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Combining stimulus-triggered release and active targeting strategies improves cytotoxicity of cytochrome c nanoparticles in tumor cells Moraima Morales-Cruz, Alejandra Cruz-Montañez, Cindy M. Figueroa, Tania GonzalezRobles, Josue Davila, Mikhail Inyushin, Sergio A. Loza-Rosas, Anna M. Molina, Laura Muñoz-Perez, Lilia Y. Kucheryavykh, Arthur D. Tinoco, and Kai Griebenow Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00461 • Publication Date (Web): 09 Jun 2016 Downloaded from http://pubs.acs.org on June 19, 2016

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Molecular Pharmaceutics

Combining stimulus-triggered release and active targeting strategies improves cytotoxicity of cytochrome c nanoparticles in tumor cells Moraima Morales-Cruz1, Alejandra Cruz-Montañez2, Cindy M. Figueroa2, Tania GonzálezRobles2, Josue Davila3, Mikhail Inyushin4, Sergio A. Loza-Rosas2, Anna M. Molina2, Laura Muñoz-Perez2, Lilia Y. Kucheryavykh3, Arthur D. Tinoco2, and Kai Griebenow2* 1

Department of Biology, University of Puerto Rico, Río Piedras Campus, San Juan, PR 00931

2

Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, PR 00931

3

Department of Biochemistry, Universidad Central del Caribe, School of Medicine, Bayamón,

PR, 00560 4

Department of Physiology, Universidad Central del Caribe, School of Medicine, Bayamón, PR,

00560

ACS Paragon Plus Environment


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ABSTRACT: Proteins often possess highly specific biological activities that make them potential therapeutics, but their physical and chemical instabilities during formulation, storage, and delivery have limited their medical use. Therefore, engineering of nano-sized vehicles to stabilize protein therapeutics and to allow for targeted treatment of complex diseases, such as cancer, is of considerable interest. A micelle-like nanoparticle (NP) was designed for both, tumor targeting and stimulus-triggered release of the apoptotic protein cytochrome c (Cyt c). This system is composed of a Cyt c NP stabilized by a folate-receptor targeting amphiphilic copolymer (FAPEG-PLGA) attached to Cyt c through a redox-sensitive bond. FA-PEG-PLGA-S-S-Cyt c NPs exhibited excellent stability under extracellular physiological conditions, whereas once in the intracellular reducing environment, Cyt c was released from the conjugate. Under the same conditions, the folate-decorated NP reduced folate receptor positive HeLa cell viability to 20% while the same complex without FA only reduced it to 80%. Confocal microscopy showed that the FAPEG-PLGA-S-S-Cyt c NPs were internalized by HeLa cells and were capable of endosomal escape. The specificity of the folate receptor-mediated internalization was confirmed by the lack of uptake by two folate receptor deficient cell lines: A549 and NIH-3T3. Finally, the potential as anti-tumor therapy of our folate-decorated Cyt c-based NPs was confirmed with an in vivo brain tumor model. In conclusion, we were able to create a stable, selective, and smart nanosized Cyt c delivery system. KEYWORDS: active targeting, biodegradable polymer, drug delivery, nanoparticle, protein drug, triggered release


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1. INTRODUCTION The inactivation of the upstream components of the signaling pathways (such as the p53 pathway) that activate the release of cytochrome c (Cyt c) from the mitochondria to the cytoplasm in response to DNA damage, disable apoptosis in many cancer cells.1 This explains the limited efficiency of many classic chemotherapeutic agents (e.g., Taxol®) that cause DNA damage leading to p53-dependent apoptosis. Such limitations and a generally low therapeutic index have spurred efforts to identify new and more effective chemotherapeutic agents that can be tolerable in high doses and act independently of the p53 pathway. For example, some previous studies have demonstrated that the delivery of an apoptotic protein, such as Cyt c, directly to the cytoplasm of various cancer cell lines induced apoptosis in them.2-5 However, due to several protein delivery and stability issues,6 the use of proteins as therapeutics is limited. A major challenge in the development of delivery systems that seek to improve protein drugs is to achieve high drug loading of the device and an efficient release of the active protein at the target site.7 Recently, we overcame payload limitations commonly seen in delivery systems with therapeutic proteins6,7 by designing protein-based nanoparticles (NPs) coated with a biodegradable polymer PLGA.8 The designed Cyt c drug delivery system incorporates a triggered release mechanism mediated by the reducing environment inside of the cells and it showed improved cytotoxicity and biocompatibility when compared to previous reported Cyt c formulations.2,5 For the in vivo application of this kind of NP delivery system additional considerations are necessary. Although the NP size takes advantage of passive targeting, i.e., through the enhanced permeation and retention (EPR) effect characteristic of tumors,9-12 it is not the sole determinant of tumor specificity in in vivo studies. The surface of the NP should be modified (e.g., by attaching PEG molecules) to increase the blood circulation half-life of the system. The at-



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tachment of targeting moieties (i.e., active targeting) also improves the internalization and intracellular delivery of NPs. Only two FDA approved nano-formulations for cancer therapy (i.e., Abraxane and Rexin-G) have combined passive targeting with active targeting. At present there are few NP formulations of drug delivery systems which combine active targeting and triggered release to treat cancer. In this work we improve our previous smart-release nano-carrier by transforming it into an active delivery system, using a folate-poly(ethylene glycol)-poly(lactic-coglycolic acid)-thiol (FA-PEG-PLGA-SH) conjugate to coat Cyt c-based NPs. This new vehicle for therapeutic proteins possesses the stability advantages of a micellar-like NP system: the hydrophobic polymer shell region (PLGA) stabilizes the protein core, whereas the hydrophilic polymer shell region (FA-PEG) facilitates the suspension in aqueous media, making the particle an appropriate candidate for intravenous administration. Typically, polymeric micelles consisting of amphiphilic polymers that are able to spontaneously self-assemble into core-shell nanostructures are used for the loading of hydrophobic drugs.13,14 In our case, a polymer/protein micelle-like NP is formed in an organic medium mediated by a cross-linking reaction between the amphiphilic polymer and the amine groups on the hydrophilic protein NP surface (Figure 1). We chose folic acid (FA) as the ligand displayed on the surface of Cyt c NPs because the folate receptor (FR) is a well-known tumor marker that binds folate-drug conjugates with high affinity and carries them into the cells via receptor-mediated endocytosis.11,15 FA is important in the formation of new cells because it is required in one carbon metabolic reactions and consequently is essential for the synthesis of nucleotide bases.10,15 The folate receptor is overexpressed by tumor cells in several tumors but in normal tissue it is low in concentration.16 In addition, folate receptors present in normal cells (i.e., lung, brain, and kidneys cells) are inaccessible to the NP delivered via intravenous routes.15,16 Our new drug delivery vehicle combines pas-


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sive and active targeting capabilities with a stabilized nanoparticle protein formulation to facilitate an intracellular reduction-triggered mechanism for the release of Cyt c.

Figure 1. Synthesis of polymer/protein Cyt c-based NPs. Cyt c NPs are made insoluble in aqueous media by a coat consisting of FA-PEG-PLGA-SH copolymer.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Reagents. Cytochrome c from equine heart, reduced glutathione ethyl ester, 3-nitro-2-pyridinesulfenyl chloride (Npys-Cl), O-methyl-O′-succinylpolyethylene glycol (PEGCOOH, 2,000 Da), fluorescein isothiocyanate isomer I (FITC), protease inhibitor cocktail, N,Ndiisopropylethylamine (DIEA), and tributhylphosphine (PBu3) were purchased from SigmaAldrich (St. Louis, MO). Acetonitrile (HPLC grade) and dimethylformamide (DMF) were purchased from Fisher (Waltham, MA). Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) was



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purchased from Proteochem (Denver, CO). Poly(lactide-co-glycolide) thiol end-capped (PLGASH, 30,000 Da, copolymer ratio 1:1) and folate-poly(ethylene glycol)-carboxylic acid (FolatePEG-COOH, 3,000 Da) were purchased from Akina, Inc (West Lafayette, IN). 4’, 6-Diamidino2-phenylindole (DAPI), propidium iodide (PI), and FM-4-64 membrane stain were purchased from Invitrogen (Grand Island, NY). All reagents were used without further purification. All other chemicals were from various commercial suppliers and were at least of analytical grade. HeLa, A549, and NIH-3T3 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and grown according to ATCC instructions. 2.2. Synthesis of FA-PEG-PLGA-SH. To protect the thiol group of PLGA-SH with Npys,17 PLGA-SH (500 mg, 0.016 mmol) was dissolved in 25 ml of anhydrous DMF and reacted with Npys-Cl (3.3 mg, 0.024 mmol) overnight at RT (Figure 2). The resultant product PLGA-Npys was dialyzed against distilled water overnight using 10,000 g/mol MW cut off dialysis tubing. The product precipitated in the tubing. The pellet was collected by centrifugation (10,000 rpm for 15 min at 4ºC in a Hermle Z323K centrifuge) and then dried by lyophilization. PLGA was attached to folate-conjugated poly(ethylene glycol) (FA-PEG-COOH) to synthesize a FA-PEGPLGA copolymer. The free alcohol group on PLGA-Npys was linked to the terminal carboxyl group on the FA-PEG-COOH. Briefly, 400 mg of PLGA-Npys (0.0133 mmol) and 120 mg of FA-PEG-COOH (0.04 mmol) were dissolved in 20 mL of anhydrous DMF. An excess of N,Ndiisopropylethylamine (DIEA) was added dropwise to the resulting polymer solution and the reaction mixture was stirred overnight. The resulting mixture was dialyzed against distilled water overnight using 10,000 g/mol MW cut off dialysis tubing. The precipitated product FA-PEGPLGA-Npys was pelleted by centrifugation and the pellet was dried by lyophilization. The thiol group in PLGA was then deprotected with tributylphosphine (PBu3). Briefly, 200 mg of FA-


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PEG-PLGA-Npys (0.006 mmol) was dissolved in 10 mL of anhydrous DMF and was treated with an excess of PBu3 overnight at RT. The resulting mixture was dialyzed against water overnight, the precipitated product FA-PEG-PLGA-SH was pelleted by centrifugation and the pellet was dried by lyophilization. All reactions were carried out using flame-dried round bottom flasks under a N2 atmosphere. The formation of folate-conjugated copolymer, FA-PEG-PLGA-SH, was monitored and confirmed by 1H NMR analysis. NMR spectra were recorded on a Bruker AV-500 MHz spectrometer using dimethyl sulfoxide-d6 as solvent (DMSO-d6). Chemical shifts (δ) were calibrated relative to the DMSO-d6 signal at 2.50 ppm 1H NMR (500 MHz, DMSO-d6) δ: 8.53 (s, FA, 1H), 7.97 (s, FA, 2H), 6.68 (s, FA, 2H), 5.22 (m, PLGA), 4.93 (m, PLGA), 4.7 (s, FA, 2H), 3.34 (s, PEG/PLGA), 1.50 (m, PLGA) (Figure 2). A control polymer PEG-PLGA-SH (DMSOd6) was synthesized following the same procedure using a modified PEG not carrying the terminal folate modification. 1 H NMR (500 MHz, DMSO-d6) δ: 5.22 (m, PLGA), 4.93 (m, PLGA), 3.34 (s, PEG/PLGA), 1.50 (m, PLGA). 2.3. Synthesis of FA-PEG-PLGA-S-S-Cyt c NP. Cyt c nanoparticles were obtained using a method developed by our laboratory (Figure 1).8 Briefly, 10 mg/ml of Cyt c in the presence of the excipient methyl-β-cyclodextrin (1:8 w/w) in nanopure water was solvent-precipitated by adding acetonitrile at a 1:4 volume ratio. Following the Cyt c nanoprecipitation, the SPDP linker (0.8 mg, 1:3 Cyt c-to-linker molar ratio) was added directly to the resulting suspension to modify the nanoparticle surface with the linker. This releases N-hydroxysuccinimide. After 30 min, 30 mg of FA-PEG-PLGA-SH (0.0009 mmol) was dissolved in 10 ml of acetonitrile and added to the mixture to form a thiol bond with the Cyt c attached linker releasing pyridine 2-thione. The mixture was allowed to react at RT for 18 h. To stop the reaction and to remove unreacted reagents, the nanoparticles were washed by multiple cycles of resuspension/centrifugation (8,000 rpm, 15



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min). The supernatant was used to determine the level of covalent modification of the nanoparticles with the copolymer by measuring the concentration of pyridine-2-thione at 365 nm using the extinction coefficient 8.08x103 M-1cm-1. 2.4. In vitro release of Cyt c. The release of Cyt c from FA-PEG-PLGA-S-S-Cyt c NP was measured as described.8 In brief, 0.5 mg of FA-PEG-PLGA-S-S-Cyt c NP powder was suspended by sonication in 1 ml of 50 mM PBS with 1 mM EDTA at pH 7.4 and glutathione (GHS) concentrations of 0, 0.001, and 10 mM simulating extra- and intracellular conditions.5 Incubation was performed for various times at 37 ºC, and the NPs were pelleted by centrifugation at 14,000 rpm for 10 min. The supernatant was removed and used to determine the concentration of released Cyt c. The protein concentration was determined by spectrophotometric analysis from the heme absorbance at 408 nm. The pellet was resuspended in GHS-PBS buffer. The amount of released protein was used to construct cumulative release profiles. The experiments were performed in triplicate, the results averaged, and the standard deviations calculated. 2.5. Zeta potential measurements. Zeta potential values of the different formulations of Cyt c NPs were determined using a Zetasizer Nanoseries from Malvern. The samples were dispersed in distilled water and subjected to ultrasonication at 240 W for 60 sec prior to the measurements. Zeta potential measurements were repeated three times for each sample, and the data are reported as averages with the standard deviations. 2.6. Dynamic light scattering (DLS) and scanning electron microscopy (SEM). Particle size of FA-PEG-PLGA-S-S-Cyt c NP were determined by dynamic light scattering using a DynaPro Titan. The samples were dispersed in DMF and subjected to ultrasonication at 240 W for 30 sec prior to the measurements. SEM of the different formulations of Cyt c NPs was performed using


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a JEOL 5800LV scanning electron microscope at 20 kV. The samples were coated with gold for 10 sec to a thickness of 10 nm using a Denton Vacuum DV-502A. 2.7. Cell-free caspase-3 assay. The cell lysate was obtained as described by us.5 Briefly, FAPEG-PLGA-Cyt c NP was reacted with 10 mM GHS, centrifuged at 8,000 rpm for 15 min and the supernatant was removed to determine the concentration of released Cyt c. The cell-free reaction was initiated by adding 100 µg/mL of Cyt c to freshly purified cytosol (3 mg/ml) in a total reaction volume of 50 µL. NPs of the non-apoptotic protein α-lactalbumin at 100 µg/mL were employed as negative control and Cyt c from the commercial source as positive control. The reaction was incubated at 37ºC for 150 min. Next, the caspase-3 and caspase-9 assays were performed following the manufacturer’s protocol (CaspACE™ assay; Promega, Madison, WI). The plate was incubated overnight at room temperature and the absorbance at 405 nm was measured in each well using a Thermo Scientific Multiskan FC. All measurements were performed in triplicate. 2.8. Cell culture. Human cervical carcinoma (HeLa), human lung carcinoma (A549), and mouse embryonic fibroblast (NIH-3T3) cells were maintained in accordance with the ATCC protocol. The GL261 glioma cell line derived from C57BL/6 mice was obtained from the NCI (Frederick, MD). The HeLa and A549 cells were cultured in minimum essential medium (MEM) and NIH3T3 and GL261 cells in Dulbecco's modified Eagle's medium (DMEM). Both media contained 1% L-glutamine, 10% fetal bovine serum (FBS), and 1% penicillin in a humidified incubator with 5% CO2 and 95% air at 37°C. All experiments were conducted before cells reached 25 passages. 2.9. MTS Assay. Mitochondrial function was measured using the CellTiter 96 aqueous nonradioactive cell proliferation assay from Promega Corporation. HeLa cells (5,000 cells/well)



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were seeded in 96-well plates and incubated with serial dilutions (12.5, 25 and 50 µg/mL of Cyt c) of FA-PEG-PLGA-S-S-Cyt c NP and PEG-PLGA-S-S-Cyt c NP for 6 h. Controls, such as, 65 µg/mL FA-PEG-PLGA-SH and PEG-PLGA-SH were also tested. T-test analysis was used for comparison of two independent groups for cell viability. A difference between folate-targeted and folate-free Cyt c NP was considered statistically significant at a p-value of

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