Synthesis,characterization,and property of biodegradable PEG-PCL-PLA terpolymers with miktoarm star and triblock architectures as drug carriers

Abstract

A series of amphiphilic terpolymers with miktoarm star and triblock architectures of poly(ethylene glycol) (PEG), poly(ε-caprolactone) (PCL) and poly(l-lactide acid) (PLLA) or poly(DL-lactide acid) (PDLLA) terpolymers were synthesized as carriers for drug delivery. The architecture, molecular weight and crystallization behavior of the terpolymers were characterized. Anticancer drug doxorubicin was encapsulated in the micelles to investigate their drug loading properties. The miktoarm star terpolymers exhibited stronger crystallization capability, smaller size and better stability than that of triblock polymeric micelle, owing to the lower CMC values of miktoarm star polymeric micelle. Furthermore, the drug-loaded miktoarm star polymeric micelles showed the cumulative DOX release account of the micelles with PDLLA blocks was 65.3% while the release account of the corresponding micelles containing PLLA blocks was 45.2%. The IC₅₀ values of drug-loaded miktoarm star polymeric micelle were lower than triblock polymeric micelle. Meanwhile, Confocal laser scanning microscopy (CLSM) and Flow Cytometry results demonstrated that the miktoarm star micelles were more favorable for cellular internalization. The miktoarm star micelles with PDLLA blocks were promising carriers for anticancer drug delivery.

Keywords

Miktoarm polymeric micelle drug delivery doxorubicin anticancer activity

Introduction

Hydrophobic antitumor drugs, including paclitaxel (PTX), doxorubicin (DOX) and 9-Nitro-20(S)-camptothecin (9-NC), are the promising broad-spectrum anticancer drug with high potency against human tumors, and it has achieved valuable success in early clinical trials.¹ However, there are large difficulties on the clinical application of these hydrophobic antitumor drugs due to its poor solubility and stability.² To avoid these shortcomings, drug delivery systems are developed. Polymeric micelles are potential carriers for antitumor delivery, and they have exhibited signiﬁcant properties in enhancing solubility and maintaining bioactivity.³ It is applicable for hydrophilic drugs such as doxorubicin hydrochloride (DOX•HCl).⁴ The hydrophobic cores of polymeric micelles are considered as drug depository, and the hydrophilic shells effectively prolong the circulation time in blood.

Many factors including composition, molecular weight, ratio of hydrophilic and hydrophobic segments, elasticity and crystallization capability affect the properties of polymeric micelles. The drug loading properties of biodegradable polymeric micelles are closely related to the architecture of the copolymers, so amphiphilic and biodegradable copolymers with different architectures including block, star and hyperbranch were designed and synthesized.^5–9 Miktoarm star copolymer micelles have attracted great interest in the past decades due to their smaller hydrodynamic radius, higher drug loading content and better stability. The property investigations between star polymers and their block counterparts were reported by many researchers.^10–13 However, most of the reported miktoarm star terpolymers were non-degradable or at least one arm was non-degradable. The researches were mainly focused on their extraordinary self-assembly or aggregation behaviors,^14–16 and the synthesis and biomedical applications of biodegradable miktoarm star terpolymers were rarely studied.

Poly(lactic acid) (PLA) is an important biodegradable polymer and has been widely used in drug delivery.^17–21 PLA has three stereoisomers: semi-crystallized poly(d-lactide acid) (PDLA), poly(l-lactide acid) (PLLA) and amorphous poly(DL-lactide acid) (PDLLA).²² The drug release kinetics were closely related to the crystallinity of polymeric carriers.^23,24 Nederberg found that the stereo complex micelle with enantiomerically mixed PLLA/PDLA blocks provided higher paclitaxel loading capacity and near zero ordered paclitaxel release.²⁵

Although PCL- and PLA-based biodegradable polymeric micelles were extensively studied for drug delivery,^26,27 nearly no researches were focused on the combination of PCL and PLA as hydrophobic segments in biodegradable micelles with different architectures as carriers for drug delivery. In this paper, we synthesized two kinds of ABC miktoarm star terpolymers based on PEG, PCL and PLLA or PDLLA segments as well as their corresponding triblock counterparts to investigate the effect of architecture on the properties of micelles. The synthetic route of miktoarm star terpolymers and triblock copolymers is presented in Figures 1 and 2, respectively. The architecture and molecular weight of the polymers were characterized, and also the crystallization behavior, CMC, size and morphology of the micelles were measured. Doxorubicin was encapsulated in those polymeric micelles as a model drug to investigate the effect of architecture and crystallization on drug release, anticancer effect and cellular uptake in vitro. We know that DOX is a hydrophobic antitumor drug and DOX•HCL is its water-solubility form. The solubility of DOX•HCL in water is 10 mg/mL and sink condition of hydrophobic DOX is around 50 µg/mL. Moreover, when the pH 7.4 is changed to pH 5, the drug changed from hydrophobic to hydrophilic due to the protonation of DOX.

Experimental

Materials

Poly(ethylene glycol monomethyl ether) (mPEG, Mn = 2000g/mol, Sigma-Aldrich), ε-Carprolactone (ε-CL; 99%, Sigma-Aldrich), l-Lactide (L-LA; 99%, Purac, Netherland), and DL-Lactide (DL-LA; 99%, Purac, Netherland) were recrystallized in toluene. Benzyl alcohol, tetrahydrofuran (THF), dichloromethane (DCM), trichloromethane (CHCl₃), dimethyl sulfoxcide (DMSO) were purchased from Chengdu Kelong Chemical Co. (Chengdu, China), dried over CaH₂ and distilled under vacuum. Tin(II)2-ethylhexanoate (Sn(Oct)₂, 99.5%, Sigma-Aldrich) was used as received. Pyridine (99%, Aldrich) and triethylamine (TEA; 99%, Asta Tech Pharmaceutical, Chengdu, China) were dried over CaH₂. Doxorubicin hydrochloride (DOX·HCl; Shanghai Yingxuan Chempharm Co. Ltd., Shanghai, China) was neutralized by adding 1 mol/L NaOH aqueous solution to obtain Doxorubicin (DOX). Maleic anhydride (MA, 99%, TCl), p-methyl benzene sulfonic chloride (TsCl, 99%, Asta Tech Pharmaceutical, Chengdu, China), ammonia (28 wt.%, Kelong Chemical Co., Chengdu, China), 4-dimethylaminopyridine (DMAP, 99%, Sigma-Aldrich), N,N′-dicyclohexylcarbodiimide (DCC, 99%, Heowns, Tianjin, China) were used without further purification. Rosewell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin and cell counting kit-8 (CCK-8) were used for cell tests.

Characterizations

¹H Nuclear Magnetic Resonance (¹H NMR) spectra were recorded on a Bruker Avance NMR spectrometer at 400MHz using CDCl₃ as solvents with 0.5% tetramethylsilane as the internal standard. Gel Permeation Chromatography (GPC) measurement was accomplished on an Agilent, 1100 Series, including a Waters 717 model auto sampler, a 2414 refractive index detector and a Waters instrument equipped with a model, 1515 pump. Chloroform was utilized as the eluent at a flow rate of 1.0 ml·min⁻¹ at 25°C. Transmission electron microscopy (TEM) (FEI Tecnai G20 F20) was utilized to characterize the morphology of blank polymeric micelles. The lyophilized micelles were redispersed in distilled water and dropped onto Quantifoil holey carbon foil (Micro Tools GmbH, Germany), and the samples were dried overnight at room temperature. Differential scanning calorimetry (DSC) (Q2000 TA Instruments) was used to study the thermal properties of polymeric micelles. The blank micelles and the drug-loaded micelles were first heated from 25°C to 180°C to eliminate the thermal history then cooled to −80°C at 10°C/min. Afterwards, the samples with PLLA segment were heated to 180°C with the heating rate of 10°C/min while the drug-loaded micelles with PDLLA segment were cooled to −80°C at 10°C/min, and then were heated to 100°C at the same rate. POM (Nikon Eclipse LV 100POL) was utilized to observe the crystal morphology. Each sample dissolved in chloroform was dropped on a coverslip and the solvent was removed by evaporation. DLS tests were recorded on a Zetasizer Nano ZS (Malvern Instrument Ltd. Co., Worcestershire, UK) to determine the size and size distribution of polymeric micelles. The results were the average values in three measurements.

Syntheses of benzyl-PCL, benzyl-PLLA and benzyl-PDLLA

Benzyl-PCL, benzyl-PLLA and benzyl-PDLLA were obtained via ring opening polymerization (ROP) with benzyl alcohol as the initiator and Sn(Oct)₂ as the catalyst according to our previously reported.²⁸ The yields were about 86.7%.

Syntheses of benzyl-PCL-MA and mPEG-NH2

Benzyl-PCL (2 g, 0.95 mmol) and maleic anhydride (0.49 g, 4.74 mmol) were dissolved in 20 mL THF under nitrogen atmosphere. The flask was put into an ice bath. Then the mixed liquid of 1 mL pyridine and 10 mL THF was added dropwise slowly under stirring vigorously. The flask was immersed in an oil bath at 30°C for 48 h. The mixture was concentrated and precipitated in excess cold methanol. During the filtration, the filter cake was washed with pure cold methanol. Finally, the residue was vacuum dried at room temperature for 12 h to remove the residual methanol. The yield of this reaction was 76.7%.mPEG-NH₂ was synthesized according to the literature.²⁹ mPEG (6 g, 3 mmol) and TsCl (2.86 g, 15 mmol) were dissolved in 50 mL DCM and put in an ice bath with stirring. 2.5 mL TEA (18 mmol) diluted in 20 mL DCM was added dropwise in the solution. The reaction was stirred at room temperature for 48 h. After the completion of reaction, the solution was washed with 1 mol/L HCl and 1 mol/L NaHCO₃ solutions for three times, then dried by MgSO₄ overnight and filtered. The filtrate was concentrated and precipitated in cold ether. The product mPEG-OTs was obtained by filtration and dried under vacuum for 12 h. mPEG-OTs and excess ammonia (100 ml) were added in a flask with stirring vigorously at room temperature for five days. The flask content was extracted with DCM for three times. The organ phase was dried with MgSO₄ over night and filtered. The filtrate was concentrated and precipitated in excess cold ether. mPEG-NH₂ was received after filtration and vacuum dried for 12 h.

Synthesis of benzyl-PCL-PEG

Benzyl-PCL-PEG was synthesized by Michael Addition reaction as reported in the literature.³⁰ Briefly, benzyl-PCL-MA (1g, 0.48 mmol) and mPEG-NH₂ (1.12 g, 0.56 mmol) were dissolved in 15 mL DCM in a flask under nitrogen atmosphere. The flask was immersed in an ice bath. DMAP (0.68 g, 5.57 mmol) dissolved in 15 mL DCM was added dropwise into the flask. After a few hours, the flask was transferred into an oil bath at 30°C for 48 h. When the reaction was completed, the flask content was concentrated, precipitated in excess cold methanol and filtered. The filter cake was dried under vacuum for 12 h.

Syntheses of miktoarm star PEG-PCL-PLA terpolymers

The synthesis of PEG-PCL-PLLA miktoarm star terpolymer (denoted as SL) was accomplished through an esterifiaction. Benzyl-PCL-PEG (0.8 g, 0.195 mmol) and benzyl-PLLA (0.78 g, 0.39 mmol) were dissolved in 5 mL chloroform. After the reaction system was immersed in an ice bath, DCC (5 g, 24 mmol) diluted in 15 mL chloroform was added dropwise to the solution of benzyl-PCL-PEG and benzyl-PLLA. The mixture was maintained at room temperature for 48 h. Then the mixture was maintained at −20°C overnight and filtered. The filtrate was concentrated and precipitated in excess cold ether for three times. After filtration, the residue was dried under vacuum. The synthetic process of PEG-PCL-PDLLA miktoarm star terpolymer (denoted as SDL) was the same except that the benzyl-PLLA was changed to benzyl-PDLLA.

Synthesis of mPEG-PCL

mPEG (2 g, 1 mmol) and ε-CL (1 g, 8.76 mmol) were dried under vacuum in a polymerization tube at 80°C for 1 h. Sn(Oct)₂ in toluene (0.1 wt.% to ε-CL) was dropped in the mixture and dried in vacuum to remove the toluene. After the polymerization tube was sealed in vacuum, the tube was immersed in an oil bath at 120°C with stirring for 48 h. The product was dissolved in DCM and concentrated. The concentrated residue was precipitated in cold diethyl ether, filtered and vacuum-dried.

Syntheses of triblock copolymers mPEG-PCL-PLLA and mPEG-PCL-PDLLA

The triblock copolymers mPEG-PCL-PLLA (denoted as BL) and mPEG-PCL-PDLLA (denoted as BDL) were synthesized via the ring opening polymerization (ROP) of L-LA and DL-LA with mPEG-PCL as macroinitiator and Sn(Oct)₂ as catalyst. mPEG-PCL (1 g, 0.25 mmol) was dried in vacuum at 80°C for 1 h. Then LLA (0.5 g, 3.47 mmol) was added in the mixture and continuously vacuum-dried with stirring. Half-hour later, Sn(Oct)₂ dissolved in toluene (0.1 wt.% to LLA) was dropped into the mixture and dried under vacuum for 20 min. The polymerization tube was sealed and put into an oil bath at 120°C with stirring. After 48 h, the product was dissolved in DCM, concentrated and precipitated in excess cold diethyl ether. mPEG-PCL-PLLA was obtained after filtration and vacuum-dried. mPEG-PCL-PDLLA was obtained using the same method except that L-LA was replaced with DL-LA.

Critical micelle concentration (CMC)

The critical micelle concentration (CMC) of SL, SDL, BL, BDL polymeric micelles were determined through a fluorescence spectrometer (F-7000, Hitachi Co., Japan) using pyrene as the fluorescence probe. 6.67 mg of pyrene was dissolved in 50 mL acetone in a volumetric flask and then 1 mL was taken into a 1000 mL volumetric flask. After acetone was volatilized, the final concentration of pyrene was 6 × 10⁻⁷ mol/L by adding 1000 mL deionized water. The concentration of polymeric micelles in aqueous solution of pyrene was varied from 0.01 μg/mL to 500 μg/mL. The excitation spectra were scanned from 300 nm to 360 nm with the emission wavelength of 393 nm. Excitation and emission band widths were both 5 nm. The ratios of peak intensities between 338 nm and 334 nm were recorded as a function of logarithm of polymeric micelle concentration. CMC is defined as the intersection between the tangent of the curve (Peak Intensity ratio vs. log (micelle concentration) at the inflection point and the tangent of the points at the low portion of the curve.

Preparation of doxorubicin-loaded micelles

The doxorubicin-loaded polymeric micelles were prepared using dialysis method. In brief, 2.5 mg of DOX was dissolved in 1.5 mL DMSO and disposed by ultrasound for 10 min before 10 mg of polymeric micelle was added in the solution. After stirring violently for 2 h, the solution was added dropwise into 10 mL deionized water with vigorous stirring overnight. The solution was poured into a dialysis tubing to dialyze against deionized water at 4°C for 12 h. The inner phase was centrifuged and the supernatant was lyophilized. The whole process was carried out in dark.

The DOX loading content was determined via UV-Vis measurement at 480 nm, which is the maximum ultraviolet absorption wavelength of DOX. The calibration curve of DOX/DMSO with different DOX concentration was measured. The drug loading content (DLC) was calculated with the following formula:

DLC (%) = \frac{(mass of drug in drug - loaded micelle}{mass of drug - loaded micelle)} \times 100 %

Drug release profile

DOX-loaded micelles dispersed in 1 mL of phosphate buffered saline (PBS, pH = 7.4) were poured in dialysis tubings (MWCO 1000), the tubings were immersed in 25 mL PBS in 50 mL centrifuge tubes. All the centrifuge tubes were placed in a shaking water bed at 37°C. In each time point, 1 mL of PBS in each tube was taken out, and 1 mL PBS was added in each tube. The released DOX was confirmed via a fluorescence detector with an excitation wavelength at 480 nm and emission wavelength scanned from 500 to 600 nm. The absorption at 550 nm was recorded to analyze. The in vitro drug release experiment was carried out in triplicate under sink condition and the results were presented as mean value with standard deviation (SD).

Cytotoxicity assay

The cytotoxicity of SL, SDL, BL and BDL polymeric micelles against 3T3 and 4TI cells was determined by CCK-8 assay. The cells were seeded in 96-well at the density of 8000 cells per well and 100 μL medium (DMEM for 3T3 cells and RPMI 1640 for 4T1 cells) containing 10% FBS and 1% penicillin-streptomycin. The cells were incubated in 5% CO₂ atmosphere at 37°C. The medium was removed after cultivation for 24 h and another 100 μL fresh medium with different concentration of blank micelles was added for 24 h incubation. The medium was removed, washed with PBS (pH = 7.4) and replaced with 100 μL of serum-free medium containing 10 μL CCK-8. After incubation for 2 h, the absorbance of each well was detected by a Thermo Scientific MK3 (Thermo fisher, USA) at the wavelength of 450 nm.

In vitro anticancer activity

The in vitro anticancer activity of DOX-loaded SL, SDL, BL and BDL polymeric micelles against 4T1 cells was studied by CCK-8 assay. The 4T1 cells were seeded in 96-well at the density of 4000 cells per well with 100 μL RPMI 1640 medium. After incubation for 24 h, the RPMI 1640 medium was replaced with the same volume of RPMI 1640 containing DOX-loaded micelles with concentrations that ranged from 3 × 10⁻³ to 60 μg/mL and incubated for another 24 h. The cell viability was measured by CCK-8 assay and the value of IC₅₀ was calculated via Graphpad Prism 5.

Cellular uptake

Confocal laser scanning microscopy (CLSM) and flow cytometry test were used to investigate qualitatively and quantitatively the cellular uptake of DOX-loaded polymeric micelles. For CLSM studies, 4T1 cells were seeded in 35 mm diameter glass dishes with 1 mL of RPMI 1640 at the density of 1 × 10⁵ cells/mL for 24-h incubation. The medium was removed and 1 mL fresh RPMI 1640 with DOX-loaded SL, SDL, BL, BDL polymeric micelles (the final DOX concentration was 10 μg/mL) was added. The cells were incubated for 2 h and 6 h, respectively. Each dish was washed with PBS (pH = 7.4) for three times after the medium was removed and the cells were immersed in 0.5 ml PBS and detected by CLSM (TCP SP5, Leica, Germany) at the excitation wavelength of 480 nm.

Flow cytometry test

4T1 cells were seeded on 6-well with 2 mL of RPMI 1640 at the density of 4 × 10⁵ cells per well and incubated for 24 h. Then 1 mL of RPMI 1640 with DOX-loaded SL, SDL, BL, BDL polymeric micelles (the final DOX concentration was 10μg/mL) was added and incubated for 2 h and 6 h. The medium was removed and washed with PBS (pH = 7.4) before being harvested by trypsinization. The cells were resuspended in 500 μL PBS after centrifugation at the speed of 1000 r/min for 5 min, and the measurement of fluorescence intensity (excitation: 480 nm; emission: 590 nm) was recorded on a BD FACS Calibur flow cytometer.

Results and discussion

Syntheses and characterizations of terpolymers

¹H NMR spectra of benzyl-PCL-OH, benzyl-PLLA-OH and benzyl-PDLLA-OH were displayed in Figures S1, S2 and S3 (Supplementary material). Their molecular weight which was calculated from ¹H NMR result was 2303, 2108 and, 2040 g/mol, respectively.¹H NMR spectra of SL and SDL as well as their intermediate products were depicted in Figures 3 and 4, respectively. In Figure 3(a), the signals at δ = 1.3(e), 1.6(d), 2.25(c) and 4.0(f) ppm are assigned to the protons of PCL segment. The signals at δ = 5.1(b), 7.3(a) and 6.3–6.4 (g and h) ppm correspond to the protons of benzyl alcohol (b in CH₂O and a in C₆H₅) and –CH = CH–. In Figure 3(b), the peaks at δ = 3.3 (j) and 3.6 (i) ppm are attributed to the protons of mPEG segment, indicating that the segments of PCL and mPEG were connected successfully. In Figure 3(c), the signals of protons in PLLA segment are at δ = 1.45(l) and 5.1 (k) ppm, which means the segment of benzyl-PLLA was conjugated to the benzyl-PCL-PEG segment. A similar result is depicted in Figure 3(d).

The ¹H NMR spectra of BL and BDL are displayed in Figure 4. In Figure 4(a), the peaks at δ = 1.4(g), 1.7(f), 2.3(e) and 4.1(h) ppm are from the protons of PCL segment while the peaks at δ = 3.35(a), 3.7(b) and 4.25(d) ppm are attributed to the protons of mPEG segment. In Figure 4(b), the characteristic signals of protons in PLLA segment are at δ = 1.6(j) and 5.2(i) ppm. In Figure 4(c), a similar result is demonstrated.

The molecular weight of terpolymers

GPC was used to characterize the molecular weight and molecular weight distribution of polymers. The designed molecular weight, molecular weight calculated from ¹H NMR, and GPC results of all the copolymers are listed in Table S1 (SI). The designed molecular weight and the molecular weight calculated from ¹H NMR spectra are close and all the four copolymers exhibited narrow polydispersity (PDI < 1.3). It is notable that the molecular weights of miktoarm star terpolymers tested by GPC were smaller than those of triblock copolymers as the former terpolymers had smaller hydrodynamic radius.

The morphology and CMC of blank polymeric micelles

TEM images show the morphology of the four blank polymeric micelles in Figure S4 (Supplementary material). The TEM results demonstrated that the four copolymers formed spherical micelles by assembling in water. To study the stability of polymeric micelles, the CMC measurement was utilized by classical pyrene probe method and the results are listed in Table 2. The results indicated that the four polymeric micelles had extremely low values of CMC, which revealed good stability. Meanwhile, CMC values of the miktoarm star polymeric micelles were lower than that of their triblock polymeric micelles. In addition, the equilibrium constant Kv calculated according to the literature³¹ listed in Table 2 displayed the difference between the polymeric micelles containing PDLLA segments and PLLA segments, indicating that the polymeric micelles containing PDLLA segments, especially SDL micelle, were more stable in aqueous solution.

The thermal properties of blank and drug-loaded polymeric micelles

The thermal properties of the four polymeric micelles and four drug-loaded micelles were investigated by DSC. The thermal history of all the micelles was eliminated by heating them to the temperature above the melting point of PLLA (175°C). The second scanning curves are presented in Figure 5 and the melting temperature (Tm) and ΔH are summarized in Table 1. In Figure 5(a), as the PEG and PCL were polymers with strong crystallization capability, the Tms of PEG and PCL was observed in all the four copolymers. The Tms of PEG and PCL could not be separated, thus more than one Tms was observed in the DSC graphs around 50°C. In the two copolymers containing PLLA segments, the Tm of PLLA appeared. The Tms of PLLA was found at 120.1°C and 129.0°C in BL and SL, respectively. The ΔH of PEG and PCL in block copolymers was much lower than that in miktoarm star copolymers, so that the triblock architecture affected the crystallization ability of PEG and PCL more serious than that of miktoarm star architecture. The reason probably was their different polymeric architectures. In detail, PCL segment was in the middle between PEG and PLA segments in BL while PCL segment was only connected with one end of chain in SL. In other words, PCL segment got more influences of other polymer chains in BL than in SL. PLLA segment had the same reason.

Table 1.

The melting temperatures and heat enthalpy of the four polymeric micelles and four drug-loaded micelles.

Sample	Tm (°C)		ΔH (kJ/mol)
Sample	T_m1	T_m2	ΔH_l	ΔH₂
BDL	51.2		216.5
BL	51.6	120.1	154.3	52.0
SDL	49.7		552.0
SL	50.1	129.0	243.3	20.9
BDL/DOX	47.5		159.5
BL/DOX	33.41	49.19	12.3	46.2
SDL/DOX	50.67		306.3
SL/DOX	48.45		247.5

Table 2.

The CMC and Kv of polymeric micelles and the IC50 values of DOX-loaded micelles and DOX·HCl calculated by Graphpad Prism 5.

Sample	SL	SDL	BL	BDL	DOX HC1
CMC (10⁻⁸mol/L)	6.0	5.2	9.2	6.8	–
K_v(10⁵)	3.8	5.5	3.3	4.0	–
IC₅₀(µg/mL)	1.84	0.72	3.92	0.61	0.10

The thermal properties of drug-loaded micelles were evaluated and shown in Figure 5(b) and Table 1. Owing to the DSC results of drug-loaded micelles and blank micelles, the same conclusion that the crystallization capacity of miktoarm star micelles was stronger than that of linear triblock micelles could be drawn. Also, since the DOX impeded the crystallization of polymers in the drug-loaded micelles, the values of heat enthalpy were obviously decreased. For SDL and BDL samples, given the ΔH values in Table 1, the gap of crystallization capacity between blank and drug-loaded micelles was narrowed because the SDL and BDL micelles loaded more DOX than SL and BL micelles as shown in the section “Drug Release Profiles.” The melting peak of PLLA segment in the SL drug-loaded micelle was rarely observed while the peak in the BL drug-loaded micelle appeared because the drug-loading content of SL micelle was higher than that of BL micelle.

The crystallization of polymeric micelles

The crystallization of the polymeric micelles was further investigated by POM. The crystals of PCL and PLLA can observed in the images of Figure 6.³² In SDL miktoarm star terpolymers, the spherulites of PCL segments with small grain size were rapidly formed with deformed topography at 40°C. Lots of voids were observed when the crystallization was completed. The same situation was observed in BDL triblock copolymer when the temperature lowered to 35°C, it implied that the PCL chains in SDL exhibited stronger crystallization capability. In SL sample, only small size crystalline grains were observed at 110°C. When the temperature fell to 30°C, large amount of deformed PCL spherulites were observed around PLLA crystalline grains, which acted as the nucleus for the growth of PCL crystals. In BL sample, the situation was similar when the temperatures were 100°C and 35°C, respectively, and it suggested that the PLLA chains in SL exhibited stronger crystallization capability while the crystallization capability of PCL chains in SL was weaker with the comparison of PLLA and PCL chains in BL.

Size of drug-loaded polymeric micelle

The size of drug-loaded micelles was measured by DLS shown in Figure S5 (Supplementary Material). The size increase sequence was SDL < BDL ≈ SL < BL, the size of SDL micelles was the smallest, which was 70.6 nm, the biggest one was BL micelles, it was 206 nm. The sizes of all the four micelles were in the range of Enhanced Permeability and Retention (EPR) effect,³³ which could be targeted to tumor tissues.

Drug release profiles

The drug loading contents of SL, SDL, BL and BDL micelles were 6.9 ± 0.98%, 8.0 ± 0.56%, 5.2 ± 0.62% and 7.4 ± 0.36%, respectively (n = 4). It revealed that the drug-loaded micelles containing PDLLA segment both in miktoarm star and triblock PEG-PCL-PLA micelles possessed relatively higher drug loading content to those micelles containing PLLA segments. It was probably because of the easier encapsulation of DOX in the core domains of micelles composed of amorphous PDLLA and soft PCL chains. The rigid PLLA crystal domains were not favorable for drug loading. In the viewpoint of architectures, the micelles formed by miktoarm star terpolymers favored drug encapsulation.

The in vitro release of DOX was tested in PBS solution with pH 5.0 and 7.4 at 37°C (Figure 7). All the drug-loaded polymeric micelles exhibited relatively faster release during the first 6 h due to the diffusion of DOX absorbed in PEG layer. The release rate slowed down in the following 66 h. It was observed that DOX was released faster from micelles in the medium of pH 5.0 than pH 7.4 as the protonation of DOX contributed to the accelerated drug release in pH 5.0. The drug release curves were fitted using Origin with the function shown in equation (1). The derivative of the function is given in equation (2).³⁴ (The parameters values are shown in Table S2 (Supplementary Material).)

y (t) = C + A_{1} (1 - e^{- \frac{t}{t_{1}}}) + A_{2} (1 - e^{- \frac{t}{t_{2}}})

(1)

\frac{dy}{dt} = \frac{A_{1}}{t_{1}} e^{- \frac{t}{t_{1}}} + \frac{A_{2}}{t_{2}} e^{- \frac{t}{t_{2}}}

(2)

From functions (1) and (2), the first-ordered release kinetics of DOX with decreasing release rate was obtained from all the samples in the medium of pH 7.4 as well as pH 5.0. According to function (1), the cumulative drug release amount at infinite time M_∞ should be the sum of C, A₁ and A₂.Therefore, the M_∞ of drug-loaded micelles at pH 5.0 were much higher than M_∞ of drug-loaded micelles at pH 7.4 and the M_∞ of drug-loaded micelles with PDLLA segments were more than those of the drug-loaded micelles with PLLA segments, comparing their values of “C + A₁ +A₂” from Table S2 (Supplementary material).The low DOX release rate in the medium of pH 7.4 revealed that the drug-loaded micelles were safe in blood transportation. Meanwhile, the four drug-loaded micelles had almost equal drug release rates probably because the two reasons that DOX was hydrophobic and the polymeric architectures were not easily broken at pH 7.4 concealed the effects of polymeric architecture and crystallization on drug release. The release properties what we desired is almost not released before reaching the tumor site and release fast after reaching the tumor site. The cumulative release was less than 30% which implies that the micelles allow for targeted delivery of the drug to the tumor environment. The weak acid microenvironment of tumor tissues and cells could trigger the fast release of DOX according to the release profile in the medium of pH 5.0. In Figure 7 (pH = 5.0), the cumulative release amount sequence of drug-loaded micelles was SDL > BDL > SL > BL. Drug-loaded micelles containing PDLLA segments showed higher cumulative release account than those micelles containing PLLA segments partly due to the difficult diffusion of DOX from the hydrophobic cores with rigid PLLA crystal domains. Another reason was that in the tumor microenvironment, generally, the drug-loaded polymeric micelles could be taken up into tumor cells via endocytosis to the endosomes where the micelles “escape” to the cytosol through “pore formation”, “membrane rupture and burst” and “membrane fusion” which are three endosomal escape mechanisms in order to achieve the purpose of cytosolic delivery.³⁵ The drug-loaded micelles with amorphous PDLLA blocks would be easily broken in weak acid environment,³⁶ especially in the endosomes, because of the loose hydrophobic cores of micelles. To the drug-loaded micelles with the same segments, drug-loaded miktoarm star micelles released more DOX than that of the triblock micelles probably due to the micelles with miktoarm star architecture are easier to be broken in the tumor weak acid microenvironment. It was noticed that the SDL drug-loaded micelle exhibited the best release properties with high and low release rates in acid and neutral conditions, respectively.

Cytotoxicity of polymeric micelles

The cytotoxicity of the micelles was tested using NIH3T3 and 4T1 cells as displayed in Figure S6 (SI). The cell viability of normal cells and tumor cells was beyond 90% in each different micelle concentration, even in the concentration as high as 500 μg/mL. It revealed that the four polymeric micelles were biocompatible and safe in drug delivery application.

In vitro anticancer activity

The in vitro anticancer effect of DOX-loaded micelles was investigated against 4T1 cell as shown in Figure 8. The half maximal inhibitory concentration (IC₅₀) value calculated by Graphpad Prism 5 is listed in Table 2. It is obvious that the IC₅₀ values of drug-loaded micelles with PDLLA segments are much smaller than those with PLLA segments, and it implies that the in vitro anticancer activity of micelles containing PDLLA segments is much better than that of micelles containing PLLA segments, which is consistent with the result of faster release of micelles containing PDLLA in the release profiles in weak acidic environment. The small difference of IC₅₀ values between SDL and BDL drug-loaded micelles was probably because of nearly the same size of the two micelles in DLS results. The extremely low IC₅₀ of free doxorubicin hydrochloride was due to its water-solubility for rapid cellular internalization via diffusion and adsorptive endocytosis.³⁷ DOX-loaded micelles would be internalized through an endocytosis process owing to its size via EPR effect,³⁸ which was different from free doxorubicin hydrochloride. In the next step, we considered that active target and photosensitizer were immobilized on the miktoarm star micelles for chemo-and photo-dynamic combination therapy, which could greatly enhance the anti-tumor efficiency and even the IC₅₀ values were lower than that of free doxorubicin hydrochloride.

Cellular internalization

The cellular uptake of drug-loaded micelles was characterized by Confocal Laser Scanning Microscopy (CLSM) and Flow Cytometry against 4T1 cells for 2 h and 6 h as shown in Figure 9 and Figure S7 (SI), respectively. The result of CLSM in Figure 9 demonstrated that the red fluorescence of DOX increased with the extension of incubation time. And the red fluorescence intensity of SDL and BDL drug-loaded micelles was stronger than that of SL and BL drug-loaded micelles, suggesting that drug-loaded micelles containing PDLLA segments showed better cell internalization efficiency due to their smaller micelle sizes and higher drug loading contents. The fluorescence of SDL drug-loaded micelles was slightly stronger than that of BDL drug-loaded micelles, and it implied that the miktoarm star architecture was more favorable for cellular internalization than triblock architecture.

The result of Flow Cytometry elucidated in Figure S7 (SI) confirmed the above conclusion. The sequence of DOX fluorescence intensity was SDL > BDL > SL > BL, therefore, SDL drug-loaded micelles possessed the most efficient internalization capacity within all the four micelles. These results were consistent with the IC50 results. With longer incubation time, all the cell-associated intensities increased, 4T1 cells treated with SDL drug-loaded micelles also showed the highest cellular uptake within all the cells treated with drug-loaded micelles. In addition, with prolonging time, as more and more drug loaded nanoparticles enter the tumor cell, the gap of intensities would become smaller than that of short time. The results agreed with the results of CLSM.

Conclusions

In this study, four biodegradable PEG-PCL-PLA amphiphilic terpolymers with the same composition, molecular weight, ratio of hydrophilic and hydrophobic segments but different architectures of triblock and miktoarm star were synthesized to investigate the architecture effect on crystallization and drug delivery. The miktoarm star terpolymers formed spherical micelles with higher stability in aqueous solution as they possessed lower CMC values. It was found the miktoarm star polymeric micelles exhibited stronger crystallization capability compared to the corresponding triblock polymeric micelles. The characteristic of PLA chain was the primary factor to affect the drug loading and release properties of PEG-PCL-PLA polymeric micelles. The miktoarm star polymeric micelles showed higher drug loading content to triblock counterparts while the micelles with PDLLA chains were more favorable for drug loading. The drug release from micelles containing PDLLA segments in the medium with pH 5.0 was much faster than that from micelles containing PLLA segments. The IC50 values of triblock and miktoarm star micelles containing PDLLA segments were comparable and much lower than those of PEG-PCL-PLLA triblock micelles. The miktoarm star PEG-PCL-PDLLA micelle exhibited favorable cellular internalization efficiency due to its relatively small size.

Footnotes

Authors' Note

Yixin Zhang and Song Luo contributed equally to this work.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors thank the National Science Foundation of China (grant no. 51573111), the Program for Changjiang Scholars and Innovative Research Team in University (IRT-15R48),the Ministry of Education of China (grant no. 20130181110038) and the Miaozi Project in Science and Technology Innovation Program of Sichuan Province (grant no. 16-YCG061), for financial support.

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