Synthesis and unimolecular micelles of amphiphilic copolymer with dendritic poly(L-lactide) core and poly(ethylene oxide) shell for drug delivery

Abstract

A novel type of amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈ with a hydrophobic third-generation dendritic PLLA core and a functionalized hydrophilic PEO shell with surface carboxylic groups was synthesized as a carrier for drug delivery. The POSS-(G₃-PLLA-OH)₈ core was first synthesized by the combination of repetitive ring-opening polymerization (ROP) of L-lactide and branching reactions. Second, the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈ was obtained by esterification coupling between allyl-PEO-COOH chains and POSS-(G₃-PLLA-OH)₈, followed by the peripheral allyl groups reacting with 3-mercaptopropionic acid (MPA). In aqueous solution, this amphiphilic copolymer exists as stable unimolecular micelles with a unique core–shell structure and uniform size distribution (99.9–102.5 nm), as detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Doxorubicin (DOX), an anticancer drug, was encapsulated into POSS-(G₃-PLLA-b-PEO-COOH)₈ micelles to evaluate the drug release profile. The result showed that the DOX-loaded micelles, with a loading content 18.5 ± 2.3 w/w%, exhibited controlled release of up to 39% loaded drug over a time period of 80 h. In addition, the surface carboxylic groups provide the opportunity for further conjugating targeting molecules, fluorescence dye or even drugs. These results indicated that the structurally stable unimolecular micelles from POSS-(G₃-PLLA-b-PEO-COOH)₈ hold potential applications as controlled drug delivery nanocarriers.

---

Introduction

Over the past few decades, extensive research has been devoted to polymeric micelles self-assembled from amphiphilic block copolymers due to their great promising applications in drug delivery.^1–10 Used as nano-sized drug carrier, polymeric micelles can offer combining advantages such as improved water solubility, higher loading capacity, sustained controlled release, prolonged circulation time in vivo and reduced uptake by the reticuloendothelial system (RES), while remaining the drug's activity.^11–14 However, it is known that conventional polymeric micelles represent thermodynamic aggregations of multi amphiphilic macromolecules above their critical micelle concentration (CMC). Once being introduced into the bloodstream, polymeric micelles might disassemble into free polymeric chains as being subjected to high dilution and other factors such as temperature, pH, ionic strength.^15,16 This drawback may heavily hinder polymeric micelles' applications in drug delivery. In sharp contrast, unimolecular micelles, in which the outer hydrophilic polymer chains are covalently tethered to an inner hydrophobic core, possess excellent in vivo stability and are highly desirable for drug delivery.^17–26

Dendrimers, a class of highly branched macromolecules, are characterized by uniform and controlled size, high degree of surface functional groups, well-defined three-dimensional architecture with narrow polydispersity.^27–31 These features make the dendrimer-based amphiphilic copolymers with the core–shell structure attractive candidate for preparing stable unimolecular micelles.^32–35 Typically, hydrophilic polymer chains of the shell are attached to the dendrimer core via a “grafting onto” or “grafting from” method. The unimolecular micelles from the core–shell dendrimer based copolymers are of very interest for the drug delivery applications due to their excellent stability, narrow size dispersity and high loading capacity of therapeutic drugs within cavities of the dendrimer core. Nevertheless, synthesis of the dendrimer core usually involves a series of tedious repetitive steps via either a divergent or convergent method and this main drawback limits their general applicability in drug delivery.

Dendritic polymers are a new class of macromolecules with a similar structure to dendrimers, but building blocks are the narrowly polydipersed polymer chains, instead of monomers.³⁶ So, dendritic polymers are an ideal alternative for preparation of the core–shell amphiphilic copolymers. In comparison with the dendrimer-based amphiphilic copolymers, the copolymers with a dendritic polymeric core have several additional advantages as following: (1) the synthesis can be easier because a few generations can offer a large enough hydrophobic core for drug encapsulation. (2) The particle size can be easily controlled by tuning the length of polymeric building blocks. (3) The flexible interior cavities in dendritic polymeric core can improve the drug-loading capacity as compared with rigid those in dendrimers. To date, a few core–shell amphiphilic copolymers with the dendritic core have been reported.^37–40 However, core–shell amphiphilic copolymers based on dendritic polymer are rather scarce, especially those with good biodegradability and biocompatibility, thus more research effort is deserved in this area.

Various topological amphiphilic copolymers composed of hydrophobic PLA and hydrophilic PEG (also refer to PEO) segments have so far been well studied for drug delivery applications because of these two polymers' complementary biodegradability and biocompatibility properties.^41–45 In this work, we reported the development of a novel amphiphilic copolymer consisting of a hydrophobic third-generation dendritic PLLA core and a hydrophilic PEO shell (Scheme 1). The multifunctional POSS-8OH was employed to initiate the polymerization of LLA monomers. Recently, the POSSes with eight groups on the vertex of the silica cage have been extensively used in the design of drug delivery systems.^46–49 This is because that the eight corner groups can be conveniently used for the preparation of multifunctional polymers with POSS cores. More importantly, they also possess good biocompatible and non-toxic properties as well. The inner PLLA core can be used to encapsulate hydrophobic drugs, while the outer PEO shell endows the micelles with “stealth” property. Due to its unique structure, this amphiphilic copolymer exists as stable unimolecular micelles in aqueous solutions, which is desirable for drug delivery applications. The structure of the copolymer was characterized by ¹H NMR, GPC and FT-IR techniques. The micellar properties of the copolymer were determined by DLS and TEM analysis. To evaluate the suitability of this system as drug delivery carrier, the drug loading and in vitro release properties were investigated using DOX as a hydrophobic model drug.

Scheme 1 Schematic illustration of the synthetic route to the core–shell structural amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈.

Experimental

Materials

L-Lactide monomer (99.5%) was purchased from Purac and recrystallized in ethyl acetate before use. (3-Hydroxy-3-methylbutyldimethylsiloxy)-polyhedral oligomeric silsequioxane (POSS-8OH) was purchased from Hybrid Plastics. α-Allyl-ω-hydroxy poly(ethylene oxide) (M_n = 5000) (allyl-PEO-OH) was provided by professor Guowei Wang's group of Fudan University. 1,8-Diazabicyclo[5.4.0]-undec-7-ene (DBU) (98%, Aldrich), 2-mercaptoethanol (≥99.0%, Aldrich), phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO) (97%, Aldrich), propargyl alcohol (99%, Aldrich), succinic anhydride (≥99%, Aldrich), 4-(dimethylamino)pyridine (DMAP) (≥98.0%, Fluka), N,N-diisopropylcarbodiimide (DIC) (99%, Aldrich) and 3-mercaptopropionic acid (≥99%, Aldrich) were used as received. Chloroform was dried over CaH₂ and distilled prior to use. 3-Propargyloxy carbonyl propionic acid (PCPA) was synthesized according to the literature procedure.⁵⁰

Methods

¹H nuclear magnetic resonance (¹H NMR) spectra were recorded on a Bruker Ultrashield 600 MHz/54 mm NMR spectrometer at room temperature using CDCl₃ as a solvent. Gel permeation chromatographic (GPC) analysis was performed on a Waters 2690 equipped with an evaporative light scattering detector (Waters 2420) and three phenomenex linear 5 mm styragel columns (500, 104 and 106 Å). THF was used as an eluent at a rate of 1.0 mL min⁻¹. Monodispersed poly(methyl methacrylate) (PMMA) was used as standards. Fourier transform infrared (FT-IR) spectra were recorded on a Perkin Elmer spectrum 2000 spectrometer at a resolution of 1 cm⁻¹. Transmission electron microscopy (TEM) images were obtained on a JEOL JEM 2010F field emission electron microscope operating at an acceleration voltage of 200 kV. The TEM samples were prepared by dropping the polymer solution onto the surface of the carbon-coated 200 mesh copper grid and were stained with phosphotungstic acid. The mean size of nanoparticles was determined by dynamic light scattering (DLS) using a Brookhaven BI-9000AT Digital Autocorrelator. The DOX loading level was measured by an UV-visible spectrophotometer (Shimadzu UV-3600) based on DOX's absorbance at 485 nm.

Synthesis of the first generation POSS-(G₁-PLLA-OH)₈

POSS-8OH (0.631 g, 0.370 mmol) was dissolved in 5 mL dry toluene and dried by azeotropic distillation. To the reaction flask, L-lactide (2.968 g, 0.021 mol) and dried chloroform (10 mL) were added under nitrogen flow protection. When the solution became clear under stirring, a catalytic amount of DBU (9 μL, 0.06 mmol) was added. The amount of the catalyst was 1/50 of the amount of the hydroxyl groups. The reaction mixture was stirred overnight at room temperature. The product POSS-(G₁-PLLA-OH)₈ was purified by pouring the reaction solution into excessive methanol. POSS-(G₁-PLLA-OH)₈ can be precipitated from methanol, while a little of unreacted LLA monomers and catalyst were remained in the solution.(yield: 90%) (M_n,theo = 9970 g mol⁻¹; M_n,GPC = 14 020 g mol⁻¹, M_w/M_n = 1.27).

¹H NMR (CDCl₃, δ, ppm): 5.09–5.22 (br, –COCH(CH₃)O–), 4.37 (t, –COCH(CH₃)OH), 1.36–1.72 (br, –COCH(CH₃)O–).

Synthesis of POSS-(G₁-PLLA-alkyne)₈

POSS-(G₁-PLLA-OH) (1.506 g, 0.151 mmol) was dissolved in 10 mL dried CHCl₃ after dried by azeotropic distillation with toluene. Then, 3-propargyloxy carbonyl propionic acid (PCPA) (0.565 g, 3.625 mmol), diisopropylcarbodiimide (DIC) (0.57 mL, 3.625 mmol) and a catalytic amount of N,N-dimethylaminopyridine (DMAP) were successively charged into the reaction flask under nitrogen atmosphere. The reaction mixture was allowed to stir at room temperature for 16 h. The product was obtained by pouring the solution into cold methanol (yield: 94%).

¹H NMR (CDCl₃, δ, ppm): 5.09–5.22 (br, –COCH(CH₃)O–), 4.71 (s, –COOCH₂C≡CH), 2.73 (t, –OCOCH₂CH₂COO–), 2.48 (s, –COOCH₂C≡CH), 1.36–1.72 (br, –COCH(CH₃)O–). FT-IR: 3290 cm⁻¹ (ν_C–H in alkyne group).

Synthesis of POSS-(G₁-PLLA-2OH)₈

POSS-(G₁-PLLA-alkyne)₈ (1.237 g, 0.113 mmol), 2-mercaptoethanol (MPE) (0.38 mL, 5.460 mmol) and phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO) (0.038 g, 0.090 mmol, 0.1 equiv. of alkyne group) were dissolved in 5 mL dimethyl formamide (DMF). The mixture was deoxygenated through purging nitrogen gas for 20 minutes. Care should be taken to keep the flask in the dark so as to prevent the decay of BAPO via incident light. Once deoxygenated thoroughly, the reaction mixture was subject to ultraviolet (λ = 365 nm) irradiation for a period of 2.5 hours. The product was then precipitated twice in methanol to remove all excess initiator and 2-mercaptoethanol (yield: 89%).

¹H NMR (CDCl₃, δ, ppm): 5.09–5.22 (br, –COCH(CH₃)O–), 4.15 and 3.92 (m, –COOCH₂CH(S–)CH₂S–), 2.65–2.82 (br, –OCOCH₂CH₂COO–, and –SCH₂–), 1.36–1.72 (br, –COCH(CH₃)O–).

Synthesis of the second generation POSS-(G₂-PLLA-OH)₈

POSS-(G₁-PLLA-2OH)₈ (1.016 g, 0.083 mmol), L-lactide monomer (1.334 g, 9.264 mmol) and a catalytic amount of DBU (5 μL, 0.027 mmol) were polymerized according to the procedure above mentioned to give POSS-(G₂-PLLA-OH)₈ as a white, crystalline powder. The yield was 2.210 g (92%) (M_n,theo = 28 341 g mol⁻¹; M_n,GPC = 24 540 g mol⁻¹, M_w/M_n = 1.26).

¹H NMR (CDCl₃, δ, ppm): 5.10–5.32 (br, –COCH(CH₃)O–), 4.38 (t, –COCH(CH₃)OH), 4.21 (t, –SCH₂CH₂OCO–), 3.04 (t, –SCH₂CH₂OCO–), 2.73 (–OCOCH₂CH₂COO–), 1.40–1.83 (br, –COCH(CH₃)O–).

Synthesis of POSS-(G₂-PLLA-alkyne)₈

POSS-(G₂-PLLA-OH)₈ (1.620 g, 0.057 mmol) and PCPA (0.285 g, 1.830 mmol) were reacted under the presence of the esterification coupling reagent DIC (0.23 mL, 1.830 mmol) and a catalytic amount of DMAP according to the procedure above mentioned to give the product POSS-(G₂-PLLA-alkyne)₈ (yield: 93%).

¹H NMR (CDCl₃, δ, ppm): 5.09–5.22 (br, –COCH(CH₃)O–), 4.71 (s, –COOCH₂C≡CH), 4.21 (t, –SCH₂CH₂OCO–), 2.48 (s, –COOCH₂C≡CH), 1.36–1.72 (br, –COCH(CH₃)O–). FT-IR: 3291 cm⁻¹ (ν_C–H in alkyne group).

Synthesis of POSS-(G₂-PLLA-2OH)₈

POSS-(G₂-PLLA-alkyne)₈ (1.340 g, 0.044 mmol) and MPE (0.20 mL, 2.807 mmol) were reacted under the presence of BAPO (0.029 g, 0.070 mmol) in DMF to produce POSS-(G₂-PLLA-2OH)₈, following the procedure for synthesizing POSS-(G₁-PLLA-2OH)₈ (yield: 90%).

¹H NMR (CDCl₃, δ, ppm): 5.08–5.26 (br, –COCH(CH₃)O–), 4.10–4.35 (br, –COOCH₂CH(S–)CH₂S–, –SCH₂CH₂OCO–), 2.61–2.83 (br, –OCOCH₂CH₂COO–, and –SCH₂–), 1.36–1.72 (br, –COCH(CH₃)O–).

Synthesis of the third generation POSS-(G₃-PLLA-OH)₈

POSS-(G₂-PLLA-2OH)₈ (1.138 g, 0.034 mmol) initiated the polymerization of L-lactide (1.112 g, 7.714 mmol) in the presence of DBU catalyst (4 μL, 0.022 mmol) to produce POSS-(G₃-PLLA-OH)₈ as a white, crystalline powder. The yield was 2.210 g (yield: 94%) (M_n,theo = 65 301 g mol⁻¹; M_n,GPC = 47 360 g mol⁻¹, M_w/M_n = 1.26).

¹H NMR (CDCl₃, δ, ppm): 5.09–5.30 (br, –COCH(CH₃)O–), 4.52–4.69 (br, –SCH₂CH₂OCO–), 4.37 (t, –COCH(CH₃)OH), 3.01–3.24 (br, –SCH₂CH₂OCO–), 2.68–2.78 (br, –OCOCH₂CH₂COO–), 1.40–1.83 (br, –COCH(CH₃)O–).

Synthesis of α-allyl-ω-carboxylic poly(ethylene oxdie) (allyl-PEO-COOH)

Allyl-PEO_5k-OH (6.420 g, 1.28 mmol), succinic anhydride (0.257 g, 2.56 mmol), and DMAP (0.313 g, 2.56 mmol) were dissolved in 200 mL dry chloroform and the reaction was stirred at 80 °C overnight. The solution was washed with a diluted HCl solution and water successively, then dried over anhydrous magnesium sulfate. The filtrate was concentrated on a rotary evaporator and precipitated twice in diethyl ether to afford the product allyl-PEO_5k-COOH (yield: 87%).

Synthesis of the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-allyl)₈

The POSS-(G₃-PLLA-b-PEO-allyl)₈ was synthesized by esterification coupling of the hydroxyl groups on POSS-(G₃-PLLA-OH)₈ with the carboxyl end group of allyl-PEO_5k-COOH in the presence of DIC and DMAP catalysts. POSS-(G₃-PLLA-OH)₈ (0.230 g, 0.004 mmol) and allyl-PEO_5k-COOH (1.336 g, 0.267 mmol) were dried by azeotropic distillation with toluene and dissolved in 5 mL dried CHCl₃. Then, DIC (42 μL, 0.267 mmol) and a catalytic amount of DMAP were added into the reaction mixture. The reaction lasted for 24 h at room temperature under N₂ atmosphere. After removal of the solvent, the crude product was dialyzed against deionized water for 48 h using dialysis tube (molecular weight cut off (MWCO), 12 000 Da) and freeze dried (yield: 62%) (M_n,theo = 175 301 g mol⁻¹; M_n,GPC = 56 680 g mol⁻¹, M_w/M_n = 1.27).

¹H NMR (CDCl₃, δ, ppm): 5.10–5.25 (br, –COCH(CH₃)O–), 3.60–3.72 (br, –OCH₂CH₂–), 1.40–1.83 (br, –COCH(CH₃)O–).

Synthesis of the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈ with surface carboxyl groups

The surface of the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-allyl)₈ could be functionalized with various groups such as amine, hydroxyl or carboxyl group via a click thio–ene reaction between the surface allyl groups and various mercaptans. As an example, the surface functionalization of POSS-(G₃-PLLA-b-PEO-allyl)₈ with carboxyl groups is described here. POSS-(G₃-PLLA-b-PEO-allyl)₈ (0.110 g, 0.017 mmol of allyl group), 3-mercaptopropionic acid (MPA) (0.018 g, 0.170 mmol) and BAPO (0.004 g, 0.008 mmol) were dissolved in 2 mL DMF, followed by bubbling with N₂ flow for 20 min to eliminate oxygen completely. Then, the mixture was subject to ultraviolet (λ = 365 nm) irradiation for a period of 2 h under stirring. The solution was precipitated twice in cold diethyl ether to provide the final product.

Preparation of DOX-loaded unimolecular micelles of POSS-(G₃-PLLA-b-PEO-COOH)₈

The blank and DOX-loaded unimolecular micelles of POSS-(G₃-PLLA-b-PEO-COOH)₈ were prepared by a simple dialysis technique. In a typical experiment, DOX·HCl (10 mg, 0.017 mmol) and 3-fold molar triethylamine (TEA) (7.2 μL, 0.052 mmol) were dissolved in DMSO (5 mL) and kept stirring for 2 h to remove hydrochloride. After this, copolymer (30 mg) was added and stirred to form a homogeneous solution. Deionized water (20 mL) was added dropwise into the solution under vigorous stirring over ∼1 h. After stirring for another 4 h, the solution was dialyzed against deionized water using a dialysis tube (MWCO, 3500 Da) for 24 h and the solution outside the tube was replaced by fresh deionized water every 6 h. Finally, the concentration of the copolymer was set to 1 mg mL⁻¹ by changing the total volume of the solution inside the tube. To determine the loading amount of DOX in micelles, 3 mL of the above solution was freeze dried and then dissolved in DMSO. The DOX loading content was calculated to be ∼18.5 w/w% based on the UV-vis spectrophotometer at 485 nm against a standard calibration curve.

In vitro release study of DOX from micelles

The in vitro DOX release profile from the POSS-(G₃-PLLA-b-PEO-COOH)₈ micelles was studied as follows: 5 mL of DOX-loaded micellar solution was placed in a dialysis tube (MWCO 3500 Da). The dialysis tube was then immersed into 50 mL PBS buffer solution under gentle stirring at 37 °C. Periodically, 3 mL external buffer solution was taken out and replaced by the equal volume of fresh medium. The amount of the released DOX was measured by a UV-vis spectrophotometer at 485 nm. The experiment was conducted in triple and the data are shown as the mean value in the final release curve.

Results and discussion

Synthesis and characterization of the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈

Unimolecular micelles from the core–shell amphiphilic copolymers have attracted increasing attention for their great potential applications in drug delivery due to their excellent in vivo stability. In this work, a novel amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈, consisting of a dendritic PLLA core and a PEO shell like structure was successfully synthesized following the steps shown in Scheme 1. As compared with the dendrimer-based amphiphilic copolymers, the synthesis of this copolymer is easier and a fewer steps are needed to produce the larger core for drug encapsulation. Meanwhile, the PLLA chains in the inner dendritic core and the PEO chains in the outside shell endow this copolymer with good biocompatibility and biodegradability.

The third-generation dendritic PLLA core was first divergently synthesized by repetitive ring-opening polymerization (ROP) of L-lactide monomers and branching reactions of PLLA chain ends. ROP of L-lactide was accomplished using organic base DBU as catalyst at room temperature and the targeted degree of polymerization (DP) of PLLA arms was set to 7 (M_n,arm = 1008) in every generation. Starting from the initiator POSS-8OH, an eight-arm polyester with hydroxyl end groups was obtained, which is the first generation polymer POSS-(G₁-PLLA-OH)₈. The hydroxyl groups of POSS-(G₁-PLLA-OH)₈ were then functionalized with 3-propargyloxy carbonyl propionic acid (PCPA) to form alkyne-terminated POSS-(G₁-PLLA-alkyne)₈. Subsequently, the alkyne groups reacted with 2-mercaptoethanol via the thio–yne click reaction to generate 16 hydroxyl groups POSS-(G₁-PLLA-2OH)₈. The combination of high efficient esterification coupling and thio–yne addition reaction simplified the branching procedure requiring no tedious protection/deprotection steps. The polymer POSS-(G₁-PLLA-2OH)₈ was then used as macroinitiator to initiate the polymerization of L-lactide to give the second-generation dendritic polymer POSS-(G₂-PLLA-OH)₈. The end of PLLA arms in second generation were successively functionalized with PCPA and 2-mercaptoethanol and produced the intermediates alkyne-terminated POSS-(G₂-PLLA-alkyne)₈ and 32 hydroxyl groups POSS-(G₂-PLLA-2OH)₈, respectively. The third-generation polymer POSS-(G₃-PLLA-OH)₈ was obtained from the macroinitiator POSS-(G₂- PLLA-2OH)₈.

The structure of each generation was confirmed by the combination of ¹H NMR, GPC and FT-IR. Fig. 1 shows the ¹H NMR spectra of the first generation PLLA and their intermediates. The peaks (a) and (b) in Fig. 1A can be assigned to the methyl and methine protons of PLLA arms, respectively, while the peak (c) is attributed to the methine proton neighboring the end hydroxyl group. After functionalization with PCPA, the peak (c) in Fig. 1A disappears and two new peaks (d) and (e) apparently appear, which can be assigned to characteristic methylene protons and alkynyl proton of end propargyl group (Fig. 1B). The characteristic peaks belonging to the protons of propargyl group disappear completely in the following thiol–yne reaction with 2-mercaptoethanol (Fig. 1C). The branching reactions were also confirmed by FT-IR analysis. From the Fig. 2, it can be seen that the absorption peak of alkyne group at 3290 cm⁻¹ appears and then disappears following the branching process of the ends of PLLA arms with PCPA first and 2-mercaptoethanol later. The results suggest that the branching reactions were performed completely. The branching process of the second generation PLLA is also well-characterized according to the methods above (see Fig. S1 and S2†). The GPC traces for the three generations of PLLA are shown in Fig. 3. Narrowly dispersed products were obtained for each of the generations POSS-(G₁-PLLA-OH)₈, POSS-(G₂-PLLA-OH)₈ and POSS-(G₃-PLLA-OH)₈. It is also observed that the GPC traces for higher generations shift to more shorter retention time region, meaning the macroinitiators POSS-(G₁-PLLA-2OH)₈ and POSS-(G₂-PLLA-2OH)₈ successfully initiated the polymerization of L-lactide. Shown in Table 1 are the general characteristics of the three generation dendritic PLLA. Noted that the molecular weight for every generation determined by GPC is apparently different from the theoretical value and this should be due to the fact that the standard for GPC is the linear PMMA while the three generations PLLA are dendritic.

Fig. 1 ¹H NMR spectra of POSS-(G₁-PLLA-OH)₈ (A), POSS-(G₁-PLLA-alkyne)₈ (B) and POSS-(G₁-PLLA-2OH)₈ (C) in CDCl₃.

Fig. 2 FT-IR spectra of POSS-(G₁-PLLA-OH)₈ (A), POSS-(G₁-PLLA-alkyne)₈ (B) and POSS-(G₁-PLLA-2OH)₈ (C).

Fig. 3 DPC traces of POSS-(G₁-PLLA-OH)₈ (A), POSS-(G₂-PLLA-OH)₈ (B), POSS-(G₃-PLLA-OH)₈ (C) and POSS-(G₃-PLLA-b-PEO)₈ (D).

Table 1 Characterization of three generations of dendritic PLLA and the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-allyl)₈

Polymer	Mn^a	Mn^b	Mw/Mn^b	Yield (%)	Coupling E.F^c (%)
a Calculated molecular weight for DP = 7 of PLLA arms in every generation.b Molecular weight and polydispersity index determined by GPC with PMMA as standard.c Coupling efficiency of POSS-(G3-PLLA-OH)8 with allyl-PEO-COOH.
POSS-(G1-PLLA-OH)8	9970	14 020	1.27	90
POSS-(G2-PLLA-OH)8	28 341	24 540	1.26	92
POSS-(G3-PLLA-OH)8	65 301	47 360	1.26	94
POSS-(G3-PLLA-b-PEO-allyl)8	175 301	56 680	1.27		69

---

The core–shell structural amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-allyl)₈ was obtained as allyl-PEO-COOH chains covalently tethered to the POSS-(G₃-PLLA-OH)₈ core via the esterification coupling reaction. Fig. 4 shows the ¹H NMR spectra of the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-allyl)₈ and the precursor POSS-(G₃-PLLA-OH)₈. It can be seen that the peak between 3.60 and 3.72 ppm, which is assigned to the methylene protons of PEO chains, is observed clearly after the coupling reaction. Meanwhile, the GPC trace of the POSS-(G₃-PLLA-b-PEO-allyl)₈ moves toward higher molecular weight region as compared with that of the precursor (Fig. 3D). The above results indicate the successful formation of the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-allyl)₈ with a hydrophobic dendritic PLLA core and a hydrophilic PEO shell. According to the relative integral values of the methylene protons (c) of PEO and methine protons (b) of PLA blocks, the coupling efficiency can be easily measured, which is about 69.2%. The surface allyl groups on POSS-(G₃-PLLA-b-PEO-allyl)₈ was further functionalized with MPA via the thio–ene click reaction to give the carboxylic acid-terminated amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈, which can be further conjugated some special molecules. Disappearance of the peak at 5.89 ppm attributed to the allyl groups indicates the successful modification of POSS-(G₃-PLLA-b-PEO-allyl)₈ with MPA (Fig. S3†).

Fig. 4 ¹H NMR spectra of POSS-(G₃-PLLA-OH)₈ (A) and POSS-(G₃-PLLA-b-PEO-allyl)₈ (B) in CDCl₃.

Preparation of unimolecular micelles from the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈

As expected, the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈ should form core–shell structural unimolecular micelles in aqueous solution. The hydrophobic dendritic PLLA will form the core of the micelles, while the hydrophilic forming the shell. TEM and DLS were employed to determine the morphology and size of the formed blank micelles. As displays in Fig. 5, the core–shell structure of the formed micelles is clearly observed. Meanwhile, the size of the formed micelles is more uniform than that of conventional micelles. This is because that the former is unimolecular, while later is the thermodynamic aggregations of multi polymeric chains, the size of which varies with the aggregation numbers of polymeric chains. The diameter of the core of the formed micelles determined from TEM image is about 30 nm, which is basically consistent with the calculated diameter (∼32 nm) of the dendritic PLLA core within a single amphiphilic copolymer molecule POSS-(G₃-PLLA-b-PEO-COOH)₈.¹⁷ These results confirm that the micelles self-assembled from the amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈ are single-molecule micelles, which are structurally stable in aqueous solution. The average hydrodynamic diameter of the micelles measured by DLS is about 101 ± 0.3 nm. Being similar to the result observed by TEM, the size also exhibits a very narrow distribution with the polydispersity index (PDI) about 0.028. It should be noted that the sizes of the micelles measured by DLS are larger than those measured by TEM because the former is the hydrodynamic diameters of the micelles in an aqueous solution, whereas later is the size of the dried particles. In order to evaluate properly the assembly behavior of the copolymer, hydrodynamic diameters and PDI of the drug-loaded micelles were also measured, which are 144 ± 1.6 nm and 0.034, respectively. The formed micelles still can keep a very narrow size distribution even though the diameter increases after drug loading, further conforming that they are structurally stable and can be considered as promising candidates for drug delivery.

Fig. 5 TEM image and size distribution of the formed blank micelles from the polymer POSS-(G₃-PLLA-b-PEO-COOH)₈.

In vitro release of DOX from POSS-(G₃-PLLA-b-PEO-COOH)₈ micelles

As the above results have demonstrated, POSS-(G₃-PLLA-b-PEO-COOH)₈ unimolecular micelles possess an inner hydrophobic PLLA core and an outer hydrophilic PEO shell. Herein, DOX, a hydrophobic anticancer drug, was encapsulated into the PLLA core to evaluate the controlled drug release ability of the micelles. The drug loading content (LC) and encapsulation efficiency (EE) of unimolecular micelles were determined to be 18.5 ± 2.3 w/w% and 68.6 ± 6.1 w/w%. As compared with the dendrimer-based micelles,³⁴ the LC of the micelles from POSS-(G₃-PLLA-b-PEO-COOH)₈ is higher. The reason should be that there are larger cavities within the dendritic PLLA core, which can encapsulate more drugs. Subsequently, in vitro release of the drug from DOX-loaded micelles was conducted under simulated physiological conditions (phosphate buffer pH 7.4, 37 °C). From Fig. 6, it was observed that the encapsulated DOX was released from the micelles with a controlled manner. About 27% DOX was released in first 24 h with a slight burst and then it slowly rose to 39% in the followed 56 h. In contrast, nearly all free DOX had released relatively faster into the medium with 8 h. These results indicate that the POSS-(G₃-PLLA-b-PEO-COOH)₈ unimolecular micelles are quite desirable as drug nanocarriers due to their controlled drug release characteristics, excellent structural stability, near uniform size distribution and biocompatibility.

Fig. 6 In vitro release profiles of the free DOX (A) and DOX loaded in the micelles from the copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈ (B) at 37 °C (phosphate buffer, pH 7.4).

Conclusions

A novel type of amphiphilic copolymer POSS-(G₃-PLLA-b-PEO-COOH)₈ was successfully synthesized as a carrier for hydrophobic drug delivery. In aqueous solutions, this amphiphilic copolymer exists as structurally stable unimolecular micelles with a hydrophobic third-generation dendritic PLLA core and a PEO shell with multi surface carboxylic groups, as observe by TEM. The micelles exhibit a narrow size distribution within the range of 99.9–102.5 nm, according to the results determined by DLS. The in vitro release profile indicates that DOX encapsulated within the unimolecular micelles was released over a sustained time period in a slow and steady release manner. The POSS-(G₃-PLLA-b-PEO-COOH)₈ unimolecular micelles can be considered as promising candidates for controlled hydrophobic cancer drug delivery due to their biocompatibility and biodegradability, excellent stability and controlled release ability.

Acknowledgements

The authors acknowledge the financial support from State Key Laboratory of Molecular Engineering of Polymers of Fudan University (Grant Number K2014-09).

References

1. G. S. Kwon and T. Okano, Adv. Drug Delivery Rev., 1996, 21, 107–116.
2. V. P. Torchilin, J. Controlled Release, 2001, 73, 137–172.
3. T. G. Park, J. H. Jeong and S. W. Kim, Adv. Drug Delivery Rev., 2006, 58, 467–486.
4. J. H. Park, S. Lee, J. H. Kim, K. Park, K. Kim and I. C. Kwon, Prog. Polym. Sci., 2008, 33, 113–137.
5. K. S. Soppimath, L. H. Liu, W. Y. Seow, S. Q. Liu, R. Powell, P. Chan and Y. Y. Yang, Adv. Funct. Mater., 2007, 17, 355–362.
6. L. E. Vlerken and M. M. Amiji, Expert Opin. Drug Delivery, 2006, 3, 205–216.
7. C. C. Zhou, M. Z. Wang, K. D. Zou, J. Chen, Y. Q. Zhu and J. Z. Du, ACS Macro Lett., 2013, 2, 1021–1025.
8. Y. Q. Zhu, L. Fan, B. Yang and J. Z. Du, ACS Nano, 2014, 8, 5022–5031.
9. Q. M. Liu, J. Chen and J. Z. Du, Biomacromolecules, 2014, 15, 3072–3082.
10. Z. G. Hu, X. S. Fan, H. J. Wang and J. J. Wang, Polymer, 2009, 50, 4175–4181.
11. M. Ferrari, Nat. Rev. Cancer, 2005, 5, 161–171.
12. D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit and R. Langer, Nat. Nanotechnol., 2007, 2, 751–760.
13. C. Oerlemans, W. Bult, M. Bos, G. Storm, J. F. W. Nijsen and W. E. Hennink, Pharm. Res., 2010, 27, 2569–2589.
14. C. Kiparissides and O. Kammona, Can. J. Chem. Eng., 2013, 91, 638–651.
15. R. Savié, T. Azzam, A. Eisenberg and D. Maysinger, Langmuir, 2006, 22, 3570–3578.
16. J. Gong, M. Chen, Y. Zheng, S. Wang and Y. Wang, J. Controlled Release, 2012, 159, 312–323.
17. X. S. Fan, Z. Wang and C. B. He, J. Mater. Chem. B, 2015, 3, 4715–4722.
18. M. Prabaharan, J. J. Grailer, S. Pilla, D. A. Steeber and S. Q. Gong, Macromol. Biosci., 2009, 9, 515–524.
19. M. Prabaharan, J. J. Grailer, S. Pilla, D. A. Steeber and S. Q. Gong, Biomaterials, 2009, 30, 3009–3019.
20. X. C. Pang, L. Zhao, M. Akinc, J. K. Kim and Z. Q. Lin, Macromolecules, 2011, 44, 3746–3752.
21. J. T. Guo, H. Hong, G. J. Chen, S. X. Shi, T. R. Nayak, C. P. Theuer, T. E. Barnhart, W. B. Cai and S. Q. Gong, ACS Appl. Mater. Interfaces, 2014, 6, 21769–21779.
22. H. X. Xu, J. Xu, Z. Y. Zhu, H. W. Liu and S. Y. Liu, Macromolecules, 2006, 39, 8451–8455.
23. X. J. Li, Y. F. Qian, T. Liu, X. L. Hu, G. Y. Zhang, Y. Z. You and S. Y. Liu, Biomaterials, 2011, 32, 6595–6605.
24. H. X. Xu, J. Xu, X. Z. Jiang, Z. Y. Zhu, J. Y. Rao, J. Yin, T. Wu, H. W. Liu and S. Y. Liu, Chem. Mater., 2007, 19, 2489–2494.
25. X. L. Hu, G. H. Liu, Y. Li, X. R. Wang and S. Y. Liu, J. Am. Chem. Soc., 2015, 137, 362–368.
26. J. Y. Liu, Y. Pang, W. Huang, X. H. Huang, L. L. Meng, X. Y. Zhu, Y. F. Zhou and D. Y. Yan, Biomacromolecules, 2011, 12, 1567–1577.
27. E. Buhleier, W. Wehner and F. Vögtle, Synthesis, 1978, 155–158.
28. G. R. Newkome, C. N. Moorefield and F. Vögtle, Dendritic Molecules: Concepts, Syntheses, Perspectives, VCH, Weinheim, 1996.
29. J. Sanchez-Nieves, P. Fransen, D. Pulido, R. Lorente, M. Á. Muñoz-Fernádez, F. Albericìo, M. Royo, R. Gómez and F. J. de la Mata, Eur. J. Med. Chem., 2014, 76, 43–52.
30. M. C. Lukowiak, B. N. S. Thota and R. Haag, Biotechnol. Adv., 2015, 33, 1327–1341.
31. J. P. Yang, Q. Zhang, H. Chang and Y. Y. Cheng, Chem. Rev., 2015, 115, 5274–5300.
32. M. J. Liu, K. Kono and J. M. J. Fréchet, J. Controlled Release, 2000, 65, 121–131.
33. H. B. Liu, S. Farrell and K. Uhrich, J. Controlled Release, 2000, 68, 167–174.
34. X. P. Ma, Z. X. Zhou, E. L. Jin, Q. H. Sun, B. Zhang, J. B. Tang and Y. Q. Shen, Macromolecules, 2013, 46, 37–42.
35. L. Zhang, Q. H. Yin, J. Su and Q. Wu, Macromolecules, 2011, 44, 6885–6890.
36. M. Z. Yin, K. Ding, R. A. Gropeanu, J. Shen, R. Berger, T. Weil and K. Müllen, Biomacromolecules, 2008, 9, 3231–3238.
37. J. L. Six and Y. Gnanou, Macromol. Symp., 1995, 95, 137–150.
38. A. Hirao and T. Watanabe, Macromolecules, 2009, 42, 682–693.
39. H. S. Yoo, T. Watanabe, Y. Matsunaga and A. Hirao, Macromolecules, 2012, 45, 100–112.
40. H. F. Zhang, J. P. He, C. Zhang, Z. H. Ju, J. Li and Y. L. Yang, Macromolecules, 2012, 45, 828–841.
41. K. J. Zhu, X. Z. Lin and S. L. Yang, J. Appl. Polym. Sci., 1990, 39, 1–9.
42. H. R. Kricheldorf and J. Meier-Haack, Makromol. Chem., 1993, 194, 715–725.
43. A. Sawhney, C. P. Pathak and J. A. Hubbell, Macromolecules, 1993, 26, 581–587.
44. C. Hiemstra, Z. Zhong, P. J. Dijkstra and J. Feijen, J. Controlled Release, 2005, 101, 332–334.
45. C. Hiemstra, Z. Y. Zhong, L. B. Li, P. J. Dijkstra and J. Feijen, Biomacromolecules, 2006, 7, 2790–2795.
46. W. A. Zhang and A. H. E. Muller, Prog. Polym. Sci., 2013, 38, 1121–1162.
47. X. Wang, D. Li, F. Yang, H. Shen, Z. B. Li and D. C. Wu, Polym. Chem., 2013, 4, 4596–4600.
48. C. McCusker, J. B. Carroll and V. M. Rotello, Chem. Commun., 2005, 996–998.
49. F. K. Wang, X. H. Lu and C. B. He, J. Mater. Chem., 2011, 21, 2775–2782.
50. W. A. Zhang and A. H. E. Müller, Macromolecules, 2010, 43, 3148–3152.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra19942e

---