Multifunctional polymer nanocarrier for efficient targeted cellular and

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Controlled Release and Delivery Systems

Multifunctional polymer nanocarrier for efficient targeted cellular and subcellular anticancer drug delivery Dimitrina Babikova, Radostina Kalinova, Denitsa B. Momekova, Iva Ugrinova, Georgi T. Momekov, and Ivaylo Vladimirov Dimitrov ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00192 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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ACS Biomaterials Science & Engineering

Multifunctional polymer nanocarrier for efficient targeted cellular and subcellular anticancer drug delivery Dimitrina Babikova†, Radostina Kalinova†, Denitsa Momekova‡, Iva Ugrinova§, Georgi Momekov‡, Ivaylo Dimitrov*,† †

Institute of Polymers, Bulgarian Academy of Sciences, Akad. G. Bonchev St., Bl 103A, 1113

Sofia, Bulgaria ‡

Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Street, 1000 Sofia, Bulgaria

§

Institute of Molecular Biology, “Acad. Roumen Tsanev”, Bulgarian Academy of Sciences,

Akad. G. Bonchev St., Bl 21, 1113 Sofia, Bulgaria * Corresponding author: Ivaylo Dimitrov, E-mail: [email protected]; Tel: +359(2) 979 3628; Fax: +359(2) 870 0309.

ABSTRACT

A multifunctional triblock copolymer intended for targeted drug delivery applications has been designed and successfully synthesized. Following various controlled polymerization and modification steps a saccharide end-functionalized polyoxyethylene block was attached through

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a cleavable in slightly acidic conditions aromatic imine bond to amphiphilic diblock copolymer comprising biodegradable hydrophobic and modified with mitochondria targeting groups polycationic blocks. The micelles formed from the triblock copolymer in aqueous media possess key functions (cleavable “stealth” shield, targeting groups) needed for the safe extracellular transport, successful cell internalization and drug delivery to the target cellular organelles. The multifunctional nanocarriers were loaded with the plant-derived anticancer drug curcumin and the in vitro analyses revealed their superior cytotoxic, apoptogenic and NF-kB-inhibitory effects on target cells over the free drug and non-functionalized polymer micelles of similar composition. Moreover, the enhanced cellular internalization and mitochondrial accumulation of the multifunctional nanocarriers compared to their non-functionalized analogues was visualized by fluorescence microscopy. The results obtained indicate that the presented multifunctional micelles have a potential for application in nanomedicine for enhanced organelle-specific drug delivery.

KEYWORDS: multifunctional; dual-targeting; pH-sensitive; polymer nanocarrier INTRODUCTION Nanomedicine, defined as nanotechnology applied to health and medicine, is an emerging method for treating cancer and other genetic diseases.1-3 The problems in conventional treatments with small molecule drugs include low specificity, rapid clearance and biodegradation, poor solubility, etc.4 Nanoparticles (NP)s used as drug carriers have several advantages over conventional chemotherapy. They offer the opportunity to deliver medication directly to the tumor while sparing to a great extent the healthy tissue and to control the release of encapsulated payload in such way that a high percentage of the trapped drug is released after the particles have


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reached their target site.5 Currently, a wide variety of platforms are being evaluated as nanocarriers for potential use in nanomedicine, including lipid-based6-8, polymer-based9,10, and inorganic nanoparticles11,12, some of them approved for clinical use.13,14 Polymeric nanoparticles are the most attractive materials for drug delivery applications since they allow the control over a number of key features, such as molar mass, biodegradability, hydrophobicity, functionality, particle size, etc.15 Nanocarriers accumulate in the tumor vicinity through the distinctive for the tumor tissue leaky vasculature, an effect known as the enhanced permeability and retention (EPR) effect achieving the so called passive targeting.16,17 To prolong NPs’ blood circulation a strategy of their surface modification has been developed by coating with hydrophilic polymers such as poly(ethylene glycol) (PEG) providing a ‘stealth’ properties that inhibit blood protein adsorption.18 Although the majority of clinically approved or under evaluation nanocarriers contain PEG, there are several issues, such as the decrease in cellular uptake and endosomal escape (‘PEG dilemma’) that need to be addressed.19 The active targeting on the other hand, is based on receptor molecules over-expressed on the cellular membranes of cancerous cells that are selectively targeted by nanoparticles decorated with the respective ligands.20 These specific interactions may also promote internalization of nanocarriers through receptor mediated endocytosis.21 It was established that the introduction of targeting ligands leads to higher efficacy through the enhanced NPs internalization into the tumor cells without affecting the overall tumor localization.22,23 Further enhancement of targeting effect and reduction of non-specific uptake could be achieved by dual and multi-targeting systems generally representing NP-surfaces decorated with two or more targeting moieties that recognize different receptors on the same or different cells.24,25 Over the last decade, subcellular drug targeting, i.e. the specific delivery of biologically active molecules to and into cell organelles has gained a significant interest and


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referred as “new frontier” or “third level” of drug targeting.26,27 The reason for that is the involvement of all subcellular organelles in the etiology and/or pathogenesis of human diseases. Moreover, most of the drugs produce their pharmacological effects on subcellular targets, located either in the cytosol, or at (or inside) the cell organelles.28 Mitochondria are membrane bound organelles that play important roles in living cells such as energy production, apoptosis induction, calcium homeostasis. Various cancer or genetic diseases originate from mitochondrial dysfunction.29 Therefore, targeting mitochondria is attracting great attention in nanomedicine and various strategies using nanocarriers for delivery of therapeutic and/or diagnostic payloads to mitochondria are being developed.30-32 Thus, in 2005 it was demonstrated that the decoration of liposomes surface with triphenylphosphonium (TPP) cations renders them mitochondriotropic.33 Since then, a variety of TPP–bearing nanocarriers for mitochondria targeting have been reported.32,34 Recent studies indicate that the plant polyphenol curcumin interacts directly with specific mitochondrial targets.35 In different cell-free systems curcumin has been shown to directly induce opening of the permeability transition pores, with subsequent increase in the mitochondrial membrane permeability, mitochondrial swelling, loss of membrane potential, and inhibition of ATP synthesis.36,37 Another emerging mitochondrial target for this compound is the mitochondrial voltage dependent anion channel (VDAC-1), whose closure by curcumin is possibly implicated in its apoptogenic mode of action.38 Curcumin has been also found to inhibit the mitochondrial enzyme hexokinase II and hence to induce glycolytic inhibition and subsequent apoptosis.39 On these grounds mitochondrial targeting using curcumin loaded nanoparticles seems as a viable strategy towards optimization of its anticancer effects.


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Overall, the rational design of complex multifunctional drug delivery nanocarriers with desired properties on different levels of transport (the systemic circulation, the tissue and subcellular levels) is still a challenge. Precise synthetic methods are required to introduce the appropriate moieties needed to overcome various extra- and intracellular barriers. Herein, we present a multistep synthetic strategy towards novel multifunctional polymer nanocarrier with detachable in slightly acidic conditions PEG outer shell bearing cellular targeting ligands, polycationic middle layer possessing the buffering capacity needed for the nanovehicle’s endosomal escape modified with pendant subcellular targeting ligands and a biodegradable hydrophobic core. The multifunctional nanocarrier was loaded with the anticancer drug curcumin and subjected to in vitro biological evaluations. Separately, a triblock copolymer with the same composition and similar molar-mass characteristics but lacking targeting and detachable moieties was also synthesized for comparative studies. The multifunctional nanocarriers show superior properties and accumulate preferably in mitochondria as compared to the non-functional analogues. EXPERIMENTAL SECTION Reagents and materials.

The chemicals used in the synthetic and evaluation procedures

were purchased from Sigma-Aldrich unless otherwise noted. Dichloromethane (DCM, ≥99.5%), N,N-dimethylformamide (DMF, ≥99.8%) and tetrahydrofuran (THF, >99%) were purified through a distillation from calcium hydride. Diethyl ether (≥99.5%) was dried over Na2SO4. The other solvents such as 2-propanol (≥99.5%), ethanol (≥99.8%), hexane (≥99%) and acetone (≥99.5%) were used as received. A pure D,L-lactide (LA) monomer was obtained via recrystallization from toluene/ethyl acetate solvent mixture (95:5 v/v) prior to use. The N,N-


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dimethylaminoethyl methacrylate (DMAEMA, 98%) monomer was purified from the inhibitor traces by passing it through a neutral Al2O3-containing column. Methoxypolyoxyethylene (MPEO-2K, Mn=2000 g mol-1) was freeze-dried from toluene. Copper (I) bromide (CuBr, 99.999%), 2-bromo-2-methylpropionyl bromide (BIBB, 98%), 4-dimethylaminopyridine (DMAP,

>99%),

1,1,4,7,10,10-hexamethyltriethylenetetramine

(HMTETA,

97%),

(N,N,N’,N”,N”-pentamethyldiethylenetriamine (PMDETA, 99%), N,N-diisopropylethylamine (DIPEA, 99.5%), 3-chloropropylamine hydrochloride (CPA, 98%), lactobionic acid (LBA, 97%), 4-formylbenzoic acid (FBA, 97%), 1-hydroxybenzotriazole (HOBT,

Multifunctional polymer nanocarrier for efficient targeted cellular and

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