CC BY
13
Научни трудове на Съюза на учените в България-Пловдив. Серия В. Техника и технологии, т. XV, ISSN 1311 -9419 (Print), ISSN 2534-9384 (On- line), 2017. Scientific Works of the Union of Scientists in Bulgaria-Plovdiv, series C. Technics and Technologies, Vol. XV., ISSN 1311 -9419 (Print), ISSN 2534-9384 (On- line), 2017.
СИНТЕЗ И МИЦЕЛООБРАЗУВАНЕ НА МУЛТИФУНКЦИОНАЛЕН ТРИБЛОКОВ СЪПОЛИМЕР ЗА НАСОЧЕНО ДОСТАВЯНЕ НА
ЛЕКАРСТВЕНИ ВЕЩЕСТВА Димитрина Бабикова, Радостина Калтнова, Ивайло Димитров Институт по полтмерт - БАН, ул. „Акад. Георги Бончев" бл. 103-А, 1113 София, България
SYNTHESIS AND MICELLIZATION OF MULTIFUNCTIONAL TRIBLOCK COPOLYMER FOR TARGETED DRUG DELIVERY Dimitrina Babikova, Radostina ICaUnova^vayloDimkrov Institute of Polymers - BAS, Acad. Georgi Bonchev Str., block 103-A, BG - 1113 Sofia, Bulgaria Abstract
Polymeric nanoparticles play a central role in the systems fot controlled drug delivery. Herein, we present a novel polymeric drug delivery system intended for both cellular and subcellular targeting. The synthetic strategy involves a multistep procedure leading to the formation of amphiphilic triblock copolymer poly(ethylene oxide)-b-poly(D,L-lactide)-b-poly(N,N-dimethylaminoethyl methacrylate), PEO-b-PLA-b-PDMAEMA bearing all the necessary functions (targeting and degradable) in the same macromolecule. Initially, amphiphilc diblock copolymer PLA-b-PDMAEMA with terminal amine group and pending triphenylphosphonium subcellular targeting ligands was synthesized applying controlled polymerization and modification techniques. In the second step, a heterobifunctional PEO-block, decorated with lactobionic cellular targeting ligand and aldehyde end group was attached to the diblock copolymer through the formation of pH-sensitive aromatic imine bond to yield the multifunctional triblock copolymer. The amphiphilic triblock copolymer self-associated in aqueous media into nanosized multifunctional micelles that were characterized with dynamic light scattering (DLS) and transmission electron microscopy (TEM).
Kyewords: multifunctional block copolymer, polymeric nanoparticles, targeted drug delivery
1 Introduction
Over the past two decades, the attention of the leading pharmaceutical companies is directed to the problem of controlled and targeted delivery of drugs into the human body to achieve therapeutic effect. Polymeric nanoparticles exhibit several features that favor their utility as nanocarriers for drug delivery applications. The most important advantages are low toxicity, various opportunities for functionalization, ability to impart desired properties, control of size, topology and microstructure (Blanco, 2015). The rational design of drug delivery nanovehicles is based on two targeting approaches. Passive targeting relies on the characteristics of the delivery vehicle itself and the pathology of the specifc disease that leads to a preferential accumulation of nanoparticles at the site of interest. The passive mechanism of nanoparticle accumulation and uptake is known as the enhanced permeability and retention (EPR) effect and was frst reported by Maeda et al.
(Matsumura 1986; Maeda, 2012). The use of stimuli responsive carriers is another option for accumulating active substances to the desired sites (Shenoy, 2005; Hoffman, 2013). The second approach is based on active targeting strategies that direct the drug delivery systems to the sites of interest on cellular and subcellular level using various targeting moieties (ligands) (Tada, 2007). The proposed drug delivery concept for polymeric nanocarriers is composed of three main elements: a hydrophobic core of PLA-block (drug carrier), a positively charged layer of PDMAEMA-block that behaves like endosomotropic agent (accumulates in endosomes, buffering the pH and promoting the nanocarrier endosomal escape) and detachable PEO-corona, which suppresses the non-specific interactions with biological components including entrapment by the reticuloendothelial system (RES), thereby leading to prolonged blood circulation time of micelles (Miyata, 2011).
2 Materials and methods
2.1 Reagents and materials. All chemicals were purchased from Sigma-Aldrich. Dichloromethane (DCM,) and tetrahydrofuran (THF) were distilled from calcium hydride prior to use. D,L-Lactide (LA) was recrystallized from toluene/ethyl acetate mixture (95 : 5 v/v). N,N-Dimethylaminoethyl methacrylate (DMAEMA) was passed through a column containing neutral aluminum oxide. Ethylene oxide (EO) was distilled over calcium hydride at subzero temperature. Propargyl alcohol (PrOH) and N,N-dimethylethanolamine were distilled under reduced pressure. Triethylamine (TEA) was distilled from potassium hydroxide. Methanol, hexane, acetone, copper(I) bromide (CuBr), 2- bromo-2-methylpropionyl bromide (BIBB), 4-dimethylaminopyridine (DMAP), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), (4-bromobutyl) triphenylphosphonium bromide (Br-Bu-TPP-Br), lactobionic acid and 4-formyl benzoic acid were used as received.
2.2 Methods. 1H NMR spectra were recorded in CDCl3 or DMSO-d6 on a Bruker Avance II+ 600 MHz instrument. Gel permeation chromatography (GPC) was performed in THF at a flow rate of
1.0 mL min-1 using Shimadzu Nexera XR HPLC chromatograph, calibrated versus polystyrene narrow molar mass standards. Infrared spectra were recorded on a IRAffinity-1 Shimadzu Fourier Transform Infrared (FTIR) spectrophotometer with MIRacle Attenuated Total Refectance Attachment. UV/Vis spectra were taken on a DU 800 Beckman Coulter spectrometer. Transmission electron microscope (TEM) images were obtained using HRTEM JEOL JEM-2100 (200 kV) instrument. Dynamic light scattering (DLS) measurements for particles' size and size-distribution determination as well as their surface zeta potential analyses were carried out on a NanoBrook 90Plus PALS from Brookhaven Instruments Corporation.
3 Results and discussion
3.1 Synthetic strategy. Controlled synthesis of the multifunctional amphiphilic triblock copolymer involves several steps. A metal-free organocatalyzed controlled ring-opening polymerization proposed by Nederberg et al. (Nederberg, 2001) was applied for the synthesis of PLA biodegradable block. A commercially available propargyl alcohol was used as a heterobifunctional initiator for D,L-lactide controlled polymerization (Babikova 2016). Thus, while the hydroxyl group initiated the polymerization process, each of the formed polymer chains was quantitatively functionalized with the "clickable" alkyne end-group with no need of additional protection/deprotection steps. The obtained heterobifunctional polyester was characterized by GPC, :H NMR and FTIR spectroscopy. The polyester's terminal hydroxyl group was reacted with BIBB followed by a controlled radical polymeization of the second polymer block of PDMAEMA with desired length. The polymerization proceeded in THF and was completed within 5 hours. The copper-containing catalytic system was removed by passing the reaction mixture through Al2O3-containing column. The amphiphilic diblock copolymer was recovered in high yield (above 75%). The final steps at this stage of the synthetic procedure involved the primary amine group modification of PLA chain end through reaction of its terminal alkyne groups with azidopropylamine followed by the introduction of subcellular targeting ligands into the
PDMAEMA-block through partial quaternization of the dimethylamino-groups with bromobutyl-derivative of triphenylphosphonium (TPP+) ligand.
Separately, a heterobifunctional PEO, bearing dimethylamino and hydroxyl end groups was synthesized by an anionic ring-opening polymerization of ethylene oxide using potassium alkoxide of N,N-dimethylethanolamine as an initiator (Berlinova, 2000). The next step was modification of the PEO terminal hydroxyl and dimethylamino groups with aromatic aldehyde and lactobionic (cellular targeting) moieties respectively. Finally, the functional PEO-block was attached to the amphiphilic PLA-b-PDMAEMA diblock copolymer through the formation of pH-sensitive aromatic imine bond yielding the multifunctional triblock copolymer (Figure 1).
Figure 1. Chemical copolymer.
structure of the multifunctional PEO-b-PLA-b-PDMAEMA triblock
The degree of each monomer polymerization and the completeness of the modification reactions were determined by 1H NMR spectroscopy. The polymers molar-mass distributions were obtained from the GPC analyses. Since only controlled and living polymerization techniques were applied the resulting homo- and block copolymers obtained at different stages of the synthetic procedure exhibit monomodal and narrow molar-mass distributions with dispersities in the 1.1-1.3 range. 3.2 Block-copolymer micelles formation. Above their critical micellization concentration (CMC) the amphiphilic triblock copolymer can spontaneously form micelles comprising hydrophobic PLA cores, polycationic PDMAEMA layer with TPP+ subcellular targeting ligands and PEO corona with lactobionic cellular targeting ligands. The polymer micelles were obtained in aqueous media by the nanoprecipitation technique. Initially, a predetermined amount of the triblock copolymer was dissolved in acetone (good solvent for all constituent blocks) and added dropwise to water or phosphate buffered saline (PBS, pH 7.4). After the organic solvent evaporation polymer micelles were formed and subjected to analyses. The CMC of the triblock copolymer in aqueous media was determined applying the dye solubilization method described by Alexandridis et al. (Alexandridis, 1994). UV measurements on increasing concentrations of triblock copolymer (0.005-2.0 mg mL-1) in the presence of the hydrophobic dye 1,6-diphenyl-1,3,5-hexatriene (DPH, 10 |L from 0.4 mM solution in methanol) in aqueous media were performed. While DPH does not dissolve in water it solubilizes into the hydrophobic micellar core, giving a characteristic spectrum with absorption maximum at 356 nm. By plotting the intensity of this maximum vs. block copolymer concentration the CMC value of 0.03 mg mL-1 was estimated from the cross-point of the obtained two straight lines. Thus, for further analyses micelles with concentration of 1 mg mL-1 were prepared.
The DLS measurements showed formation of particles with an average diameter of approx. 50 nm with monomodal and relatively narrow size-distribution (Figure 2a). Thus, the obtained mutifunctional micelles are with optimal for potential use in nanomedicine sizes. The formation of
spherical nanoparticles was visualized by TEM analysis (Figure 2b). The data concerning average particle sizes are in good agreement with those obtained from the DLS measurements. The particles suface charge is reduced significantly after the attachment of the PEO-block. The surface zeta potential drops from 17 mV for the micelles of PLA-b-PDMAEMA diblock copolymer to 5,7 mV for the multifunctional PEO-b-PLA-b-PDMAEMA triblock copolymer micelles. This is as an evidence for the "stealth" effect of the PEO-block (Figure 2c).
Diameter (nm) Zeta potential (mV)
Figure 2. Characteristics of the functional PEO-b-PLA-b-PDMAEMA block copolymer micelles: (a) size-distribution curve measured by DLS in PBS, pH 7.4 (d = 46.3 (±1.9) nm, Pdl: 0.253); (b) TEM-image (d = 61 (±11) nm); and (c) zeta potentials measured in PBS, pH 7.4 (16.9 mV for the amphiphilic PLA-b-PDMAEMA diblock copolymer micelles and 5.7 mV for the PEO-containing multifunctional triblock copolymer micelles).
4 Conclusions.
Novel, multifunctional amphiphilic triblock copolymer was designed and successfully obtained applying a multistep synthetic pocedure. The copolymer consists of biodegradable hydrophobic block of poly(D,L-lactide), polycationic poly(N,N-dimethylaminoethyl methacrylate) block with weakly basic amino groups having sufficient buffering capacity and detachable poly(ethylene oxide) block. Furthermore, the system was decorated with lactobionic cellular and triphenylphosphonium subcellular targeting ligands. The amphiphilic triblock copolymer self-associated in aqueous media into multifunctional nanosized micelles intended for targeted drug delivery applications..
Acknowledgements
This research was financially supported by the National Science Fund of Bulgaria through project DFNI T02-21/2014.
References
Alexandridis P., Holzwarth J., Hatton T. Macromolecules 1994, 27, 2414-2425. Babikova D., Kalinova R., Zhelezova I., Momekova D., Konstantinov S., Momekov G., Dimitrov I. RSC Adv. 2016, 6, 84634-84644.
Berlinova I., Dimitrov I., Vladimirov N. Polymer 2000, 41, 6431-6438. Blanco E., Shen H., Ferrari M., Nat. Biotechnol. 2015, 33, 941-951. Hoffman A. Adv. Drug Deliv. Rev. 2013, 65, 10-16. Maeda H. J. Control. Release 2012, 164, 138-144. Matsumura Y., Maeda H. Cancer Res. 1986, 46, 6387-6392. Miyata K., Christie R., Kataoka K. React. Funct. Polym. 2011, 71, 227-234. Nederberg F., Connor E., Moller M., Glauser T., Hedrick J. Angew. Chem., Int. Ed. 2001, 40, 2712-2715.
Shenoy D., Little S., Langer R., Amiji M. Pharm. Res. 2005, 22, 2107-2114. Tada H., Higuchi H., Watanabe T., Ohuchi N. Cancer Res. 2007, 67, 1138-1144.
