoparticles, followed by the remote loading of Dox into the same nanoparticle by a transmembrane pH gradient. The subsequent self-assembly of apo A-I proteins reconstructed the co-loaded liposomes and formed rHDL nanoparticles. Theoretically, the


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resultant self-assembled rHDL allows precise co-loading of PTX and Dox at the designed ratios, improving the ratio-dependent synergistic effects and reducing toxic side reactions. For proof of our hypothesis, the configurations, stability and in vitro release behavior of co-loaded rHDL were investigated. Cytotoxicity assays against HepG2 and MCF-7 cells in vitro revealed that various combinatorial effects could be obtained by adjusting the PTX-to-Dox ratios, thereby maximizing the synergistic efficiency at the optimal ratios. More importantly, this rHDL nanoparticle not only transferred drug cargos directly into the cytoplasm through SR-BI pathway but also translated in vitro synergy effect into antitumor efficiency under in vivo situations, while significantly improving the therapeutic outcomes compared to free drug solutions. In this study, co-loaded rHDL nanoparticles were exploited as a potent delivery system for efficacious combination chemotherapy.

MATERIALS AND METHODS Materials. Doxorubicin hydrochloride (Dox∙HCl) and paclitaxel (PTX) were purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). Egg phosphatidylcholine (EPC) and dimyristoyl phosphatidylcholine (DMPC) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). Fluorescein isothiocyanate (FITC), BLT-4 and 4’,6-diamidino-2phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, USA). The fluoresceinlabeled

lipid

N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-

phosphoethanolamine triethylammonium salt (FITC-DHPE), LysoTracker Deep Red and DiD cell-labeling solution were purchased from Life Technologies (Carlsbad, CA). Dulbecco's modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were obtained from GIBCO, Invitrogen Corp. (Carlsbad, USA). Trypsin was provided by Sango Co.,


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Ltd, (Shanghai, China). Other materials were purchased from various suppliers and were reagent grade or high performance liquid chromatography (HPLC) grade. The plasmid pNFXex containing human apolipoprotein A-I cDNA, was a generous gift from Dr. Oda (Children’s Hospital Oakland Research Institute, USA)38, then apo A-I recombinant protein was expressed in Escherichia coli and purified using His-Trap nickel affinity chromatography, as described by Ryan et al39.

Synthesis of fluorescein-labeled apo A-I protein. Conjugation of apo A-I with fluorescein isothiocyanate (FITC) was performed according to the manufacturer’s instructions. Briefly, the buffer of apo A-I proteins was first exchanged with 0.1 M carbonate buffer (pH8.0) via dialysis at 4 °C. Then, FITC was dissolved in anhydrous DMSO at 1 mg/mL. One hundred microliters of FITC solution was slowly added into 1 mL of apo A-I solution (2.5 mg/mL in 0.1 M carbonate buffer) under gentle, continuous stirring for 8 h at 4 °C. After conjugation, the solution was dialyzed against PBS (20 mM, 150 mM NaCl, pH7.4) to thoroughly remove unreacted agents. The labeled apo A-I solution was stored at -20 °C until further use.

Preparation of co-loaded rHDL nanoparticles. The preparation of co-loaded rHDL consisted of the construction of drug-loaded liposome and subsequent incubation with apo A-I. Firstly, liposomes were prepared by conventional lipid hydration method. Briefly, 15 mg of lipid (EPC or DMPC) and 0.3 mg of PTX were dissolved in chloroform, and the organic solvent was removed by rotatory evaporation until dried thin lipid films were formed. The resultant film was hydrated in 2 mL of 300 mM citrate solution at pH 4.0 for 30 min at ambient temperature. To obtain small and homogeneous vesicles, the liposomes were sonicated on ice for 30 min with 10s


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intervals (JY 92II, Ningbo, China), followed by 11 extrusion cycles through polycarbonate membranes with 0.2 and 0.1 µm pores using a Mini-extruder (Avanti, USA). The liposome solution was then dialyzed in HEPES buffer (20 mM HEPES, 150 mM NaCl, pH7.5) to exchange the external phase. Doxorubicin was then remotely loaded into the rHDL via a pH gradient method. The PTXloaded rHDL nanoparticles were pre-heated at 37 °C, and the proper amount of 5 mg/mL doxorubicin solution was added to the rHDL solution and then incubated for 30 min at 37 °C while stirring gently to remotely load doxorubicin into the inner phase of the rHDL nanoparticles. After the preparation of co-loaded nanoparticles, apo A-I solution (2.5 mg/mL) was added to the liposomes solutions at an apo A-I/lipid weight ratio of 1:10, which was followed by incubation for 6 h at 4 °C. Free PTX and Dox were then removed by dialysis. The co-loaded rHDL nanoparticles were prepared with a defined PTX/Dox molar ratio. Then, the rHDL nanoparticles and liposomes were stored at 4 °C. For the fluorescence microscopy experiments, 0.6 mol% of FITC-labeled lipid (FITC-DHPE) was incorporated into the co-loaded rHDL formulation. Meanwhile, co-loaded rHDL nanoparticles containing FITC-labeled apo A-I proteins were also prepared using the same method.

Determination of the encapsulation efficiency. To study the loading capacity, rHDL or liposome samples were dissolved in 1% Triton X-100 solution to release the cargo. The Dox concentration was measured by Shimadzu RF-5301PC spectrofluorometer (Ex=470 nm, Em=590 nm). Meanwhile, PTX loaded in the nanoparticles was determined by C-18 reverse-phase highperformance liquid chromatography (HPLC). PTX was measured at 227 nm using a mobile


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phase consisting of a mixture of acetonitrile and water (3:1, v/v) at flow rate of 1.0 mL/min. The encapsulation efficiency was calculated as follows: Encapsulation efficiency% =

× 100%

Characterization of the co-loaded rHDL nanoparticles. One hundred microliters of rHDL nanoparticles was diluted with 2 mL of HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.5). Then, the hydrodynamic size, size distribution and zeta potential of the rHDL nanoparticles were measured by dynamic light scattering method with a 90Plus Particle Size Analyzer (Brookhaven Instruments Corporation, Holtsville, New York).

The morphology of the rHDL nanoparticles was observed via transmission electron microscopy (TEM) using the negative staining technique. Following Forte’s method40, a drop of rHDL solution was dissolved in an aqueous solution consisting of 0.125 M ammonium acetate and 2.6 mM ammonium carbonate at pH7.4, which was subsequently mixed with an equal volume of 2% sodium phosphotungstate solution. The samples were deposited onto a copper grid with a carbon film for approximately 1 min, and then the excess of the sample was blotted with filter paper. The rHDL nanoparticles were visualized using a Tecnai 12 transmission electron microscope (FEI Company, Netherlands).

To determine the stability of co-loaded rHDL nanoparticles under physiological conditions, the changes in the particle diameter of rHDL and corresponding liposomes were monitored over times. The samples was mixed with equal volume of 20% FBS (Sijiqing, Hangzhou, China) cell culture medium and then incubated at 37 °C for predesigned time intervals (0 h, 1 h, 4 h, 12 h, 24


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h, 48 h and 72 h). After incubation, 100 µL of sample was diluted in 1 mL of HEPES buffer and then the diameter was measured by the DLS method.

In vitro drug release assay. The release profiles of PTX and Dox from rHDL nanoparticles or liposomes were investigated in PBS or 10 % FBS (Sijiqing, Hangzhou, China) cell culture medium by dialysis. Briefly, 100 µL aliquots of samples were placed into a variety of 3.5K Slide-A-Lyzer Mini dialysis tubes (Thermo Scientific, MA), dialyzed against 500 mL of PBS or 10% FBS cell culture medium, respectively, followed by incubation at 37 °C with continuous shaking. At predetermined times, one tube was withdrawn. The amount of PTX and Dox was quantified by HPLC and fluorescence method, respectively. The release experiments were conducted in triplicate.

Cell culture. The HepG2 cells (human hepatocellular carcinoma cell line) and MCF-7 cells (human breast cancer cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose containing 10% fetal bovine serum (FBS) and supplemented with 50 U mL-1 penicillin and 50 U mL-1 streptomycin. All cells were cultured at 37 °C with 5% CO2 before use. The inoculated density was 5 × 104 cells/well for a six-well plate and 5 × 103 cells/well for a 96well plate.

Evaluation for in vitro cell viability. HepG2 cells were seeded in 96-well plates at a density of 5 × 103 cells per well in 100 µL of medium and cultured at 37 °C in a 5% CO2 atmosphere for 24 h. After the removal of the culture medium, the cells were then exposed to a series of drug concentrations of rHDL containing single drug alone or a combination of drugs at different molar


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ratios for 48 h. The free drugs were used as a control and underwent the same procedure. The medium was removed, and the cells were subjected to the Cell Counting Kit-8 assay (CCK-8, Dojindo, Japan) according to the manufacturer’s instructions. The absorbance of the solution was measured on a BioTek microplate reader at 450 nm with a reference wavelength of 600 nm. The relative

cell

viability

was

calculated

as

follows:

Cell

viability

=

(OD450(sample)/OD450(control))×100%. Data are presented as the mean ± SD (n=5). The inhibitory concentration (ICx) values were analyzed using Origin 8.0 (OriginLab, Northampton, MA) by nonlinear regression, where x represent x% proliferation inhibition . The combination index (CI) was calculated by the equation41: CI =

, , + , ,

CA,x and CB,x are the concentrations of drug A and drug B used in combination to achieve x% proliferation inhibition. ICx,A and ICx,B are the inhibitory concentrations for the individual drugs alone to achieve the same effect, respectively. Thus, a CI=0.9-1.1 represents additive activity, a CI>1.1 indicates antagonism, and CI

Targeted Biomimetic Nanoparticles for Synergistic Combination

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