Thermosensitive tribrachia star-shaped s -P(NIPAM- co -DMAM) random copolymer micelle aggregates: Preparation,characterization,and drug release applications

Materials and reagents

N-isopropylacrylamide (NIPAM, 98%) and N,N-dimethylacrylamide (DMAM, 99.5%) were purchased from the Aladdin and Alfa Aesar, respectively, without further purification. The trimesic acid (95%, Aldrich) and mercaptoethylamine (95%, Sigma) were used as received. 2,2′-Azobisisobutyronitrile (AIBN, Xi’an Chemical Reagents Corp, China) was recrystallized from ethanol before use. Tetrahydrofuran (THF), ethanol, concentrated sulfuric acid (H₂SO₄), sodium bicarbonate (NaHCO₃) and benzene, all of analytical grade, were provided from the Sinopharm Chemical Reagent Co. Ltd, and without further purification.

Synthesis procedure

The synthesis of thermosensitive tribrachia star s-P(NIPAM-co-DMAM) polymers was accomplished via the following three-step reaction, and the synthesis strategies were demonstrated in Scheme 1.

▸ Synthesis of cores (Triethyl 1,3,5-benzenetricarboxylate, TBTC): 6.3 g (0.03 mol) trimesic acid, 30 ml (ca. 0.51 mol) ethanol, and 20 ml benzene were added into a 150-ml one-neck flask, followed by a complete dissolution in a 50℃ water bath. Then, 3 ml (0.056 mol) H₂SO₄ was dropwise added into the mixture solution and the water segregator, a glassware that is usually used to remove water produced in the organic preparation experiments, was mounted on the flask. After 5 ml water was drawn off from the side pipe of the water segregator, the mixture was refluxed for 12 h. After most of the ethanol was evaporated at 78.3℃, the residual solution in the flask was poured into a beaker filled with 60 ml distilled water. Afterward, sodium bicarbonate powder was added in batches with stirring until no CO₂ gas produced. Subsequently, vacuum filtration was performed, and the precipitate was rinsed with water till neutrality and dried in vacuum at 50℃. The average yield was 95.02%. Fourier transform infrared spectra (FT-IR) (ν, cm⁻¹): 2937–3100 (Ar–H and –CH₂–CH₃ stretch), 1709–1736 (C = O stretch), 1024 (C–O stretch), 1392–1445 (C–H in-plane bending), at 1255–1268 (C–H in-plane bending in aromatic skeleton), 860 and 730 (C–H out-of-plane bending of 1,3,5-trisubstituted benzene ring). ¹H NMR (300 MHz, CDCl₃): δ (ppm) = 8.85 (signal a, s, 3H), 4.46 (signal b, q, 6H), and 1.44 (signal c, t, 9H).

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Scheme 1

Synthetic scheme of representative thermosensitive s-P(NIPAM-co-DMAM) star-shaped polymer.

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Table 1.

Formula designation and the GPC data of the as-synthesized star polymers.

[S]:[M] a	Codes	[NIPAM]: [DMAM] b		Mn	Mw	PDI	Copolymer compositions [NIPAM]:[DMAM] c
							Theoretical	Experimental
0.1	Arm-1	9	0	21,900	33,000	1.51
	Star-1			42,400	57,200	1.35
	Arm-2	8	1	11,400	20,900	1.83	6.4:1	6.5:1
	Star-2			26,700	42,800	1.44	6.4:1	6.3:1
	Arm-3	7	2	8,700	15,800	1.82	3.0:1	3.3:1
	Star-3			21,700	35,400	1.63	3.0:1	3.4:1
0.06	Arm-4	7	2	10,700	19,400	1.81	3.0:1	3.4:1
	Star-4			22,500	34,500	1.40	3.0:1	3.5:1

PDI: polydispersity indexes.

a

[S]:[M] stands for molar ratios of chain transfer agents to monomers and [M] = 20 mmol.

b

[NIPAM]:[DMAM] represents the molar ratios of the two monomers.

c

The theoretical and experimental [NIPAM]:[DMAM] composition ratios in the resultant copolymers were calculated from the mole fraction copolymerization equation and NMR data, respectively.

Preparation of the copolymer micelles

Star-shaped polymer micelles were prepared by a dialysis method following our previous work. ²⁷ Specifically, 50 mg s-P(NIPAM-co-DMAM) was dissolved in 10 ml dimethylformamide (DMF) to make the polymer solution with a concentration of 5000 mg L⁻¹, and then deionized water was dropwise added into the above-mentioned polymer solution under vigorous stirring until the opalescence appeared, indicating the formation of polymer micelles. The solution was stirred overnight and then dialyzed against 1000 ml deionized water (MWCO: 8000–14000) for 48 h to remove DMF.

Characterization and determinations of the star copolymers

FT-IR of the resultant star polymers were recorded on an AVATAR 360 ESP FT-IR spectrometer (Nicolet, USA), and samples were pressed into KBr pellets. ¹H NMR spectra of the resultant star polymers were obtained on an Avance 300 MHz NMR spectrometer (Bruker Avance, Germany) with tetramethylsilane as internal standard and CDCl₃ as solvent at 25℃. The molecular weight and polydispersity index were determined by gel permeation chromatography (GPC, Waters, USA). THF, high-performance liquid chromatography grade, was used as a mobile phase at a flow rate of 1.0 ml min⁻¹ at 35℃. The samples were dissolved in THF, and the polymer solutions were filtered through a 0.45 -µm needle-type filter (organic, Φ13) prior to the measurement. The calibration curve was carried out using polystyrene standards with the molecular weight range from 1 to 500 kDa.

Measurements of critical aggregate concentration

Critical aggregate concentration (CAC) is a measure describing the physical properties of the micelles and refers to the micelle’s stability. The most common ways to determine CAC of polymer micelles include fluorescence spectroscopy, UV spectroscopy, surface tension, and so on. In this work, the micellization and CAC of the star polymers in aqueous solutions were studied by means of a surface tension technique. ²⁷ The surface tension of each solution was determined individually by DCAT21-Tensiometer (DataPhysics, Germany) and using the Wilhelmy plate method. In brief, 40 ml of a series of aqueous solutions containing different concentrations (1 × 10⁻⁵→1 × 10⁰mg ml⁻¹) of the polymers were prepared, and then the sample solutions were left overnight to equilibrate at room temperature.

Determination of CP

Optical transmittance of the micellar solution (250 mg L⁻¹) with temperature was measured on a U-3900/3900H UV–vis spectrophotometer (Hitachi, Japan) at λ = 500 nm to examine the thermosensitive phase transition behavior of the prepared micelles. The heating rate was set at 0.5℃ min⁻¹, and the apparatus was calibrated using deionized water. The CP value of the polymer micelles was defined as the temperature producing a 50% decrease in the turbidity or optical transmittance.

Transmission electron microscope observations

The morphology and size of the polymer micelles were observed by a transmission electron microscope (TEM) using a JEM-2100 instrument (Japan). TEM images were obtained at an accelerating voltage of 200 keV, and the sample was prepared by dipping a drop of micelle dispersion on a copper grid with carbon film. Excess copolymer solution was wiped off with filter paper, and the grid was dried under ambient atmosphere for 1 h. It was necessary to stain with 2% phosphotungstic acid aqueous solution before the measurement.

Dynamic light scattering measurements

The hydrodynamic size and polydispersity of the micelles prepared were evaluated by dynamic light scattering (DLS, BI-90Plus, Brookhaven Instruments, USA) equipped with an argon ion laser operating at λ = 660 nm and at a fixed scattering angle of 90°. All of the sample measurements were performed at 25℃, and the polymer concentration was 250 mg L⁻¹ unless otherwise noted.

PTX loading and in vitro PTX release

To prepare micelle-based drug formulation, PTX (4 mg) and star copolymer (15 mg) were dissolved in 5 ml DMF, and then the solution was vigorously stirred at room temperature for 2 h. Subsequently, the deionized water was added into the mixture solution until the opalescent solution is formed. The mixture solution was transferred into a dialysis bag (MWCO: 2000 Da) to dialyze against deionized water for 12 h to remove DMF. The dialysate was dried by lyophilization, and the drug-loaded micelles were collected. To determine the loading capacity (LC) and entrapped efficiency (EE) of drugs, 4 mg lyophilized drug-loaded micelles was dissolved in DMF, and the drug concentration was determined with a UV–vis spectrometer (U-3900/3900H, Titachi Corp., Japan) at a wavelength of 228 nm using a standard calibration curve experimentally obtained with PTX/acetonitrile solutions. The LC and EE were calculated according to the following formulas.^28,29

The in vitro drug release experiments were carried out at simulated physiological media of pH 7.4 and different temperatures. The lyophilized drug-loaded copolymer micelles (4 mg) were dissolved in phosphate-buffered saline (PBS) solution at a concentration of 1 mg ml⁻¹ and then were placed into dialysis bag (MWCO = 2000). The dialysis bag was directly immersed into 500 ml PBS solutions of pH 7.4 at 25℃, 37℃, and 43℃. After predetermined period, 4 ml of PBS solutions was drawn out from the analysis system. Meanwhile, 4 ml fresh PBS was added into the release system for supplement. The concentration of the PTX in the release samples was determined by UV absorbance at 228 nm. The cumulative drug release (wt%) was calculated on the basis of the following formula

where M_t is the amount of drug release at t time, and M₀ stands for the amount of drug loaded in the copolymer micelles.

MTT assay

MTT (3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), a yellow tetrazole, is reduced to purple formazan in living cells. MTT assay is a colorimetric assay for assessing cell viability. In this work, MTT assay was employed to evaluate the in vitro cytotoxicities of the blank copolymer micelles toward L929 cells and bioactivity or therapeutic potency of PTX after loaded into the copolymer micelles against MDA-MB231 human breast cancer cells, as described in the literature. ³⁰ The cells were seeded in a 96-well plate at 1 × 10⁴ cells well⁻¹ in a complete Dulbecco’s modified eagle’s medium containing 10% hyclone fetal bovine serum (high glucose DMEM) and incubated at 37℃ in 5% CO₂ atmosphere for 24 h. The culture medium was then removed, and the blank and PTX-loaded micelle solutions with various concentrations in complete DMEM were used to replace the culture medium. The cells were subjected to MTT assay after being incubated for additional 48 h at below (37℃) and above CP (43℃). The optical densities (ODs) were monitored by a 96-wells universal microplate reader (Model 680, Bio-Rad laboratories Ltd, UK) at 490 nm. The cell viability was calculated using the following equation

where OD_control and OD_samples stand for the ODs of the blank and PTX-loaded micelles solutions, respectively.

Synthesis and characterization of the tri-armed star-shaped polymers

Triethyl 1,3,5-benzenetricarboxylate as a core was prepared through esterification reaction between excess ethanol and trimesic acid in the case of H₂SO₄ as catalyst. On the other side, the amino-terminated P(NIPAM-co-DMAM)-NH₂ copolymers were synthesized by free radical chain transfer polymerization. Finally, tri-armed star-shaped s-P(NIPAM-co-DMAM) polymers were synthesized via ammonolysis reaction, as shown in Scheme 1. To confirm the structure of the resultant polymers, FT-IR, ¹H NMR, and GPC analyses were carried out, and the results are displayed in Figures 1 to 3 and Table 1. The FT-IR assignations of TBTC core, P(NIPAM-co-DMAM)-NH₂ arm, and the resultant polymers were provided in the experimental section. The data show that the major characteristic vibration peaks of these samples emerge in their separate FT-IR spectrum. In particular, the disappearance of the stretch band of esterified carbonyl groups at 1709–1736 cm⁻¹ in TBTC due to the ammonolysis reaction preliminarily reveals synthesis of the target product.

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Figure 1.

FT-IR spectra of representative core, arm, and star polymer products.

¹H NMR spectra were further adopted to confirm the chemical structure of the resultant polymers, as depicted in Figure 2. It is noticed from Figure 2(a) that TBTC as core produces three characteristic chemical shift signals of hydrogen protons at 8.85, 4.46, and 1.44 ppm, which are ascribed to the hydrogen proton in 1,3,5 - three substituted benzene rings (Ar–H), methylene protons (–COOCH₂CH₃), and methyl protons (–COOCH₂CH₃), respectively. These shift signals have no impurity peaks, and the integral area ratios are about 1:2:3, almost identical to the proton number ratios of a, b, and c in the TBTC molecule, suggesting that highly purified samples were prepared. The P(NIPAM-co-DMAM)-NH₂ as arm has chemical shift signals at 2.90 ppm assigned to –N(CH₃)₂ in DMAM structural units; at 1.14, 3.99, and 6.35 ppm ascribed to –NHCH(CH₃)₂, –NHCH(CH₃)₂, and –NHCH(CH₃)₂ in NIPAM structural units, respectively; at 2.14 and 1.65 ppm attributable to –CH–CH₂– and –CH–CH₂– in main repeating units, respectively. Three weak shift signals at 3.72, 3.49, and 2.63 ppm originate from –SCH₂CH₂NH₂, –SCH₂CH₂NH₂, and –NH₂ end group, respectively. By contrast, the star polymer in Figure 2(c) displays almost the same ¹H NMR spectra as those of the arm, and the corresponding shift signals at 2.86, 1.07, 3.93, 6.45, 2.08, and 1.59 ppm, as described above. Considering high molecular weight of the resultant polymers and overlapping, the shift signals from –SCH₂CH₂NH– cannot be readily perceived. Whereas the shift signals of the benzene ring protons in Figure 2(c) are found to appear at 8.80 ppm, but fairly weak, compared with the isopropyl proton shift signals, also as shown in the inset in Figure 2(c). The difference of 0.05 ppm is reasonable within a margin of error. All these corroborate the synthesis of starlike s-P(NIPAM-co-DMAM) polymers. The NIPAM and DMAM composition ratios in copolymers were further determined by estimating the integral area ratios of the methyl proton signal at 2.90 ppm assigned to –N(CH₃)₂ in PDMAM to the shift signal at 1.14 or 3.99 ppm ascribed to –NHCH(CH₃)₂ or –NHCH(CH₃)₂ in PNIPAM. For comparison, the [NIPAM]:[DMAM] composition ratios in the copolymers are also theoretically calculated from the mole fraction copolymerization equation

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Figure 2.

¹H NMR spectra of (a) triethyl 1,3,5-benzenetricarboxylate core, (b) P(NIPAM-co-DMAM)-NH₂ arm, and (c) s-P(NIPAM-co-DMAM) star polymer.

GPC was employed to measure the apparent number-average molecular weight (M_n), weight-average molecular weight (M_w), and polydispersity indexes (PDI), and the results are tabulated in Table 1. It is clearly noticed that the molecular weight is decreased with increasing the mass ratios of DMAM to NIPAM and molar ratios of chain transfer agents to monomers. In the meanwhile, it is found that the M_n values of the star polymers are less than three times those of the linear analogues of the same molar mass, as expected, because of their small radius of gyration and hydrodynamic volume. The PDI values, especially in the case of the precursors (P(NIPAM-co-DMAM)-NH₂ arms), are relatively high, and in the range from 1.35 to 1.80, which is characteristic of the free radical polymerization mechanism and arm-first method. It is interesting to notice that the PDI decreases from the arm to the corresponding star, which is in good agreement with the star formation. The low PDI of the arm (1.5–1.8) for a radical polymerization may be ascribed to the reasonably control over the process parameters, for example, a stationary reaction temperature. In this process, there may be no automatic acceleration process, no branching and cross-linking reaction resulting from the transfer reaction to macromolecules. Especially, the chain transfer agent plays an important role in modulating the molecular weight. Figure 3 further depicts GPC traces of the three-armed starlike s-P(NIPAM-co-DMAM) polymers. It is clear that almost monomodal and symmetrical elution peaks appear, and there is no shoulder peak, no tailing in higher molecular weight regions for the four kinds of star copolymers. Slight tailing in lower molecular weight areas signifies that a little amount of remnants of one-arm or two-arm copolymers, even free arms are detected. The wide distribution of arm numbers is due to steric hindrance, as expected. By the combination of FT-IR and ¹H NMR results, it can be inferred that the three-armed starlike s-P(NIPAM-co-DMAM) copolymers are synthesized in our work.

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Figure 3.

GPC traces of the synthesized star polymers: (a) Star-1, (b) Star-2, (c) Star-3, (d) Star-4, and (e) P(NIPAM-co-DMAM)-NH₂ arm: Arm-2.

Self-assembly micellization of s-P(NIPAM-co-DMAM) copolymers

The homopolymers and (amphiphilic) random copolymers are capable of forming micelles, as has been reported previously.^31–33 Inspired by the concept, the copolymers consisting of PNIPAM and DMAM repeating units are synthesized and anticipated to self-assemble into micelle aggregates by the aid of the strong interchain interactions at a suitable concentration. Considering that the reactivity ratios of PNIPAM and DMAM are less than 1 (r_NIPAM = 0.82 and r_DMAM = 0.977), the random copolymers are reasonably formed in the reaction. It was reported that the block copolymers containing PNIPAM were once observed to form the copolymers nanoparticles at 26℃–26.5℃. ³⁴ The phase transition of the grafted PNIPAM layer starts at pretty low temperatures of ca. 15℃–20℃ owing to the strong interchain interactions. ³⁵ Therefore, we assume that the PNIPAM short chains in the random copolymers tend to moderately shrink during the micellization at about 15℃–27℃, exhibiting slight hydrophobicity. Given that the resulting random copolymers contain a very small hydrophobic TBTC molecule at its inner core, thermosensitive PNIPAM, and hydrophilic PDMAM, certain selective polar solvents such as water can trigger aggregation of the copolymers, forming core-shell micelle aggregates with the hydrophobic TBTC and slight hydrophobic PNIPAM as a core, and hydrophilic PDMAM as a shell. However, since these chains are very short based on the unique structural feature of the random copolymers, the core-shell structure of the random copolymer micelle aggregates may not have clear boundaries like general block copolymer micelles. It is thus inferred that the star s-P(NIPAM-co-DMAM) random copolymers constructed by the certain hydrophobic PNIPAM and the hydrophilic PDMAM may form micelle aggregates with an irregular core-shell structure through hydrophobic interactions among the isopropyl groups of PNIPAM chains and inter-star association above some polymer concentration, where the hydrophobic TBTC and slight hydrophobic PNIPAM construct an imprecise or loose core, and the hydrophilic PDMAM short chains act as an outer shell. In the self-assembly micelle aggregates, the hydrophilic PDMAM parts do their best to be exposed to the bulk solvent (water), and the hydrophobic TBTC and the PNIPAM with certain hydrophobicity at room temperature of about 15℃–27℃ are tucked in the interior of an assembly. The typical diagram of formation of random polymer micelle aggregates is illustrated in Scheme 2. In the sketch, PNIPAM short chains emerge in the form of linear chains at the ends or looped chains between two PDMAM short chains. Likewise, PDMAM short chains also present linear chains at the ends or looped chains between two PNIPAM short chains.

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Scheme 2.

Scheme illustrating formation of polymer micelles and the loading and release process of drug.

To investigate the micellization of s-P(NIPAM-co-DMAM) copolymers and decide the critical concentration producing micellization, surface tension technique was used to determine the CAC values of the starlike s-P(NIPAM-co-DMAM) polymers, as illustrated in Figure 4(a). It is clear that the surface tension is almost unchangeable when the polymer concentration is below 1 mg L⁻¹ and abruptly decreases as the concentration surpasses the value; and finally tends to be stable, hinting the formation of micelle aggregates. The CAC value is taken from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the point at the highest concentration and is calculated to be as low as 4.24 mg L⁻¹. Similarly, the CAC values of other prepared star polymer micelle aggregates are estimated in the same method and summarized in Table 2. It is noticed that the CAC values of these polymer micelle aggregates are in the range from 4.06 to 6.55 mg L⁻¹, far less than the CAC values of linear PNIPAM polymer micelle aggregates with identical or comparable molecular weight.^36,37 This signifies that the polymer micelle aggregates are considerably stable even in an infinite dilution aqueous phase and can easily be used in the systematic circulation through intravenous injection into a large volume of blood. Further investigations disclose that the CAC values increase with increasing DMAM mass ratios in that the incorporation of stronger hydrophilic DMAM chains than NIPAM chains leads to increase in solubility, and thus the formation of the micelle aggregates is restrained. By comparing Star-3 with Star-4, it is found that the CAC values increase with increasing the molar ratios of chain transfer agents to monomers. This suggests that longer arms or higher molecular weights of the arms are favor of formation of the micelle aggregates due to decreased hydrophilicity.

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Figure 4.

Surface tension dependence on logarithmic concentrations at room temperature (a) and UV–vis transmittance change with temperature (b), (the concentration of Star-2: 250 mg L⁻¹), of representative s-P(NIPAM-co-DMAM) polymer micelle.

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Table 2.

Physicochemical properties of the resultant star polymer micelle aggregates.

Codes	CAC (mg L−1)	CP (℃) a	Dh (nm)b
Star-1	4.06	32.3	191.76
Star-2	4.24	36.6	148.14
Star-3	5.31	44.7	137.49
Star-4	5.01	41.0	136.55

CAC: critical aggregate concentration; CP: cloud point.

a

Polymer concentration is 250 mg L⁻¹.

^bDetermined by DLS at 25℃ and the polymer concentration is 250 mg L⁻¹.

Thermo-induced micellization behavior

PNIPAM homopolymers and amphiphilic block copolymers containing PNIPAM blocks have so far been widely used in biomedical fields as drug target release carriers. However, in most cases, their low thermosensitive phase transition temperature in aqueous solution (CP or LCST is less than 37℃) leads to the early leakage of the drug encapsulated in polymer micelles during blood circulation in the body. To eliminate the defect and keep most payloads in the drug-loaded micelles under the condition of the normal physiological temperature, it is necessary to modulate the CP or the phase transition temperature to a desired temperature by copolymerizing a more hydrophilic comonomer with various proportions.^27,28,38 Under this case, once circulating to tumor tissues where the environmental temperature is above the CP, the PNIPAM shell layers of these micelles will become hydrophobic, and the thermosensitive polymer micelles are destabilized and undergo a coil-to-globule transition in aqueous media, delivering the encapsulated drug at target sites. ³⁹ Conversely, the structure of the polymer micelles is stable below the CP, and the drug loaded in the core is not released. Of course, the early leakage of payloads from polymer micelles is not simply tackled by modulating the CP of the polymers but also prevented by mediating the CAC values, etc. When the micelles solutions are diluted below CAC, polymer micelles are gradually disintegrated. ⁴⁰ A lower CAC signifies slower dissociation behavior, allowing the retention of loaded drugs for a longer duration, ⁴¹ as stated before.

In this study, hydrophilic DMAM monomers were incorporated into the star-like polymers, and the thermo-triggered volume phase transition was investigated by UV–vis transmittance change with temperature. The UV–vis transmittance versus temperature curve of representative Star-2 micelle aggregates is depicted in Figure 4(b). The CP value of the Star-2 micelle aggregate can be estimated by a 50% decrease in transmittance in the transmittance–temperature plot and is about 36.6℃, very close to the nominal body temperature. Therefore, when this kind of polymer micelle aggregates is adopted to entrap and delivery hydrophobic drug, the premature leakage of drug can be avoided and further targeted drug release can be attained by the microenvironmental temperature change at lesion sites, tissues, or cells. The CP results of other star polymers with various monomer compositional ratios are tabulated in Table 2. The CP values are obviously found to depend on the amount of the chain transfer agents and the feed ratios of NIPAM to DMAM. With increasing the mass ratios of hydrophilic DMAM components from Star-1 to Star-2 to Star-3, the hydrophilicity of the star polymers is enhanced, and then the hydrophobic phase separation produces at higher temperature and the CP values increase. This trend corresponds to the decreased molecular weights of the star polymers. Low chain transfer agents result in high molecular weights (comparing Star-3 with Star-4), especially high PNIPAM molecular weight or arm length (comparing Arm-3 with Arm-4), and thus the CP values decrease. Considering that the starlike PNIPAM micelle (Star-1) has almost identical CP value to the linear PNIPAM homopolymer one, ⁴² it is therefore inferred that the CP values do not change with geometrical morphology or architecture but can be tailored by hydrophilic/hydrophobic copolymer compositions and/or molecular weights and chain length, as expected.

Morphologies and size distribution

The morphology of representative copolymer micelle aggregates (Star-4) in aqueous solution at room temperature was observed by TEM, as demonstrated in Figure 5. It is obvious from Figure 5(a) that the micelles are almost spherical in shape and have an average diameter of 51 nm. However, a relatively broad size distribution between 35 and 110 nm is found in the case of the Star-4 polymer micelle aggregates. The morphology of the star polymer micelle aggregates in PBS solution of pH 7.4 at 37℃ was also visualized to investigate whether the micelle aggregates would be disassociated or would maintain their stability, as shown in Figure 5(b). No perceptible change in morphology and size is seen, and the mean diameter is about 52 nm. Thus, it is inferred that the polymer micelle aggregates should be stable in the PBS solution of pH 7.4 at 37℃, which lays a foundation for further biomedical applications. To determine the size and size distribution of the prepared star polymer micelle aggregates, DLS measurements were carried out, and the results are shown in Table 2. The hydrodynamic diameters (D_h) of the copolymers micelle aggregates are typically below 150 nm, which is in favor of drug target release applications. The polymer micelle aggregates with this size level can avoid glomerular filtration in the kidneys. Meanwhile, small particle sizes of the polymer micelle aggregates make for the retention and accumulation of drug at tumor tissues and thus passive targeting can achieved at tumor tissues because the tumor blood vessels has enhanced permeability and retention.^43,44 The thermo-triggered D_h alterations are further discussed in Figure 6. At room temperature (<CP), the D_h values of the copolymer micelle aggregates are in a broad size range from 67 to 265 nm centered at 133 nm. As the medium temperature is heated above CP (50℃), the D_h value is obviously decreased, with a size range from 22 to 116 nm centered at 50 nm, and the particle size distribution also obviously narrows down. This finding is in contrast to the results from other researcher.^34,45 In their investigations, the size was found to increase above the CP of the polymers due to increased hydrophobic interactions of the polymer chains. On the contrary, there are also scholars who found the same results as our work, where no temperature-induced aggregation generated and the decreased size above CP is believed to be ascribed to the shrinkage or collapse of the thermosensitive polymer segments as shells.^46–48 When the temperature is below the CP, the arms of the star polymers in aqueous solution are in a stretched state because the arms are water-soluble based on the hydrogen bonding interactions between the –CONH– groups in the arms and water molecules,^49,50 and thus the polymer molecular chains have stronger mobility. Hence, the micelles have larger particle size and wider size distribution. In contrast, the hydrophobicity of PNIPAM segments is enhanced above the CP value. As a result, the interaction between P(NIPAM-co-DMAM) arms and water molecules recedes while the hydrophobic interaction between polymers is enhanced. The arms of the star polymers are in a contraction state, and the mobility of the arms abates in solution, which leads to the decreased size of the micelle aggregates and narrower particle size distribution.

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Figure 5.

TEM images of Star-4 copolymer micelle aggregates with the concentration of 250 mg L⁻¹ in various conditions of preparation: (a) aqueous solution at room temperature and (b) PBS solution of pH 7.4 at 37℃.

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Figure 6.

Histograms of particle size and size distribution of representative Star-4 micelle aggregates measured by DLS at 25℃ (<CP) and 50℃ (>CP). Polymer concentration is 250 mg L⁻¹.

Thermo-triggered PTX release behavior

PTX is an important water-insoluble anticancer drug and has broad-spectrum anticancer potency. On the other hand, the copolymers may form the micelle aggregates with an irregular or loose core-shell structure through hydrophobic interactions and inter-star association,^31,33 as stated before. Therefore, PTX should predominantly be encapsulated in the core formed by the hydrophobic TBTC and thermosensitive PNIPAM through hydrophobic interactions between polymers and drug, where the polymer moieties possess high affinities for specific PTX drugs and retain them. ⁸ Strong drug–polymer interactions can decrease the micelle’s hydrophilicity and cause the micellar structure to collapse, irreversibly entrapping the drug molecules within the shrunken structure. Of course, it cannot be ruled out that some PTX may be entrapped through the hydrogen bonding between functional groups (–CONH–) in polymer moieties including PNIPAM and PDMAM and drug molecules, and a few in the small hydrophobic TBTC molecules. Their large surface area allows bioconjugation among micelle moieties and drug. The PTX loading in the star copolymer micelle aggregates is illustrated in Scheme 2. To confirm the feasibility of the star random copolymer micelle aggregates as novel drug targeted release carriers, PTX was loaded into the copolymer micelle aggregates to demonstrate the sustained drug release. The LC values of the PTX-loaded “Star-2” and “Star-4” micelle aggregates are 14.5% and 15.8%, and the EE values are about 45.7% and 51.3%, respectively.

Furthermore, thermo-triggered PTX release behavior from “Star-2” and “Star-4” copolymer micelle aggregates was investigated, as depicted in Figure 7. It is clear that the release rate of PTX from the “Star-4” micelle aggregate is markedly faster at temperature of 43℃ than at 37℃. After 12 h, the drug release amount reaches to about 12.9% and 64.5% at 37℃ (<CP) and 43℃ (>CP), respectively. After 72 h, the release amount is about 22.8% and 90.8% at 37℃ (<CP) and 43℃ (>CP), respectively. Since pH values vary in normal tissues (pH 7.4) and cancer cells (pH 4.5–6.5), the drug release behavior of the “Star-4” micelles is investigated at pH 5.6 and the above two temperatures. Our findings show that the PTX-loaded copolymer micelle aggregates remain more swift release rate at 43℃ than at 37℃ in PBS solution of pH 5.6. This conclusion is in agreement with the thermo-triggered PTX release behavior at pH 7.4. Moreover, it is found that the variation of pH values does not significantly influence the drug release behavior of the micelle aggregates as there is no pH-sensitive group or component. Nevertheless, it is interesting to notice that the PTX-loaded copolymer micelle aggregates only respond to the temperature change at pH either 7.4 or 5.6. Therefore, it is inferred that the micelle-based drug formulation can quickly deliver most of the entrapped drug in cancer cells (43℃ and pH 5.6), and meanwhile avoid premature leakage of PTX in normal tissues (37℃ and pH 7.4) and reduce harm to normal cells, achieving targeted therapy effect. This thermo-induced PTX release is reported to be correlated with temperature-induced structural alterations of copolymer micelle aggregates or the conformation alterations of polymer chains, especially the PNIPAM chains.^{39,42,46–48,51} Below CP, PNIPAM chains are at a relatively extended state albeit they slightly shrink into the micelle shell, and thus the drug is delivered only by diffusion, resulting in a low release amount. Above CP, the copolymers produce contraction and aggregation, undergoing a coil-to-globule transition. The sharp conformation transition leads to fast drug release. Of course, the temperature effect is also responsible for the release in that increased temperature makes for diffusion. The drug release process triggered by temperature is illustrated in Scheme 2. Dong et al. ²² once well modulated the uptake and release of sophoridine by the reversibly opening and closing of PNIPAM layers at various temperatures and accordingly achieving the controlled and targeted release of drugs. The “Star-2” copolymer micelle aggregate produces remarkably faster drug release than “Star-4” at 37℃ in that the release temperature is slightly higher than its CP value. Therefore, the “Star-4” copolymer micelle aggregate is more applicable for the specific-targeting controlled release, which can be attained by mediating the compositional ratios of the two monomers.

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Figure 7.

Thermo-triggered in vitro PTX release profiles from PTX-loaded “Star-2” (a) 25℃ and (d) 37℃ in PBS of pH 7.4 and “Star-4” copolymer micelle aggregates in various PBS solutions of pH 7.4 and 5.6 (b) 37℃; pH 5.6, (c) 37℃; pH 7.4, (e) 43℃; pH 5.6, and (f) 43℃; pH 7.4.

Cytotoxicity evaluation

The cytotoxicity of the blank “Star-4” copolymer micelle aggregates against L929 and therapeutic potency or bioactivity of the PTX-loaded “Star-4” copolymer micelle aggregates toward MDA-MB231 human breast cancer cell lines were detected, respectively, as shown in Figure 8. In the case of the blank “Star-4” copolymer micelle aggregate (Figure 8(a)), cell viabilities are detected to be more than 92% in a high concentration range up to 1000 mg L⁻¹, and thus the copolymer micelle aggregate itself is nontoxic at normal body temperature. The PTX-loaded “Star-4” copolymer micelle aggregates with various concentrations (Figure 8(b)) exhibit no detectable cytotoxicities at normal physiological temperature of 36.3℃–37.2℃ (<CP 41℃), whereas at 43℃ (>CP or at cancer sites) decreased cell viabilities produce, reflecting thermo-triggered drug toxicity of the “Star-4” micelle. This property is equivalent to or even slightly superior to bioactivity or therapeutic potency of free PTX against MDA-MB231 cells. The enhanced cytotoxicity at 43℃ is due to a high cumulative drug release amount from the PTX-loaded “Star-4” copolymer micelle aggregates after 48 h, resulting in an enhanced intracellular drug dose. Drug uptake by cells is also responsible for this result.^52,53 Active interaction between the collapsing PNIPAM chains above the CP and cells would produce high drug uptake by cells, and thus higher cell cytotoxicity emerges. The cytotoxic activity of the copolymer micelle aggregates matches well with micelle structural changes and PTX release from the corresponding micelle aggregate. It is worth noting that although cells are incubated at 43℃, the slightly high temperature does not affect the biology of the cells and the experimental results, which has been justified by investigating the cytotoxicity of the blank “Star-4” copolymer micelle aggregate against L929 cells after 48 h culture at 43℃ (Figure 8(a)). The results disclose that there is no significant difference in cytotoxicity, and more than 90% cells survive. Guo et al. ⁴⁶ reported that the low hyperthermia could not kill the cells at low concentrations of DOX. Therefore, it is inferred that the high temperature does not affect the biology of the cells in a short time, and the conclusion of the thermo-triggered cytotoxicity is reliable. In fact, the temperature at tumor issues is generally higher than that at normal cells, and it does not further kill more cancer cells. Consequently, the prepared copolymer micelle formulations can effectively reduce damage to normal cells and attain the desired antitumor activities and the targeted therapy effectiveness.

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Figure 8.

Cytotoxicities of blank micelle aggregate against L929 cells and PTX-loaded “Star-4” copolymer micelle aggregates (the concentration: 1–1000 mg L⁻¹; PTX concentration: 0.2–187.6 µg ml⁻¹) as well as free PTX against MDA-MB231 human breast cancer cell lines after incubation at 37℃ (<CP) and 43℃ (>CP).