A nano-sized blending system comprising identical triblock copolymers with different hydrophobicity for fabrication of an anticancer drug nanovehicle with high stability and solubilizing capacity

Abstract

Background:

A very common and simple method (known as the blending method) to formulate drug delivery systems with required properties is to physically mix amphiphilic block copolymers with different hydrophobicity. In addition to its simplicity, this blending strategy could help avoid the time and effort involved in the synthesis of block copolymers with the desired structure required for specific drug formulations.

Purpose:

We used the blending strategy to design a system that could overcome the problem of high hydrophobicity and be a good candidate for drug product development using PEG-PLA-PEG triblock copolymers.

Methods:

Two types of PEG-PLA-PEG triblock copolymers with similar (long) PLA molecular weights (MWs) and different PEG MWs were synthesized. The micellar formulations were prepared by blending the two block copolymers in various ratios. The size and stability of the blending systems were subsequently investigated to optimize the formulations for further studies. The loading properties of doxorubicin or paclitaxel into the optimized blending system were compared to that in mono systems (systems composed of only a single type of triblock copolymer). In vitro and in vivo anti-cancer effects of the preparations were evaluated to assess the use of the blending system as an optimal nanomedicine platform for insoluble anticancer agents.

Results:

The blending system (B20 system) with an optimized ratio of the triblock copolymers overcame the drawbacks of mono systems. Drug uptake from the drug-loaded B20 system and its anticancer effects against KB cells were superior compared to those of free drugs (doxorubicin hydrochloride and free paclitaxel). In particular, doxorubicin-loaded B20 resulted in extensive doxorubicin accumulation in tumor tissues and significantly higher in vivo anti-cancer effects compared to free doxorubicin.

Conclusion:

The blending system reported here could be a potential nanoplatform for drug delivery due to its simplicity and efficiency for pharmaceutical application.

Keywords:

• blending system
• block copolymer
• triblock copolymer
• drug delivery
• nanomedicine
• cancer

Acknowledgments

This research was supported by a grant (16173MFDS542) from the Ministry of Food and Drug Safety in 2018 and by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2014R-1A2A1A11050094 and 2015R1A5A1008958).

Disclosure

The authors report no conflicts of interest in this work.

Supplementary material

Methods

Characterizations of block copolymers

¹H-NMR spectroscopy and gel permeation chromatography (GPC) were used to determine the MW and the composition of the block copolymers. ¹H-NMR was performed using Varian, Gemini 2000 (NMR 300 MHz) instrument (Varian, Palo Alto, CA, USA). CDCl₃ was used as solvent for analysis of block copolymers. The molecular weight of the PLA segment was determined from ¹H-NMR spectrum by examining the peak intensity ratio of the methane proton of the PLA segment (COCH(CH₃)O: δ=5.2 ppm) and the methylene protons of the PEG segment (OCH₂CH₂: δ=3.6 ppm) based on the number-average molecular weight of PEG.¹ Number- and weight-average molecular weights (M_n and M_w, respectively) as well as polydispersity index (PDI=M_w/M_n) of the copolymers were determined by GPC using Agilent Technology series-1200 instrument, equipped with the refractive index detector. THF was used as the mobile phase at 1.0 mL/min of flow rate. Column temperature was set at 30°C. The copolymers were dissolved in THF, filtered, and injected into PLgel 10 μm MIXED-B column (Agilent). The molecular weight of block copolymers was calculated based on the calibration curve made from a series of polystyrene standards (Scientific Polymer Products Inc., Ontario, NY, USA).²

Doxorubicin release from micelles

For the DOX release test, 1 mL of DOX-loaded micelle solutions was transferred into dialysis membrane tubes (Spectra/Por^®, MWCO 3.5 kDa). The dialysis membrane tubes were subsequently immersed in a vial containing 10 mL of PBS pH 7.4 and incubated in shaker water bath at a speed of 70 rpm and 37°C. At predetermined time points (1 hr, 3 hrs, 6 hrs, 9 hrs, 12 hrs, 24 hrs, and 48 hrs), the media in the vials were collected to determine the amount of DOX released and the vials were replenished with 10 mL of fresh PBS pH 7.4. The amount of DOX released from the micelles was quantified by using UV-VIS spectrometer (GENESYS 10 UV, Thermo Scientific, Waltham, MA, USA) at wavelength λ=481 nm.

PTX release from micelles

For the PTX release test, 1 mL of PTX-loaded micelle solutions was transferred into dialysis membrane tubes (Spectra/Por^®, MWCO 3.5 kDa). The dialysis membrane tubes were subsequently immersed in a vial containing 10 mL of PBS pH 7.4 and incubated in shaker water bath at a speed of 70 rpm and 37°C. At predetermined time points (1 hr, 3 hrs, 6 hrs, 9 hrs, 12 hrs, 24 hrs, 48 hrs, and 72 hrs), the media in the vials were collected to determine the amount of PTX released and the vials were replenished with 10 mL of fresh PBS pH 7.4. The collected PTX solutions were then lyophilized, and the amount of PTX released from the micelles was quantified by liquid chromatography (Agilent) with a UV detector at the wavelength of 230 nm.

Results and discussion

Characterization of PEG-PLA-PEG

PEG-PLA-PEG (5K-10K-5K) triblock copolymer and functional PEG-PLA-PEG (2K-10K-2K) were synthesized with the methods as reported.³−⁵ The success of triblock copolymer synthesis was confirmed by ¹H NMR and GPC (Table S1, Figure S1). The peak at 3.6 ppm was assigned to proton b of PEG. The peaks at 5.2 ppm and 1.6 ppm were assigned to protons a and c of PLA, respectively. According to GPC analysis, the MW of PLA in two types of triblock copolymers were similar with the values in the range of 9–11 KDa. Since the two block copolymers had a similar length of PLA and a big difference in MW of PEG, they would probably possess different physicochemical characterizations, resulting in the unique properties of micelles.

Figure S1 ¹H NMR spectrum of (A) PEG-PLA-PEG (2K-10K-2K, R=-CH₃) and (B) functional PEG-PLA-PEG (5K-10K-5K, R= -H). CDCl₃ was used as solvent.

Abbreviation: PEG-PLA-PEG, poly(ethylene glycol)-poly(lactic acid)-poly(ethylene glycol)

Figure S2 Drug-release profile of blending system (B20) and original micelles (mono 2 and mono 5). (A) DOX release and (B) PTX release. For information on DOX release from Mono 2 see Hoang et al.⁶

Abbreviations: DOX, doxorubicin; PTX, paclitaxel.

Figure S3 Quantitative fluorescence intensities of tumor compared to those of different organs after 24 hrs from i.v. injection of the micelles.

Figure S4 In vivo tumor regression: (A) Balb/c nu mice 27 days after treatment. Arrows: tumors. (B) Extracted tumors in mice 27 days after treatment.

Table 1 Characteristics of different blending systems depending on the composition of T5 and T2 polymers

Table 2 CMC and characterization of drug-loaded micellar systems

Table S1 Characterizations of triblock copolymers

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