Poly(lactic acid)/poly(ethylene glycol) block copolymer based shell or core cross-linked micelles for controlled release of hydrophobic drug

Abstract

To improve the stability of micelles and decrease the burst release behaviours of hydrophobic drugs, poly(lactic acid)/poly(ethylene glycol) (PLA/PEG) block copolymer based shell or core cross-linked micelles are successfully fabricated. First, PLA–PEG diblock and PLA–PEG–PLA triblock copolymers terminated with acryloyl end groups are synthesized and characterized by ¹H NMR and Fourier Transform Infrared (FTIR). These PLA/PEG block copolymers can spontaneously form micelles, exposing hydrophilic PEG segments outside while hiding hydrophobic PLA segments inside the micelles. The methacryloyl groups, exposed on the outer of shell in the PLA–PEG methacrylate copolymer micelles, are copolymerized with N-vinylpyrrolidone and lead to the formation of shell cross-linked (SCL) micelles. On the contrary, the core cross-linked (CCL) micelles are fabricated through the photo-crosslinking reaction of acryloyl end groups inside the core of PLA–PEG–PLA diacrylate copolymer micelles using poly(ethylene glycol) diacrylate as cross-linker. TEM and DLS are used to investigate the morphology and size of SCL and CCL micelles. Results suggest that the size of these micelles depends on the length of PLA segments in the PLA/PEG diblock micelles and the cross-linking degree. Besides, the shell cross-linking increases the size of the micelles, while the core cross-linking decreases the size of the micelles. Notably, both SCL and CCL micelles retain higher stability than that of uncross-linked micelles. Based on these results, hydrophobic tetrandrine (TED), as the model drug, is used to evaluate the controlled release behaviours of SCL or CCL micelles. Results show that both SCL and CCL micelles can decrease the burst release phenomenon in the initial period. The release performance can be controlled via changing the length of PLA segments in the copolymers. It is indicated that these SCL or CCL micelles are useful for a hydrophobic drug-carrier system.

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1. Introduction

Drug controlled delivery technology is one of the most prominent areas of human health care science. Amphiphilic block copolymer based micelles have attracted significant attention due to their unique core–shell architecture. Generally, polymeric micelles are assembled by macromolecular amphiphiles due to the hydrophobic interaction between core-forming segments in an aqueous solution. The hydrophobic core of the polymeric micelle serves as a container to improve the solubility of water-insoluble drug.¹ While the outer hydrophilic shell composed of flexible segments offers the micelles excellent dispersibility in an aqueous solution, mask the hydrophobic core and prevent reticuloendothelial uptake. When the drug was enwrapped into these micelles, these micelles become payload reservoirs that help to regulate the dose and prolong the bioavailability from each topical application of various encapsulated drugs.^2–4 Moreover, the size of polymeric micelles ranged from 10 to 200 nm, which are suitable for site-specified delivery by injection⁵ and could carry hydrophobic drug through a blood stream, and even target lesions tissue (especially tumour) by the enhanced permeation and retention (EPR) effect during the circulation in vivo.^1,6 The application of polymeric micelles mainly depends on the properties of the core and the shell.⁷

In the fabrication of micelles, hydrophobic poly(lactic acid) (PLA) segment and hydrophilic poly(ethylene glycol) (PEG) segment are widely applied because their biodegradable and non-toxicity.^8,9 In addition, PEG segments could protect the micelle from unspecific protein binding, a process known as the “stealth effect”.¹⁰ Thus, the interest in the study of PLA/PEG block copolymers as drug delivery systems greatly increased in the last decades. Generally, amphiphilic PLA/PEG block copolymers can be synthesized via chemical ring-opening copolymerization or enzymatic synthesis.¹¹ The PLA/PEG di- or triblock copolymer has been used for the preparation of polymeric micelles with core–shell architecture in aqueous environments, which was recognized in the early 1990s.^12,13 Since then, PLA/PEG block copolymer based micelles system has been used to deliver a variety of hydrophobic drug. Tsutomu Ishihara et al.,¹⁴ found that prostaglandin E1 can be efficiently and stably embedded in the PLA/PEG micelles and gradually release from the micelles at various rates into diluted serum in vitro. Kwon GS et al.,¹⁵ successfully combine up to three poorly water-soluble drugs (Paclitaxel, Etoposide, Docetaxel) in one PEG/PLA micelles at clinically relevant concentrations. The hydrophobic PLA cores accommodate drugs with a high loading efficiency, and their brush-like PEG shell ensure long circulation of micelles in vivo.¹² The loading efficiency and release rate could be modulated by the architecture of PLA/PEG micelles, which depends on the ratio of hydrophobic to hydrophilic segments.¹⁶ In addition, some chemical functional groups can be incorporated into the PLA/PEG micelles to improve the targeting and loading efficiency, Z. Hami et al.,¹⁷ synthesize DTX-conjugated polymeric micelles having the ability to target folate receptor of tumour cells. In our previous research, in order to synthesize a novel drug carrier with a better release profile, carboxymethyloxysuccinic acid (CMOSA) was combined with the end group of PLA–PEG–PLA tri-block copolymer, which could provide higher drug encapsulation efficiency and better release profiles.¹⁸

Despite the recent progress in the research of PLA/PEG micelles, some shortcomings are gradually revealed which may limit their application in clinic. The PLA/PEG based micelles are susceptible to dilution by large volume gastric juice, blood, or other body fluids, which could lead to the dissociation of micelles into unimers when the concentration of PLA/PEG copolymer is below the critical micelle concentration (CMC).¹⁹ The shear effects of blood circulation also accelerate the dissociation of micelles into individual polymer chains. Moreover, the micelles may early disintegrate or aggregated under blood proteins and lipoproteins microenvironments, further resulting in the premature drug release.^20,21 Cross-linking strategy has been utilized to solve the above mentioned stability problems following the pioneer work by Wooley and co-workers in 1996.²² Recently, shells cross-linking (SCL) or core cross-linking (CCL) approaches have been developed. Wooley et al.,²³ prepared the poly(acrylamidoethylamine)₉₀-block-PLA₄₀ SCL micelles using ethylene glycol bis[succinimidylsuccinate] as the cross-linker, which has good stability and could efficiently bind siRNA. Chu IM et al.,²⁴ prepared the CCL PLA/PEG micelles via photo-crosslinking, results found these CCL micelles exhibit excellent stability as a suspension in aqueous media at ambient temperature as compared to the micelles, achieving high encapsulation efficiency and offering a steady and long-term release mechanism for the hydrophobic anticancer drug, CPT.

The aim of the present work is to prepare the PLA–PEG diblock and PLA–PEG–PLA triblock copolymer with acryloyl groups at PEG end or PLA end, respectively. These PLA/PEG block copolymers can form micelles in aqueous solution by self-assembly with a hydrophobic PLA core and a hydrophilic PEG shell. Moreover, in order to improve the stability of micelles, the PLA–PEG SCL micelles and PLA–PEG–PLA CCL micelles were prepared via chemical cross-linking or photo-crosslinking, respectively. The encapsulation efficiencies and release behaviours of hydrophobic tetrandrine (TED), selected as model drugs, were also studied.

2. Materials and methods

2.1 Materials

L-Lactide was purchased from Purac Biochem, Gorimchem, Netherlands. PEG (M_n: 2000, 6000, 10 000) was obtained from Fluca. Tetrandrine (TED) was provided by China Biomedicine Company. All other reagents were of analytic grade.

2.2 Synthesis of PLA/PEG block copolymers

2.2.1 PLA–PEG methacrylate diblock copolymer. Methacryloyl chloride were added into the mixed solution of PEG and hydroquinone (1 wt%). The mixture was stirred vigorously for 24 h at the presence of N₂ environment. Then the mixed solution was processed as follows. (1) 10 ml of mixed solution were added into 40 ml water and it was extracted using 10 ml diphenyl ether, (2) the water phase was extracted using 50 ml mixed solution of dichloromethane/n-hexane (3 : 1 (V/V)) two times, (3) separated dichloromethane/n-hexane organic phase were distilled at 39–40 °C. The remaining is the mono-methacrylate PEG which shows light yellow viscous liquid.

The PLA–PEG methacrylate diblock copolymer was prepared by ring-opening polymerisation of L-latide in the presence of mono-methacrylated PEG with stannous octoate as catalyst. L-Latide, mono-methacrylated PEG and stannous octoate (Sn(Oct)₂) were added into a dried polymerization tube and kept under vacuum at 40 °C overnight to remove volatiles. Afterwards, the mixture was purged with N₂ three times and sealed under vacuum. The reaction was carried out at 140 °C for 24 h. The product was dissolved in chloroform and precipitated in petroleum ether. At last, the PLA–PEG methacrylate diblock copolymers were obtained after drying under vacuum at 40 °C for 24 h.

2.2.2 PLA–PEG–PLA diacrylate triblock copolymers. The PLA–PEG–PLA triblock copolymer was prepared by ring-opening polymerisation of L-latide and PEG with Sn(Oct)₂ as catalyst. L-Latide, PEG and Sn(Oct)₂ were added into a dried polymerization tube and kept under vacuum at 40 °C overnight to remove volatiles. Afterwards, the mixture was purged with N₂ for three times and sealed under vacuum. The reaction was carried out at 140 °C for 30 h. The product was dissolved in acetone and precipitated in water. A white powder was obtained and dried under vacuum at 40 °C for 24 h.

The PLA–PEG–PLA copolymer was dissolved in dichloromethane in ice bath. After N₂ were given by inhalation for 30 min, acryloyl chloride was added drop-wise into the mixed solution. The reaction was kept under N₂ and in an ice bath for 10 h, and then further reaction was carried out for 12 h at room temperature. During this process, the HCl gas was absorbed by alkaline solution. Afterwards, the precipitates were removed and the filtrate was added into iced n-hexane to precipitate the copolymer. The PLA–PEG–PLA diacrylate triblock copolymers were obtained after drying under vacuum at 40 °C for 24 h.

2.2.3 Characterization of copolymer. ¹H NMR was used to quantitatively examine the connecting structure of PLA/PEG block copolymers using Varian Mercury Vx300 spectrometer. Fourier transform infrared (FTIR) spectra were used to investigate the end groups of acrylate groups in the copolymers by BIO-RAD 3000 IR adsorption spectra analyzer.

2.3 Preparation of PLA–PEG shell cross-linked micelles

40 μl of PLA–PEG methacrylate diblock copolymer solution in acetone (4 mg ml⁻¹) was added into 4 ml of distilled water. The PLA–PEG uncross-linked micelle solution was obtained via evaporating the acetone at magnetic stirring. Then, 400 μl ammonium persulfate solution (0.5 M) and tetramethyl ethylenediamine solution (5 × 10⁻³ M) were added into the PLA–PEG uncross-linked micelle solution under N₂ condition, respectively. Finally, N-vinylpyrrolidone (NVP) was added into this solution at room temperature and stirred for 4 h, the PLA–PEG SCL micelles were obtained.

2.4 Preparation of PLA–PEG–PLA core cross-linked micelles

PLA–PEG–PLA diacrylate copolymer was dissolved in 2 ml of tetrahydrofuran (THF). The mixture was then added into 100 ml deionized water to a final concentration of 0.1, 0.2, 0.4 and 0.8 mg ml⁻¹. After sonicated for 20 min at room temperature, the PLA–PEG–PLA uncross-linked micelles formed. Then, the photoinitiator (2,2-dimethoxy-2-phenyl acetophenone, BDK, 3 wt%) and cross-linker (polyethylene glycol diacrylate, PEGDA (M_n: 200), 4 mg ml⁻¹) in THF were added into the PLA–PEG–PLA uncross-linked micelle solution. The mixture micelle solution was further exposed to UV-light irradiation (400 W) to initiate the cross-linking reaction. After 65 min, the PLA–PEG–PLA CCL micelles were formed. A series of CCL micelles with different core cross-linked degree (6%, 12%, 24% and 50%) were prepared via adjusting the content of PEGDA in the reaction system.

2.5 Characterization of micelles

The morphology of micelles was investigated by transmission electron microscope (TEM, JEM-100CX II, Japan). The size and size distribution of micelles were evaluated by dynamic light scattering (DLS, BI200SM, USA) at 532 nm. Furthermore, the stability of micelles was evaluated by monitoring the size change after being processed by increasing temperature, diluting and freezing–thawing.

2.6 Drug loading and release

2.6.1 Preparation of TED-loaded micelles. The PLA–PEG methacrylate or PLA–PEG–PLA diacrylate copolymer and TED were dissolved in acetone at different ratio. This mixed solution was dropped into water at stirring. Afterward, the acetone was volatile at low pressure and the TED/PLA–PEG or TED/PLA–PEG–PLA uncross-linked micelles were formed. Finally, these uncross-linked micelles were cross-linked according to the method of part 2.3 and 2.4 and the TED-loaded PLA–PEG SCL or TED-loaded PLA–PEG–PLA CCL micelles were finally prepared, respectively.

2.6.2 Evaluation of TED loading capacity of micelles. The TED loading capacity of micelles was investigated as previously described.²⁵ Briefly, the solution containing TED-loaded micelles was dialyzed against deionized water over 24 h. The concentration of free TED in the dialysis solution was measured by UV spectrophotometry (UV, WFZ-26A, China) at 283 nm. The encapsulation efficiency (EE) and the loading content (LC) were calculated from eqn (1) and (2), respectively.

2.6.3 Release behaviours in vitro. TED-loaded micelles (10 ml) were placed in dialysis bag and immersed them in 90 ml phosphate-buffered saline (PBS, pH = 7.4), incubated at 37 °C in vibrating air bath. 5 ml of the supernatant solution was taken out replenished by 5 ml of the fresh PBS at appropriate intervals (0.5 h, 1.0 h, 2.5 h, 4 h, etc.). The concentration of TED was analyzed by UV spectrophotometry (WFZ-26A, China) at 283 nm. Accumulate release percentage of TED from micelle was calculated according to eqn (3). Where, E_r is the accumulated releasing amount of TED (%), m_drug denote the amount of TED enwrapped in micelles, V_e and V_o represent the volume of replacement PBS (5 ml) and releasing liquid PBS (100 ml), respectively, C_i and C_n is the concentration of TED in releasing liquid when times i and n times replaced the PBS.

3. Results and discussion

3.1 Synthesis and characterization of PLA/PEG block copolymers

3.1.1 PLA–PEG methacrylate diblock copolymer. The mono-methacrylated PEG is synthesized by the reaction between the hydroxyl group of PEG and methacryloyl chloride.²⁶ The ¹H NMR spectrum of mono-methacrylated PEG in CDCl₃ is presented in Fig. 1A, the chemical shifts at 6.6, 5.5 ppm (a′) are the characteristic of methylene protons (CH₂=) of mono-methacrylate (MMA), the peak at 1.9 ppm (b′) is attributed to the methyl protons (–CH₃) of MMA. The chemical shifts at 3.7 ppm (d′) are the characterization of methylene protons (–CH₂–) in PEG.²⁷ The peaks at 4.3 ppm (c′) is referred to the –COOCH₂– originated from mono-methacrylated PEG. These data suggest that the main chemical composite of the herein production of the reaction between PEG and methacryloyl chloride is mono-methacrylated PEG. Besides, the C=C absorption peaks of MMA at 1620 cm⁻¹ are also observed in the FTIR spectroscopy of mono-methacrylated PEG (Fig. S1†), which means the PEG has been modified by MMA. LC-MS analysis of mono-methacrylated PEG also be performed (Fig. S2A†), the ion peaks at 500.3 and the fragment ion peaks at 456.2 in the mass spectroscopy of mono-methacrylated PEG also is a strong indication of the successful synthesis of the mono-methacrylated PEG (Fig. S2B†). The PLA–PEG methacrylate diblock copolymer was synthesized via ring-opening polymerization of lactide (LA) in the presence of mono-methacrylated PEG. As shown in Fig. 1B, the chemical shift of mono-methacrylated PEG also appears in the ¹H NMR spectrum of PLA–PEG methacrylate diblock copolymer. In addition, the peak at 5.2 ppm (f) and 1.6 ppm (g) are attributed to the methine protons (–CH–) and methyl protons (–CH₃) of PLA segments, respectively. These data indicate that the PLA–PEG methacrylate diblock copolymers are synthesised. As expected, the molecular weight of the copolymer increases with the increase of the feed ratio of LA and mono-methacrylated PEG. As shown in Table 1, the number average molecular weights of PLA–PEG methacrylate diblock copolymer ranges from 1672 to 59 668 and the molecular weight distribution is narrow (M_w/M_n is ca. 1.5).

Fig. 1 ¹H NMR spectra of (A) mono-methacrylated PEG, (B) PLA–PEG methacrylate, (C) PLA–PEG–PLA and (D) PLA–PEG–PLA diacrylate copolymers.

Table 1 Molecular weight and distribution of PLA–PEG methacrylate diblock copolymer

Copolymer	Percent of LA in reactants (%)	Mn	Mw/Mn
PLA822–PEG methacrylate	98.06	59 668	1.46
PLA91–PEG methacrylate	88.50	7036	1.39
PLA66–PEG methacrylate	85.97	5236	2.45
PLA30–PEG methacrylate	75.37	2644	1.62
PLA17–PEG methacrylate	60.24	1672	1.66

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3.1.2 PLA–PEG–PLA diacrylate triblock copolymer. The PLA–PEG–PLA triblock copolymer is also synthesized via ring-opening polymerization of lactide (LA) in the presence of PEG. Diacrylation of the PLA–PEG–PLA copolymers are achieved by the reaction of PLA–PEG–PLA with acryloyl chloride. In comparison to the PLA–PEG–PLA triblock copolymer (Fig. 1C), new characteristic chemical shifts appear in ¹H NMR spectrum of PLA–PEG–PLA diacrylate copolymer (Fig. 1D). The peaks at δ = 6.10–6.18 (d) belong to the –COOCH=C–. The peak at δ = 6.45–6.50 (e) and δ = 5.85–5.95 (f) represents the hydrogen's chemical shift of –COCH=CH₂ (trans) and –COCH=CH₂ (cis).²⁸ The FTIR spectra (Fig. 2) exhibit characteristic absorption bands of ether bond at 1085 and 1190 cm⁻¹, and the peak at 1756 cm⁻¹ is the characterization of ester carbonyl group belonged to PLA segment. In addition, the new absorption band of the C=C at 1631 cm⁻¹ appear in the PLA–PEG–PLA diacrylate copolymer. However, the absorption band of OH (3500 cm⁻¹) becomes weaker in PLA–PEG–PLA diacrylate copolymer than that in PLA–PEG–PLA copolymer, because the end OH of PLA–PEG–PLA has been partly reacted. This indicates that the terminal groups of the PLA–PEG–PLA triblock copolymer are derivatized into acrylate groups. In previous study, the triethylamine was used to neutralize the reaction products (HCl) when OH reacted with acryloyl chloride. But the polymer–triethylamine complex may be formed via the reaction between triethylamine and C=C, which make the products become yellow. In this work, in order to reduce this phenomenon, the HCl gas is directly adsorbed by alkaline solution and the N₂ were injected in order to carry the HCl gas out.^29,30

Fig. 2 FTIR spectra of PLA–PEG–PLA and PLA–PEG–PLA diacrylate copolymer.

3.2 Formation and structure of SCL and CCL micelles

Copolymers consisting of both hydrophobic and hydrophilic blocks tend to form micelles in aqueous phase to reduce free energy from hydrophobic interactions among hydrophobic chains.³¹ Most polymeric micelles are composed of hydrophobic segments as the internal core and hydrophilic segments as a surrounding shell. In this work, the acryloyl groups are grafted on the end of PEG of PLA–PEG diblock copolymer and PLA of PLA–PEG–PLA triblock copolymer, respectively (cf. Fig. 3). Upon contacting with an aqueous phase, these block copolymers spontaneously form nano-sized assembly, exposing hydrophilic PEG segments outside while hiding hydrophobic PLA segments inside of the micelles. The acrylate groups are exposed on the outer of shell in the PLA–PEG uncross-linked micelles, while they are packed inside of the core in the PLA–PEG–PLA uncross-linked micelles (Fig. 3).

Fig. 3 Schematic formation and structure of PLA–PEG shell cross-linked micelles and PLA–PEG–PLA core cross-linked micelles.

The PLA–PEG SCL or PLA–PEG–PLA CCL micelles are prepared by cross-linking the acryloyl groups at the ends of copolymers in the micelles. For PLA–PEG SCL micelles, the cross-linking reaction occurs between methacryloyl end groups of PLA–PEG methacrylate and NVP. The polyvinylpyrrolidone (PVP) segments are formed on the surface of micelles (Fig. 3). In addition, the shell cross-linking reaction is carried out at high dilution (less than 40 mg L⁻¹, cf. Table 2) in order to avoid undesirable inter-micellar cross-linking according to previous report.³² On the other hand, PLA–PEG–PLA CCL micelles are prepared by photo-crosslinking the acryloyl groups at the ends of PLA–PEG–PLA diacrylate and PEGDA in the micelles (Fig. 3). After cross-linking, the double bond functional-group absorption peaks at 1630 cm⁻¹ of PLA–PEG–PLA diacrylate copolymer disappears (Fig. S3†), which indicates that the core of micelles are cross-linked.

Table 2 DLS analysis of PLA–PEG uncross-linked and SCL micelles

Concentration (mg L^−1)	Uncross-linked micelles		SCL micelles
	Size (nm)	Dispersion coefficient	Size (nm)	Dispersion coefficient
5	95.6	0.16	219.1	0.96
10	95.0	0.08	285.0	0.06
20	92.8	0.20	258.6	0.86
30	97.7	0.07	270.1	0.05
40	110.2	0.26	280.7	0.09

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3.3 Size and morphology of micelles

Table 2 shows the change in the intensity-average micelle diameter with copolymer concentration of PLA–PEG uncross-linked and SCL micelles measured by DLS. Uncross-linked micelles of approximately 92–98 nm are created with relatively low polydispersity values (<0.20) when the concentration of PLA–PEG methacrylate copolymer is lower 30 mg L⁻¹, there are no significant difference among them. However, the size of the uncross-linked micelles with 40 mg L⁻¹ obviously increases to 110.2 nm. Moreover, after cross-linked by NVP, the size of PLA–PEG SCL micelles significantly increases (moreover 219 nm) (Table 2 and Fig. S4†). TEM images of micelles exhibit similar phenomenon. As shown in Fig. 4A and B, the PLA–PEG uncross-linked micelles are light spherical entities with uniform size (30–70 nm). In addition, the micelles solution maintains the homogeneous and transparent after shell cross-linking. Moreover, few aggregation phenomena can be observed in TEM images, and the SCL micelles maintain the sphericity with ca. 80 nm (Fig. 4C). One can observe the cross-linked shell layer outside of micelles (Fig. 4D). The results of DLS and TEM indicate the shell cross-linking could lead to the increase of micelle's size.

Fig. 4 TEM images of PLA–PEG uncross-linked micelles (A and B) and PLA–PEG SCL micelles (C and D).

On the other hand, the size of PLA–PEG–PLA CCL micelles is smaller than that of uncross-linked micelles. As shown Fig. 5A, the PLA–PEG–PLA uncross-linked micelles exhibit good sphericity and the average size is ca. 110 nm. Notably, the core cross-linking lead to the decease of micelles' size and the size decreases with the enhancing of core cross-linked degree. As show in Fig. 5B and C, the size of PLA–PEG–PLA CCL micelles ranges 70–90 nm and 20–80 nm when the core cross-linked degree is 6% and 24%, respectively. This may be caused by the inside core of CCL micelles become compactness after cross-linking. DSL examination also exhibits similar results (Fig. 6), but the size measured by DLS is larger than the size measured by TEM, which is attributed to the shrinkage caused by the water evaporation under vacuum. In addition, Fig. 6A also suggests that the size of PLA–PEG–PLA uncross-linked micelles increase with the increasing of PLA segment length in copolymer, however, it has little effects on the size of PLA–PEG–PLA CCL micelles. Furthermore, the size of PLA–PEG–PLA CCL micelles obviously increases with the enhancing of concentration of PLA–PEG–PLA copolymer (Fig. 6B).

Fig. 5 TEM micrographs of PLA–PEG–PLA uncross-linked micelles (A) and CCL micelles with 6% cross-linked degree (B) and 24% cross-linked degree (C).

Fig. 6 Size of PLA–PEG–PLA uncross-linked and CCL micelles by DLS as the function of LA/EO mole ratio (A) and copolymer concentration (B).

It is well known that the stability of micelles plays important roles in the cellular internalization process as well as their in vivo performance. To investigate the stability of shell or core cross-linked micelles, the size was evaluated by DLS at different conditions, such as, increasing temperature, diluting and freezing–thawing (Fig. S5 & S6†). For the uncross-linked micelles, the micelles could be dissociated into unimers when the concentration of PLA/PEG block copolymer is below the CMC. Therefore, the uncross-linked micelles cannot be detected after dilution with 100 times. In addition, we also find that elevated temperature and freezing–thawing also can destroy the structure of uncross-linked micelles. As expected, shell and core cross-linking of PLA/PEG based micelles assists in the maintenance of their micelle-like structure, and enhances their stability due to the nature of chemical cross-linking. Our study finds that the size of PLA–PEG–PLA CCL micelles only declines by 20 nm after dilution with 1000 times. In addition, elevated temperature and freezing–thawing has little effects on the size of PLA–PEG SCL micelles. Meanwhile, PLA–PEG–PLA CCL micelles can keep the micelle-like structure although their size increases obviously after freezing–thawing. These results suggest that both shell and core cross-linked micelles exhibit superior structural stability compared to their uncross-linked counterpart.²⁰ The stability of micelles takes very important roles when they were injected in vivo, which can prevent premature dissociation of micelles due to dilution in blood, better retain the drug contents, prolong circulation time of micelles in blood and decrease drug premature release properties.^20,33,34

3.4 Drug encapsulation capacity of micelles

Tetrandrine (TED), obtained from the roots of stephania tetrandria, is one of a large number of known plant derived bisbenzylisoquinoline alkaloids. It could be incorporated into core of PLA–PEG SCL or PLA–PEG–PLA CCL micelles due to its hydrophobic nature. This study finds that the shell or core cross-linking can obviously improve the LC and EE of micelles. For PLA–PEG SCL micelles, LC and EE is 22% and 88%, respectively, which is higher than that of PLA–PEG uncross-linked micelles (20% and 80%) (Fig. 7A). For PLA–PEG–PLA CCL micelles, core cross-linked degree and LA/EO ratio have little effects on the LC of micelles (Fig. 7B and C), however, the EE lightly decrease with the increases of core cross-linked degree and LA/EO ratio. In addition, we also find the average size of PLA–PEG–PLA CCL loaded with TED micelles is ca. 100 nm when the cross-linked degree is 24% (cf. Fig. 7D), which is larger than that of PLA–PEG–PLA CCL micelles unloaded TED (20–80 nm) (cf. Fig. 5C). The introduction of TED makes the hydrophobic segments of micelles become stretch and loose, whereas the hydrophilic segment (PEG) remains unchanged, which leads to the increase of average size of micelles.^35,36

Fig. 7 LC and EE of PLA–PEG uncross-linked micelles and SCL micelles (A), PLA–PEG–PLA CCL micelles with different LA/EO mole ratio (B) and different core cross-linked degree (C); TEM images of TED loaded PLA–PEG–PLA CCL micelles with cross-linked degree of 24% (D).

3.5 Drug release behaviours from micelles

Drug release from carriers typically shows a triphasic profile: (1) an initial burst release of surface and pore associated drug, (2) a lag phase until sufficient polymer erosion has taken place and (3) a secondary burst with approximately zero order release kinetics.³⁷ The initial burst release is controlled by diffusion, whereas the lag phase and secondary burst phase are dependent on polymer erosion as well as diffusion.³⁸ From Fig. 8, one can find that initial burst phase from both PLA–PEG uncross-linked and SCL micelles occur within 6 h. The next 6–70 hours belong to the polymer erosion phase, the drug enwrapped in the core of micelles release due to the degradation of copolymer carriers. Moreover, one can find that the release rate of TED form SCL is significantly lower than that form uncross-linked micelles within initial 13 h (p < 0.01) and between 13 and 36 h (p < 0.05), respectively, indicating that the shell cross-linking can decrease the burst behaviour to some extent. Because the shell layer become thickness and compactness after shell cross-linking, and it can further hinder TED release form micelles. In addition, the release total amount arrive equivalent at 55.5 hours. After 215 hours, the drug release total amount is moreover 97% from both of them, suggesting the shell cross-linking do not hinder the accumulated releasing amount but it could decrease the initial burst release behaviours.

Fig. 8 Releasing behaviours of TED from PLA–PEG uncross-linked micelles and SCL micelles in PBS (pH = 7.4, 37 °C) solution. The micelles concentration is 20 mg L⁻¹ and TED concentration is 5 mg L⁻¹ accordingly. (*p < 0.01, SCL micelles compared with uncross-linked micelles within 13 h; **p < 0.05, SCL micelles compared with uncross-linked micelles between 13 and 36 h).

There is a burst phenomenon within initial 6 h for both uncross-linked micelles and CCL micelles (Fig. 9A). The TED continues release from the core of micelles between 6 and 20 h, however, the release rate decreases. After 20 h, there is almost no release of the drug. Moreover, results also suggest that CCL micelles show a significantly delayed drug release compare with the uncross-linked micelles (Fig. 9A), because the core region of the PLA–PEG–PLA CCL micelles is much more tightly packed. The releases of TED depend on the PLA segment length in PLA–PEG–PLA diacrylate copolymer. For example, the accumulated releasing amount is only ca. 17.9% at the beginning of 0.5 h when LA/EO ratio is 10, while the accumulated release amount is 64.4% when LA/EO ratio is 2 (Fig. 9B). The hydrophobic TED is mainly enwrapped in the hydrophobic PLA core of PLA–PEG–PLA CCL micelles. The interactions between TED and PLA become more strength when the PLA segment length increases, resulting in the TED release difficult from micelles with longer PLA segments. It indicates that PLA could reduce the burst behaviours at the beginning period. Meanwhile, the longer PLA segments in PLA–PEG–PLA copolymer may lead to lots of TED remain in the core and cannot release from micelles at the end. For example, the more than 93.2% TED can release from the micelles with LA/EO is 2 after 20 h, while only ca. 72.85% TED release from the micelles when the LA/EO is 10. In addition, the molecular weight of PEG segment and concentration of CCL micelles has little effects on the release performance of TED (cf. Fig. 9C and D). However, the increase of TED concentration accelerates the burst release phenomena (cf. Fig. 9E).

Fig. 9 Releasing behaviours of TED from PLA–PEG–PLA CCL micelles in PBS (pH = 7.4, 37 °C). Effect of core cross-linking (A), LA/EO ratio (B), concentration of PLA–PEG–PLA CCL micelles (C), molecular weight of PEG (D) and concentration of TED (E).

Above all, the stability and minimal drug premature release properties of SCL and CCL micelles provide platform for drug combination delivery, which is promising for enhanced intracellular delivery efficiency of many hydrophobic drugs. Van Nostrum CF et al.³⁹ believe the cross-linked micelles can enhance tumour accumulation when compared to uncross-linked micelles. Therefore, it may potentially be an effective drug carrier to effectively treat lesions tissue (especially tumour) via oral administration or intravenous.

4. Conclusions

The acryloyl groups are introduced on the end of PEG and PLA chain in PLA–PEG diblock and PLA–PEG–PLA triblock copolymer, respectively. These amphiphilic PLA/PEG block copolymers can form micelles in solution, exposing hydrophilic PEG segments outside while hiding hydrophobic PLA segments inside of the micelles. Moreover, the acryloyl groups are exposed on the outer of shell in PLA–PEG uncross-linked micelles, while they are packed inside of the core in the PLA–PEG–PLA uncross-linked micelles. These acryloyl groups can react with NVP and PEGDA to form the PLA–PEG SCL or PLA–PEG–PLA CCL micelles, respectively. Results suggest that the shell cross-linking increases the size of micelles, while the core cross-linking decreases the size of micelles. Moreover, the stability of micelles is significantly improved after either shell or core cross-linking. In addition, both PLA–PEG SCL and PLA–PEG–PLA CCL micelles can decrease burst release phenomenon at the initial period. The release performance could be controlled via changing the length of PLA segment in copolymer.

Acknowledgements

This work is supported by National Nature Science Foundation of China (Grant no. 51073119, 31271016, 31370975 and 31100674) and Natural Science Foundation of Hebei Province (H2012401017).

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Footnote

† Electronic supplementary information (ESI) available: Chemical characteristics of PLA/PEG block copolymer and micelles, stability of shell and core cross-linked micelles. See DOI: 10.1039/c4ra14376k

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