Enhanced drug resistance suppression by serum-stable micelles from multi-arm amphiphilic block copolymers and tocopheryl polyethylene glycol 1000 succinate

Abstract

Aim: To develop a robust drug-delivery system using multi-arm amphiphilic block copolymers for enhanced efficacy in cancer therapy.

Materials & methods: Two series of amphiphilic polymer micelles, PEG-b-PCL_m and PEG-b-PCL_m/TPGS, were synthesized. Doxorubicin (DOX) loading into the micelles was achieved via solvent dialysis.

Results: The micelles displayed excellent biocompatibility, narrow size distribution, and uniform morphology. DOX-loaded micelles exhibited enhanced antitumor efficacy and increased drug accumulation at tumor sites compared with free DOX. Additionally, 4A-PEG₄₇-b-PCL₂₁/TPGS micelles effectively suppressed drug-resistant MCF-7/ADR cells.

Conclusion: This study introduces a novel micelle formulation with exceptional serum stability and efficacy against drug resistance, promising for cancer therapy. It highlights innovative strategies for refining clinical translation and ensuring sustained efficacy and safety in vivo.

Plain language summary

Article highlights.

• Multi-arm amphiphilic block copolymer synthesis: prepared various multi-arm amphiphilic block star copolymers with different numbers of arms and molecular weights. Coupled with carboxyl-terminated monomethoxy poly(ethylene glycol) to form PCL-b-PEG₄₇ block copolymers.

• Micelle formation and size: assembled micelles from multi-arm block copolymers (2A-25/47, 4A-m/47, and 6A-21/47) through dialysis. Controlled particle size (50–500 nm) based on PCL length and arm number, influencing drug release and loading.

• Drug-loading capacity (DLC): Investigated DLC of DOX-loaded micelles, showing higher values for four-arm copolymers with higher N_PEG/N_PCL ratio. Four-arm copolymer micelles demonstrated higher DLC than two-arm or six-arm counterparts.

• Micelle stability: determined critical micelle concentration (CMC) values, indicating low concentrations for enhanced stability. Evaluated serum protein stability using dynamic laser light scattering (DLLS), demonstrating distinct particle sizes.

• Biocompatibility assessment: confirmed good biocompatibility of synthesized block copolymers through cell toxicity experiments on breast cancer cells (MCF-7).

• In vitro antitumor assay: demonstrated effective antitumor activity against both MCF-7 and MCF-7/ADR cells using DOX-loaded TPGS mixed micelles. Revealed the reversal of drug resistance in tumor cells, enhancing therapeutic efficacy.

• Cellular uptake of DOX: investigated cellular uptake of DOX, showing improved endocytosis in doxorubicin-resistant breast cancer cells with TPGS-containing micelles.

• Drug resistance suppression mechanism: explored the mechanism of drug-resistance suppression, highlighting enhanced apoptosis and decreased P-glycoprotein expression with TPGS.

• In vivo distribution and antitumor profiles: evaluated in vivo tumor accumulation of micelles using BALB/c nude mice, demonstrating higher FITC accumulation in tumors. Conducted in vivo antitumor assay, indicating remarkable efficacy in chemotherapy for drug-resistant tumors.

• Therapeutic potential: established the therapeutic potential of DOX-micelle-TPGS, significantly impeding further tumor growth and maintaining tumor volume within a specific range.

1. Background

Chemotherapy, a widely employed treatment for clinical cancer [1–3], is associated with various side effects that restrict its clinical utility [3–5]. For instance, most chemotherapeutic drugs, such as doxorubicin (DOX) and paclitaxel, are small hydrophobic molecules exhibiting poor stability in plasma and rapid clearance by the reticuloendothelial system (RES) in vivo [5]. Fortunately, the utilization of nanoparticles (NPs) as carriers for drug delivery presents an alternative approach featuring tumor-specific targeting ability, enhanced permeability retention (EPR) effects, and reduced toxicity [5–7]. This strategy enables increased intratumor drug concentration and improved therapeutic efficacy of diverse nanoformulations in clinical or preclinical trials [8]. Micelles, liposomes, and prodrug NPs represent common vectors utilized in drug-delivery systems [9–14]. Among these systems, biodegradable polymeric micelles (PMs), incorporating specific therapeutic agents that actively or passively target cancer cells/microenvironments, emerge as optimal carriers for delivering hydrophobic chemotherapeutic drugs [15–17]. These biodegradable polymer micelles self-assemble through amphiphilic block copolymers to encapsulate drugs within their hydrophobic core while shielding them from first-pass metabolism. Simultaneously, the hydrophilic shell stabilizes the micelle structure and enhances aqueous solubility [18].

Polymer micelles, composed of US FDA-approved polymers such as polyethylene glycol (PEG) and aliphatic biodegradable polyester (PCL, PLA, etc.) [11], have been specifically engineered to target cancer cells/microenvironments [18]. Notably, clinical trials have already commenced for micelles loaded with potent anticancer agents like NK911 and Genexol-PM. However, it is intractable that the micelle structure is comparatively unstable in vivo owing to the complicated hemato-microenvironment. These micelle constructs, before being delivered to targets, easily fall apart into fragments and are sensitive to increased ionic strength, resulting in premature drug release [19]. For example, due to the more complex environment (high blood viscosity, biomacromolecule interaction, and altered physiological conditions) in vivo, micelles might undergo structural deformation and/or disassociation, followed by premature drug release after administration into the human body. An ideal drug-delivery system should be stable and reliable; that is, the drug should not leak during delivery, which means that therapeutic toxicity is generated at the tumor site, with intratumor drug accumulation as high as possible. As a result, many efforts have been made to stabilize these constructs by bridging polyanion PMs to polycation molecules or using dendrimers, such as PAMAM [20]. Meanwhile, star-shaped polymers with a certain number of arms extending from a single point are also good candidates [21], and their structural stability, drug loading capability (DLC), solution viscosity, and encapsulation efficiency (EE) are rather excellent [22–24]. Moreover, a deeper understanding of the relationships between compositions [25,26], the number of arms, and amphiphilic block length ratios plays an important role in promoting the clinical application of micelle-based formulations.

Refractory drug resistance, however, remains a formidable challenge to be surmounted [27]. The expression of P-glycoprotein on the cell surface is considered to account for cancer's multiple drug resistance [28] and is an ATP-dependent transporter that can transport hydrophobic small molecules and exogenous substances to the extracellular space with the consumption of ATP [29,30]. Many carriers have been used, including siRNA-loaded micelles [31], TPGS mixed micelles and drug-loaded PCL-b-TPGS nanoparticles, etc. [32,33]. Among these, binding to TPGS has been widely reported as a good strategy. TPGS can inhibit P-gp to improve drug permeability through cell membranes, thus enhancing drug absorption and reducing multidrug resistance (MDR) [34]. Consequently, it is a novel strategy to apply star-shaped polymers that bind TPGS as prospective nanocarrier preparation materials to reverse drug resistance.

In this study, we synthesized a series of multi-arm amphiphilic block copolymers composed of polyethylene glycol (PEG) and polycaprolactone (PCL) with varying chain lengths. Our investigation focused on elucidating the intricate relationship between composition, structure, and the efficacy of enhanced drug therapy in nanomedicine's in vivo antitumor effects. Through meticulous adjustment of the hydrophobic/hydrophilic block length ratio, we achieved enhanced micellar serum stability, facilitating heightened DOX endocytosis. Notably, micelles with shorter PCL chain lengths demonstrated robust drug-loading capacity (DLC) and encapsulation efficiency (EE). The introduction of TPGS proved to be pivotal in overcoming drug resistance in MCF-7/ADR cancer cells, attributed to its disruption of cellular surface P-glycoproteins and suppression of DOX pumping. This led to a significant enhancement in serum stability and potent suppression of drug resistance, ultimately resulting in improved in vivo tumor growth. Our findings underscore the importance of tailoring the composition and structure of micellar formulations to enhance their in vivo performance, particularly in the context of drug-resistant chemotherapy scenarios. This work presents a novel strategy for the development of nanomedicines for clinical cancer treatment, offering promising avenues for future research in the field.

2. Materials & methods

2.1. Materials

ϵ-Caprolactone (CL) was purchased from Sigma-Aldrich (MO, USA), stirred with CaH₂ to remove water, and vacuum-distilled after 24 h. Poly(ethylene glycol) methyl ether (mPEG, Mn = 550, 750 and 2000 g/mol) was purchased from Sigma-Aldrich. Tocopheryl Polyethylene Glycol 1000 Succinate (TPGS) was provided by the Jiangsu University Hospital Lab (Jiangsu University Hospital, Jiangsu, China). Doxorubicin hydrochloride (DOX) was used as received. Acetonitrile, acetone, sodium phosphotungstate, stannous octoate (Sn(Oct)₂, Sigma-Aldrich, MO, USA), N,N-dimethylacetamide (DMAC) and triethanolamine (TEA) were of analytical grade and were used without further purification. Water was purified using a Milli-Q Synthesis A10 system (Millipore, MA, USA), with a resistivity of w18.2 MU cm. For the in vitro cell culture, Dulbecco's modified Eagle's medium (DMEM; Sigma, USA), fetal bovine serum (FBS; Gibco, NY, USA), and Cell Counting Kit-8 were used as received. Dipentaerythritol, diisopropylcarbodiimide (DIC) and 4-(dimethylamino)pyridine were purchased from Sigma-Aldrich and used as received.

2.1.1. Synthesis of amphiphilic single molecular multi-arm block copolymers

The synthetic equations for the star-shaped block copolymers are shown in Figure 1. Herein, the 4-arm copolymer 4a (PEG-b-PCL_m) is used as an example to illustrate the detailed process.

Figure 1.

Synthetic route of the na (PCL_m-b-PEG₄₇) copolymers. (A) Synthesis of carboxyl-terminated monomethoxy poly(ethylene glycol) (mPEG-COOH). (B) Synthesis of na-PCL_m. (C) Synthesis of amphiphilic na (PCL_m-b-PEG₄₇) copolymers with varying numbers of arms.

2.1.2. Synthesis of carboxyl-terminated monomethoxy poly(ethylene glycol) (mPEG-COOH)

mPEG (Mn = 2000 g/mol, 30 g, 15 mmol) was dissolved in dry toluene (150 ml) for azeotropic distillation for 12 h at 150°C to remove any traces of water. Maleic anhydride (14.7 g, 150 mmol) was then added upon cooling to 75°C. The reaction mixture was stirred for 24 h at 75°C, dropped into an excess of cold diethyl ether, and filtered. The obtained product (mPEG-COOH) was dried under vacuum at 25°C to a constant weight (yield = 94%).

2.1.3. Synthesis of 4-arm hydrophobic poly (ϵ-caprolactone) (4a-PCL_m)

The 4a-PCL_m was synthesized via the ring-opening polymerization (ROP) of CL at various monomer/initiator (M/I) ratios using Sn(Oct)₂ as a catalyst. A typical ROP procedure was conducted as follows: pentaerythritol (0.065 g, 0.5 mmol), CL (4.56 g, 40 mmol) and dry toluene (8 ml) were added to an oven-dried sealed tube under nitrogen atmosphere, followed by the addition of Sn(Oct)₂ (0.04 mmol, (Sn(Oct)₂)/(CL) = 0.001). The reaction mixture was stirred for 24 h at 80°C and subsequently dissolved in a small amount of methylene chloride after cooling to room temperature. It was then poured into excess cold petroleum ether and filtered to remove impurities. The crude product was further purified by dissolving it in methylene chloride and precipitating it thrice with cold methanol. Finally, the white powder was collected after drying under vacuum at room temperature for 48 h with a yield of 94%.

2.1.4. The coupling reaction of 4a-PCL_m with mPEG-COOH

A Schlenk tube (100 ml) was charged with 4a-PCL_m, mPEG-COOH, disopropylcarbodiimide (DIC) and 4-(dimethylamino)pyridine (DMAP) ((-OH):(-COOH):(DIC):(DMAP) = 1:5:1:10), followed with the addition of anhydrous dichloromethane under argon. The reaction mixture was stirred for 48 h at 30°C, precipitated in cold petroleum ether, filtered, and dried to a constant weight under vacuum at room temperature. Subsequently, the obtained product was dissolved in distilled water and stirred for 5 h. Finally, the resultant product was obtained using centrifuge separation and vacuum drying.

2.1.5. Synthesis of amphiphilic na (PCL_m-b-PEG₄₇) copolymers

na-PCL_m was synthesized by ROP of CL with different initiators (EG or TMP) following a previously described procedure. The execution of the experimental procedure here is almost the same as above, with the only difference being that 6a-PCL₂₁ was obtained from DPE by bulk polymerization of CL at 80°C. The coupling reaction of na-PCL and mPEG-COOH was performed to obtain na (PCL_m-b-PEG₄₇) using the approach presented above.

2.1.6. Preparation of micelles & DOX-loaded TPGS micelles

The amphiphilic block polymers and TPGS (w/w = 1:1) were weighed to approximately 4 mg in total and dissolved in 1 ml of DMAC. DOX was also dissolved in DMAC at a concentration of 4 mg/ml, followed by a dropwise addition of 1.5 molar equivalents of TEA solution under stirring for approximately 5 min and then tenfold dilution with DMAC. The mixture was then mixed with the same volume of polymer solution and stirred for 2 min. The mixture was dialyzed against PBS in the dark at room temperature for 1 d, and the DOX-loaded TPGS mixed micelles were collected and stored for further use. The blank and DOX-loaded micelles were fabricated in a similar manner.

2.2. Methods

2.2.1. Size & size distribution

Hydrodynamic diameters were determined by dynamic light scattering (DLS) analysis using a ZetaSizer (Nano-ZS, Malvern Instruments, Worcestershire, UK) equipped with a He-Ne laser (633 nm) at a scattering angle of 173°. Stock micelle solutions were diluted with MilliQ-H₂O to 0.1 mg/ml and filtrated with 0.22 μm Millipore filters (Millipore, MA, USA) of characterization. The Z-average diameter of the micelles was calculated by the instrument using cumulant analysis.

2.2.2. Morphology characterization

Samples for transmission electron microscopy (TEM) measurements (H-7000 Electron Microscope, Hitachi, Tokyo, Japan) were prepared by depositing 1 ml of micelle solution (0.5 mg/ml) on 200-mesh Formvar-free carbon-coated copper grids (Ted Pella Type-A; nominal carbon thickness of 200 nm), followed by air drying at room temperature. They were then negatively stained with a 2% hydrodated phosphotungstate (PTA) solution. The TEM images were recorded using a JEOL2100F microscope operating at an accelerating voltage of 100 kV.

2.2.3. ¹H nuclear magnetic resonance spectroscopy

¹H (300 MHz) nuclear magnetic resonance (NMR) spectra were recorded on a Varian Mercury 300 spectrometer and a Varian 400 NMR spectrometer at room temperature, using acetone-d or chloroform-d as the solvent and tetramethylsilane as the internal standard. The number-average molecular weights (Mn) of both polymers were calculated based on the integration areas of the methylene protons at 4.07 p.p.m. from PCL, methylene protons at 3.65 p.p.m. from PEG, and methylene protons at 4.14 p.p.m. from the initiator. The CL/EG molar ratio, which balances the hydrophobic and hydrophilic segments, was estimated from the peak area of PCL against PEG.

2.2.4. Gel permeation chromatography

Gel permeation chromatography (GPC) was used to calculate the relative molecular weight and molecular weight distribution, equipped with a Waters 1515 Isocratic High-Performance Liquid Chromatography (HPLC) pump, a Waters 2414 refractive index detector, and a set of Waters Styragel columns (7.8 × 300 mm, 5 mm bead size; 103, 104 and 105 Å pore size). GPC measurements were performed using chloroform as the eluent at a flow rate of 1.0 ml/min and were calibrated using a polystyrene standard. All air- or moisture-sensitive operations were performed under a nitrogen atmosphere using Schlenk techniques or in a glove box. Mn, Mw, and dispersity (Đ) (PDI = Mw/Mn) values were obtained using Agilent Technologies PL-GPC 40 chromatography with one mixed B column and two mixed D columns at 35°C.

2.2.5. Kinetic stability

Bovine serum albumin (BSA) was used to investigate the stability of the micelles. The interaction between the blank micelles and BSA was examined using DLS. The size and size distribution of micelles (different chain units self-assembled in aqueous solution) with a concentration of ∼0.5 mg/ml and a BSA solution with a concentration of ∼5.0 mg/ml in saline were measured. The micelles (∼0.5 mg/ml in water) were mixed with BSA (with C ∼5.0 mg/ml in saline) under stirring for approximately 5 min to form a mixture solution to obtain their size and size distribution.

2.2.6. Thermodynamic stability

The critical micelle concentration (CMC) was determined using fluorescence spectrometry with pyrene as a probe. A fixed pyrene concentration of 6.0 × 10^-7 M in acetone solution was employed. The copolymers were prepared at concentrations ranging from 10^-4 to 4 mg/ml. Subsequently, 1 ml of the pyrene solution in acetone was mixed with 1 μl of polymer aqueous dispersions at various concentrations. Fluorescence measurements were conducted with an excitation wavelength of 374 nm, and emissions were monitored within the range of 300–360 nm. The CMC value was determined by analyzing the relationship between the intensity ratio (I₁/I₃) in the excitation spectrum and the logarithm of polymer concentration, identifying it as the intersection point between the tangent at the inflection point and that at low concentration.

2.2.7. Drug-loading capacity

High-performance liquid chromatography (HPLC) instrument (Shimadzu SCL-10A, Shimadzu, Kyoto, Japan) equipped with a plus autosampler (ShimadzuSIL-10A, Shimadzu) and UV detector (Shimadzu SPD-10A, Shimadzu) was used to determine the DOX concentration in the micelles. Chromatographic separation was performed on a reversed-phase C18 column (4.6 mm × 250 mm, 5 μm; Fluophase PFP, Thermo Fisher Scientific, MA, USA). The eluent consisted of acetonitrile and water (50/50, v/v) and was maintained at a flow rate of 1 ml/min. The detection wavelength was 227 nm, and the injection volume was 20 μl. An amount of 5 μl of 100 μg/ml DOX-acetonitrile solution was used as the internal standard for calibration (correlation coefficient, R² = 0.996). The limit of quantification was 0.3 mg/ml, and coefficients of variation (CV) were all within 4.3%. The micelles were dissolved in acetonitrile and vortexed vigorously to obtain a clear solution. Finally, the drug-loading capacity (DLC) and encapsulation efficiency (EE) of DOX-loaded micelles were calculated using the following equations:

$$\mathrm{DLC} (\%)=\frac{\text{Weight of}\mathrm{DOX} \text{in the micelles}}{\text{Total Weight of micelles}}\times 100\%$$

$$\mathrm{EE} (\%)=\frac{\text{Weight of}\mathrm{DOX} \text{in the micelles}}{\text{Initial Weight of}\mathrm{DOX} \text{used}}\times 100\%$$

2.2.8. Cell culture

The human breast cancer cell line (MCF-7) and doxorubicin-resistant derivative cell line (MCF-7/ADR) were purchased from the American Type Culture Collection (ATCC, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco BRL, NY, USA), 50 units/ml penicillin, and 50 mg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. To maintain the resistant phenotype of MCF-7/ADR cells, cells were incubated in DMEM/FBS with 1.0 mg/ml doxorubicin. The cells were precultured overnight until 75% confluency was reached before the experiments.

2.2.9. Cellular internalization evaluation

Flow cytometry (FM): Two groups of cells (MCF-7 and MCF-7/ADR) were seeded in 24-well microplates (0.5 ml/well, 2.0 × 10⁵ cells/ml) and cultured overnight to reach 70% confluence. The cells were treated with free DOX, DOX-loaded micelles, and DOX-loaded TPGS-mixed micelle solutions for approximately 12 h, gently rinsed twice with DMEM/FBS, and digested with trypsin for approximately 3 min. Subsequently, the cells were rinsed with PBS and centrifuged twice at 800 r.p.m. for 5 min. Flow cytometry was used to characterize the fluorescence intensity of free DOX, DOX-loaded micelles, DOX-loaded TPGS mixed micelles, and untreated cells (negative control).

Confocal laser scanning microscopy (CLSM): Prior to analysis, two groups of cells (MCF-7 and MCF-7/ADR) were seeded in special dishes (0.5, 2 × 10⁵ cells/ml). The following day, cells were incubated with free DOX, DOX-loaded micelles, and DOX-loaded TPGS mixed micelles for approximately 12 h, then rinsed with DMEM/FBS and fixed with 4% paraformaldehyde. Cell nuclei were stained with 2.0 μg/ml DAPI, and images were obtained using a TCS SP confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany).

2.2.10. Cellular level cytotoxicity evaluation

MCF-7 and MCF-7/ADR cells were seeded in 96-well transparent plates (100 μl/well, 4.0 × 10⁴ cells/ml) and incubated overnight at 37°C to prepare for cytotoxicity assessment. The medium was then removed and replaced with free DOX in DMEM/FBS, DOX-loaded micelles, or DOX-loaded TPGS mixed micelles with different polymer concentrations at equivalent drug concentrations or different drug concentrations at the equivalent polymer concentration. A Kit-8 assay was used to confirm cell viability at a given time. The absorbance of the samples in each well was recorded using a microplate reader at 450 nm. The percentage of surviving cells was calculated using Equation 1:

$$\mathrm{Surviving} \mathrm{cells} (\%)=\frac{O{D}_{\mathrm{sample}}-O{D}_{\mathrm{control}}}{O{D}_{\mathrm{negative}}-O{D}_{\mathrm{control}}}\times 100\%$$ (1)

where OD_sample, OD_control and OD_Neg are the absorption at 450 nm from cells incubated with micelles, the culture medium and without micelles, respectively.

2.2.11. Apoptosis evoking

MCF-7/ADR cells were seeded in 6-well plates at 3.0 × 10⁵ cells/well in 2.0 ml medium. After 24 h, the cells were treated with free DOX, DOX-loaded micelles, DOX-loaded TPGS mixed micelle solution (2.0 mg/ml), or equivalent blank micelles for 24 h. MCF-7/ADR cells were stained using the Annexin V-PI Apoptosis Detection Kit (Invitrogen, MA, USA) according to the manufacturer's protocol. Thereafter, the samples were analyzed using the FACSCa-Libur System (BD Biosciences, CA, USA). Untreated cells were used as negative controls.

2.2.12. Western blotting

MCF-7 and MCF-7/ADR cells were treated with free DOX, blank micelles, DOX-loaded micelles, or DOX-loaded TPGS micelles for approximately 24 h. The cells were then harvested, washed and lysed in cell lysis buffer for Western blotting. Cellular lysates were resolved using SDS-PAGE and immunoblotted with anti-P-glycoprotein antibodies (Cell Signaling Technology, MA, USA).

2.2.13. ATP enzyme level testing

An ATP assay was performed as described by the literature [35]. Cells were seeded in 48-well plates at a density of 2.0 × 10⁵ cells/well and incubated overnight. Various doses of the test particles in EBSS buffer were added to the cells and incubated for 2 h at 37°C. Cells were then washed twice with ice-cold PBS and lysed with PBS containing 1% Triton X-100 for 30 min at 37°C. ATP levels in cell lysates were measured using an ATPlite 1step^® Assay Kit (PerkinElmer, MA, USA) and normalized to protein content.

2.2.14. Animal studies

All experiments were carried out using BALB/c nude mice (female, 4–6 weeks old, ∼20 g) purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). BALB/c nude mice were maintained in a pathogen-free environment and acclimated for at least 1 week before tumor implantation.

All animal studies were performed in accordance with the Regulations for Care and Use of Laboratory Animals and Guideline for Ethical Review of Animals (China, GB/T 35892-2018). All animal procedures were reviewed and ethically approved by the Shanghai General Hospital Medical Ethics Committee (2020SQ159-3).

2.2.15. Tumor implantation

Subcutaneous inoculation of 1.0 × 10⁷ MCF-7/ADR cells (in 100 μl culture medium) was performed on the right dorsal region of BalbBALB/c nude mice to induce xenograft tumor formation. The volume of the tumors reached approximately 50 mm³ after approximately 2 weeks. A successful tumor xenograft model was demonstrated using luminescent tumor images. Mice were intraperitoneally injected with 150 mg/kg luciferin (Promega, WI, USA). The tumor xenograft model was viewed using the IVIS^® Lumina II Imaging System (Xenogen, CA, USA), which was used to capture visible-light and luminescent images. Immunofluorescence revealed high EGFR expression in MCF-7/ADR tumor tissues, indicating that the tumor was successfully xenografted in vivo.

2.2.16. In vivo distribution

In the in vivo distribution study, mice bearing MCF-7/ADR tumors were randomly and evenly divided into four groups with three mice/group. An equivalent dose of 5 mg/kg micelles loaded with fluorescein isothiocyanate (FITC) was administered via the tail vein. After 24 h, the mice were anesthetized with 1.5% isoflurane in 1:2 O₂/N₂. Mice administered with FITC were used as controls. Mice injected with PBS were used as the background. The in vivo images were observed using the IVIS^® imaging system (excitation, 500 nm) and recorded using a built-in CCD camera. Mice were sacrificed after 24 h and excised to obtain hearts, livers, kidneys, tumors and spleens, which were also imaged using the same excitation wavelength.

2.2.17. Antitumor activity assay

Tumor implantation was performed as described in the section ‘Tumor implantation’. The mice were randomly and evenly divided into four groups of five mice/group (for the control, free DOX, DOX-loaded micelles and DOX-loaded TPGS mixed micelles). Subsequently, 100 μl of saline, free DOX in PBS, DOX-loaded micelles, and DOX-loaded TPGS mixed micelle solution (5 mg/kg per mouse) were injected into the tail vein three times at 3-day intervals. Tumor size was recorded every 2 days using a digital Vernier caliper. The tumor volume (V_Tumor) was calculated according to (Equation 2), where L and W are the longest and shortest diameters, respectively. Tumor progression was evaluated in terms of relative tumor volume (on Day 0) over 14 days. On day 14, at the pharmacodynamic end point, mice were anesthetized by inhaling carbon dioxide followed by cervical dislocation to ensure humane and rapid euthanasia.

$$V_{\mathrm{Tumor}}=\frac{L\times W^2}{2}$$ (2)

2.2.18. Statistical analysis

Data were analyzed using an independent t-test for comparisons between two groups and one-way ANOVA followed by a suitable post hoc test for comparisons among more than two groups. A significant difference was considered when p < 0.05. Results are presented as mean ± SD (standard deviation).

3. Results

3.1. Synthesis & fabrication of multi-arm amphiphilic block polymeric micelles

Several multi-arm amphiphilic block star copolymers were prepared in this study (Figure 1). Star-shaped copolymers with varying numbers of PCL arms and molecular weights were synthesized (2a-PCL₂₅, 4a-PCL_m and 6a-PCL₂₁). These were then coupled with carboxyl-terminated monomethoxy poly(ethylene glycol), which comprised 47 repeating units, to form the PCL_m-b-PEG₄₇ block copolymers (Figure 1). GPC testing confirmed a uniform distribution of PCL with low Đ, which then shifts uniformly to a lower elution time after the coupling reaction (suppl & suppl), and the coupling efficiency was validated by a shift to a lower elution time and confirmed by ¹H NMR spectra (suppl). Micelles assembled from multi-arm block copolymers (2A-25/47:2a [PCL₂₅-b-PEG₄₇], 4A-m/47:4a [PCL_m-b-PEG₄₇] and 6A-21/47:6a [PCL₂₁-b-PEG₄₇]) were prepared by dialysis. DOX-loaded micelles and DOX-loaded TPGS mixed micelles were prepared by incorporating DOX and TPGS, respectively, into the micelles.

As shown in Figure 2, the average hydrodynamic diameters of blank micelles, DOX-loaded micelles, and DOX-loaded TPGS mixed micelles were in the range of 50–500 nm (Figure 2A & B & suppl). Comparing the micelle sizes of the four types of four-arm block polymers, the sizes of DOX-loaded micelles increased with the shortening of PCL length (Figure 2C). However, the number of arms had little influence on micelle size (Figure 2D). The drug-loading capacity (DLC) of DOX-loaded micelles and DOX-loaded TPGS mixed micelles was also investigated and showed similar trends to their sizes (Figure 2E & suppl). After drug loading, the potential of the 4A-186/47 + TPGS was measured to be -20.38 mV, indicating a notable decrease compared with the pre-drug loading potential of 1.06 mV (suppl). Furthermore, the DLC value of micelles from the four-arm polymer was higher than that of micelles from the two-arm or six-arm copolymers (Figure 2F).

Figure 2.

Synthesis and fabrication of multi-arm amphiphilic block polymeric micelles: The correlation between arm lengths and particle sizes (A) and the schematic representation of the relationship between arm numbers and particle sizes (B) are presented. Particle sizes of micelles assembled from (C) 4-arm block copolymers with different N_PEG/N_PCL ratios and (D) block copolymers with different arms and the same N_PEG/N_PCL ratios. Drug-loading capacity of DOX-loaded and DOX-loaded TPGS mixed micelles assembled from (E) 4-arm block copolymers with different N_PEG/N_PCL ratios and (F) block copolymers with different arms and the same N_PEG/N_PCL ratios. (G) Representative transmission electron microscopy (TEM) images of 4A-23/47 + TPGS, 4A-46/47 + TPGS, 4A-93/47 + TPGS and 4A-186/47 + TPGS. (H) Cytotoxicity of blank micelles at different concentrations (0.01, 0.05, 0.1, 0.25 and 0.5 mg/ml) after 48-h incubation in MCF-7 cells. Data are expressed as mean ± standard deviation (n = 3).

DOX: Doxorubicin. 

Transmission electron microscopy (TEM) images of 4A-23/47 + TPGS, 4A-46/47 + TPGS, 4A-93/47 + TPGS and 4A-186/47 + TPGS showed spherical morphology (Figure 2G). The hydrophilic shell increases the stability of micelles and prolongs the cycle time in the body, and this spherical formulation may promote cellular or tumor uptake. To verify the biocompatibility of the synthesized block copolymers, different concentrations (0.01, 0.05, 0.1, 0.25 and 0.5 mg/ml) of samples were used to carry out cell toxicity experiments in breast cancer cells (MCF-7). The cell viability remained over 85% in all cases after 48 h, even with polymer concentrations of up to 0.5 mg/ml (Figure 2H).

3.2. In vitro micelle stability

The critical micelle concentration (CMC) of amphiphilic block copolymers is an important parameter for describing the physicochemical properties of the polymer assemblies. CMC values were evaluated by fluorescence excitation spectroscopy using pyrene as a hydrophobic spectroscopic probe. Figure 3A shows the relationship between the intensity ratio (I1/I3) of the excitation spectra and the logarithm of the polymer concentration. CMC values were obtained from the intersection of lines drawn through the points of flat regions at low concentrations and steeply increasing regions at high concentrations, which fell in the range of 0.6–5.8 mg/l. The CMC values for the star-shaped block copolymer (six arms) were lower than those of their linear counterpart (two arms) (Figure 3A). The reduced CMC was beneficial for stability when diluted in the blood as a drug carrier.

Figure 3.

In vitro micelle stability. Determination of the critical micelle concentration (CMC) of 6A-21/47, 4A-23/47 and 2A-25/47 (A). The serum protein stability evaluation by dynamic light scattering (DLS) of (B) serum protein, (C) 6A-21/47 micelle in serum protein and (D) 2A-25/47 micelle in serum protein.

BSA: Bovine serum albumin.

The in vitro stability profiles of the two- and six-arm micelles were evaluated (Figure 3B–D) using dynamic laser light scattering (DLLS). The size of serum proteins, such as BSA, was approximately 30 nm (Figure 3B), and the sizes of both the two- and six-arm micelles were in the range of 100–300 nm (Figure 3C & D).

The four-arm micelles, characterized by a well-balanced molecular structure, moderate CMC concentration and optimal particle size, exhibit an ideal combination of low CMC concentration for enhanced stability and suitable particle size for efficient biological interactions. Therefore, 4A-23/47 was selected for subsequent in vivo and in vitro biological experiments.

3.3. In vitro antitumor assay

MCF-7 and MCF-7/ADR cells were used to construct a tumor model to further evaluate their in vitro antitumor ability. First, MCF-7 and MCF-7/ADR cells were treated with free DOX, DOX-loaded micelles, or DOX-loaded TPGS mixed micelles for 48 h at the same doxorubicin concentration. The results show that the viability of sensitive cells in different treatments was clearly reduced to approximately 20% of their initial viability, and different treatment methods seem to have little influence on the results (suppl). However, more than 60% of MCF-7/ADR cells exhibit viability even after exposure to free DOX or DOX-loaded micelles, indicating that the cytotoxicity may vary based on the length of PCL. Importantly, these resistant cells showed a significant decline in viability after treatment with TPGS-DOX-loaded micelles (Figure 4).

Figure 4.

In vitro antitumor assay: The in vitro cell viability of MCF-7/ADR cells treated with 4-arm block copolymers with different N_PEG/N_PCL ratios (A) without TPGS, (C) with TPGS and block copolymers with different arms and the same N_PEG/N_PCL ratios (B) without TPGS/(D) with TPGS after 48 h of incubation with the nanoparticles at 0.5 μg/ml adriamycin. Data are expressed as mean ± standard deviation (n = 3).

DOX: Doxorubicin.

3.4. Cellular uptake of DOX

To study the cellular uptake of different groups (free DOX, DOX-loaded micelles, and DOX-loaded TPGS mixed micelles), a four-arm block copolymer (4A-23/47) was selected for the study, and confocal laser scanning microscopy was used (Figure 5A). As MDR is a problem in cancer therapy that can cause reduced drug uptake and unsatisfactory therapeutic effects, the evaluation was conducted in both doxorubicin-sensitive breast cancer cells (MCF-7) and doxorubicin-resistant breast cancer cells (MCF-7/ADR). Although the internalization of DOX-loaded micelles and DOX-loaded TPGS mixed micelles in MCF-7 cells was lower than that of free DOX, they exhibited high cellular uptake in MCF-7/ADR cells, particularly DOX-loaded TPGS mixed micelles. To further illustrate the different endocytosis of micelles in DOX-sensitive/DOX-resistant cells, flow cytometry was used, and the results are the same as those obtained using confocal laser scanning microscopy (Figure 5B & C).

Figure 5.

Cellular uptake of doxorubicin. (A) Confocal laser scanning microscopy (CLSM) images of micelle (4A-23/47) internalization in MCF-7 cells and MCF-7/ADR cells after 12 h, respectively. (B) Count of micelles (4A-23/47) by endocytosis in MCF-7 cells and MCF-7/ADR cells using flow cytometry. (C) Mean fluorescence intensity of micelles (4A-23/47) in MCF-7 cells and MCF-7/ADR cells. Statistical analysis was performed by unpaired t-test (**p < 0.01).

DOX: Doxorubicin.

3.5. Drug resistance suppression mechanism

DOX-TPGS-micelles intensified the apoptotic effect, whereas cells underwent inconspicuous apoptosis after treatment with blank micelles, free DOX, and DOX-loaded micelles, regardless of the arm number and PCL length (suppl). TPGS significantly enhanced the proapoptotic efficacy of doxorubicin. According to previous studies, chemotherapeutic resistance in tumor cells is mainly determined by the high expression of surface P-glycoprotein. Hence, P-glycoprotein expression was evaluated in DOX-loaded micelles with and without TPGS, which revealed a decreased expression of P-glycoprotein in DOX-loaded TPGS mixed micelles (Figure 6A). It can be predicted that the P-glycoprotein is remarkably disrupted by TPGS. Generally, such disruption of P-glycoprotein leads to a decrease in ATP inside cells due to the decrease in the ATP enzyme level as a result of the reduction in ATP-dependent P-glycoprotein transference. This was successfully evaluated by measuring fluorescence intensity, as shown in Figure 6B.

Figure 6.

In vivo distribution and antitumor profiles. (A) Western blot and (B) ATP analysis for P-glycoprotein expression in resistant cells treated with DOX, DOX-loaded micelle, and DOX-loaded TPGS mixed micelle; the nontreated cells serve as controls. (C) Fluorescence image of mice and (D) ex vivo fluorescence image of the excised organs of mice at 24 h after treatment with DOX, DOX-loaded micelle, and DOX-loaded TPGS mixed micelle. (E) Time-dependent tumor growth curves of different groups of mice under various treatments (DOX, DOX-loaded micelle and DOX-micelle-TPGS) in vivo. The data demonstrate the effective antitumor responses of micelle-TPGS-DOX. Micelle: 4A-23/47. Data are presented as the mean ± SD (n ≥ 3). Statistical significance was calculated using Student's t-test.

**p < 0.01, ****p < 0.0001.

DOX: Doxorubicin; PBS: Phosphate buffered saline. 

3.6. In vivo distribution & antitumor profiles

To investigate the potent in vivo antitumor effects, the biodistribution of DOX-micelle-TPGS was evaluated using classic BALB/c nude mice bearing MCF-7/ADR breast tumors. An equivalent dose of 5 mg/kg micelles loaded with fluorescein isothiocyanate (FITC) was administered via the tail vein. After 24 h, the mice and their corresponding organs, such as the heart, liver, tumor, spleen and kidney, were observed using the IVIS^® imaging system at 500 nm (Figure 6C & suppl). The higher accumulation of FITC in tumors in both nude mice and tumor tissues (Figure 6D) might be attributed to the higher cellular uptake by P-glycoprotein suppression, as illustrated above.

An in vivo antitumor assay was used to evaluate the therapeutic potential of micelles in Balb/c nude mice bearing breast cancer (MCF-7/ADR cells) (suppl). DOX-loaded TPGS mixed micelles were formulated, and tumor implantation was performed according to the procedures in Materials and Methods. When the tumor grew to approximately 50 mm³, the mice were randomized into four groups (PBS, DOX, DOX-micelle and DOX-micelle-TPGS) for iv. administration at a dose of approximately 5 mg/kg. The tumor volumes of the different groups were recorded daily. As shown in Figure 6E, the tumor volume in the control (PBS) group rapidly increased within 14 days, reaching the largest size in these groups. In addition, the tumor volumes of the DOX-micelle group were smaller than those of the DOX group, indicating the enhanced in vivo toxicity of DOX by the micelles (suppl). Importantly, the tumor growth of DOX-micelle-TPGS remains largely unaltered, indicating that our treatment significantly impedes further tumor growth and effectively maintains the tumor volume within a specific range. The volume of this group represents the lowest therapeutic index among all dosage forms.

4. Discussion

The synthesis and characterization of multi-arm amphiphilic block star copolymers in this study lay the groundwork for the development of effective drug-delivery systems. Specifically, the multi-arm amphiphilic block star copolymer PCL_m-b-PEG₄₇ with varying PCL arms (Figure 1) and molecular weight was successfully synthesized, employing a series of characterization techniques, including GPC and ¹H-NMR (suppl).

The physicochemical characterization of multi-arm amphiphilic block polymeric micelles has provided valuable insights into their efficacy as drug-delivery systems. First, the observed size range of the micelles demonstrates interesting characteristics influenced by different arms and PCL lengths (Figure 2A & B). Interestingly, DOX-loaded micelles from four-arm polymers exhibit larger sizes compared with those from two-arm or six-arm copolymers, indicating that it is the PCL length rather than the number of arms that determines physical size (Figure 2C & D). Second, regarding drug-loading capacity, micelles assembled from four-arm copolymers with a higher N_PEG/N_PCL ratio display the highest DLC. This phenomenon can be attributed to the larger size of these micelles associated with a higher N_PEG/N_PCL ratio, enabling more efficient drug loading (Figure 2E).

The CMC and serum stability are crucial parameters that determine the physicochemical properties of polymer assemblies, such as amphiphilic block copolymers. In this study, the CMC values obtained were significantly lower than those of conventional low-molecular-weight amphiphiles (i.e., CMC 10 mg/l), indicating strong aggregation and entanglement of hydrophobic polymer segments. These values were comparable to those observed in clinical studies using PEG-b-PBLA micelles (5–10 mg/l) (Figure 3A). Furthermore, the similarity in CMC values with clinically studied PEG-b-PBLA micelles emphasizes the stability and potential clinical relevance of the synthesized micelles. In terms of serum stability, interactions between serum proteins and micelles can greatly influence their stability in biological environments. A key indicator of stability is the comparison of particle sizes between micelles and serum proteins, such as BSA. Notably, even after the interaction, distinct particle sizes were maintained for both BSA and micelles in our study, indicating the stability of these micelles in solution. This can be attributed to the presence of a hydrophilic shell on the micelles which not only enhances their stability but also prolongs their circulation time within the body. Additionally, due to their spherical formulation, these micelles may further promote cellular uptake or tumoral uptake leading to increased effectiveness as drug-delivery systems (Figure 3B–D). These findings highlight the potential use of these synthesized micelles for efficient and stable delivery of therapeutic agents within biological systems.

Furthermore, the delivery system containing TPGS has shown efficacy in killing both sensitive and resistant cells. The addition of TPGS significantly reversed drug resistance and enhanced the cell-killing efficacy of antitumor drugs. These findings underscore the potential of TPGS-mixed micelles as effective strategies for overcoming drug resistance and improving the therapeutic outcomes of anticancer drugs (Figure 4). Based on the aforementioned experiments, we have observed that 4A-23/47 micelles exhibit promising attributes, including a well-balanced molecular structure, moderate CMC, and optimal particle size. Due to these favorable characteristics, 4A-23/47 micelles were selected as the preferred formulation for subsequent investigations involving anti-drug-resistant tumor cells and mouse models, both in vitro and in vivo. Consequently, our choice of utilizing 4A-23/47 micelles establishes a solid foundation for further exploration of their therapeutic potential in combating drug-resistant tumors.

In vitro investigations were conducted to elucidate the mechanism of action of resistant tumor cells. The results demonstrated that DOX-loaded micelles, particularly those combined with TPGS, exhibited enhanced cellular uptake, indicating their ability to circumvent efflux transporters and enhance drug accumulation in resistant cells (Figure 5A & B). This augmented cellular uptake of DOX-loaded micelles containing TPGS, especially in doxorubicin-resistant breast cancer cells, underscores the potential of multi-arm block copolymer-assembled micelles mixed with TPGS as promising candidates for promoting doxorubicin-resistant cancer therapy. Furthermore, apoptosis experiments performed on drug-resistant cells treated with micelles mixed with TPGS showed a significant induction of apoptosis. These findings highlight the potential of these micelles to overcome chemotherapeutic resistance and improve therapeutic outcomes (Figure 5C & suppl).

The evaluation of P-glycoprotein expression in DOX-loaded micelles, with and without TPGS, revealed a significant decrease in P-glycoprotein expression in DOX-loaded TPGS mixed micelles (Figure 6A). This suggests that TPGS disrupts the function of P-glycoprotein, leading to reduced intracellular ATP levels and consequently increasing the accumulation of anticancer drugs within cells. The enhanced intracellular drug accumulation contributes to improved therapeutic efficiency, as demonstrated by fluorescence intensity measurements (Figure 6B). Furthermore, fluorescence intensity measurements of FITC in the micelle groups indicated significantly higher tumor accumulation compared with the control group, highlighting the ability of these micelles to promote in vivo tumor accumulation of small hydrophobic molecules such as FITC or DOX (Figure 6C & D & suppl). These findings further emphasize the potential efficacy of these investigated micelles as effective drug-delivery systems for cancer therapy, particularly in overcoming drug resistance mechanisms.

In vivo studies have provided further evidence of the promising antitumor effects of micelles, as demonstrated by their ability to effectively inhibit tumor growth and induce regression in mouse models of drug-resistant breast cancer (Figure 6E). The enhanced tumor accumulation and sustained release of therapeutic agents by micelles underscore their potential for targeted and efficacious cancer therapy. The observed efficacy of DOX-micelle-TPGS in chemotherapy for drug-resistant tumors highlights its promise as a viable treatment option, effectively targeting resistant tumors and promoting intratumoral drug accumulation, thus overcoming resistance mechanisms and improving therapeutic outcomes in breast cancer.

5. Conclusion

The in vivo antitumor efficiency of a nanomedicine is influenced by its stability and drug resistance, which are determined by the composition and structure of its carrier. In this study, we investigated the detailed relationship between composition, structure, and enhanced drug therapy. Two strategies were applied for formulation optimization: the use of multi-arm block copolymers and the introduction of TPGS to suppress drug resistance. Initially, micelles were prepared using star-block copolymers via fine chemical synthesis and self-assembly. In this study, we optimized micellar composition and structural stability for in vivo antitumor therapy, especially for drug-resistant chemotherapy, by varying the arm length and arm number. Finely tuning the hydrophobic/hydrophilic block length ratio with different arm numbers and arm lengths increased the serum stability of the micelles, promoting high DOX endocytosis. Micelles with short arm lengths exhibited a strong drug-loading capacity and encapsulation efficiency.

TPGS was introduced into the micelles, and the addition of TPGS effectively overcame drug resistance in MCF-7/ADR cancer cells, as demonstrated by in vitro cellular cytotoxicity and molecular-level evaluation. TPGS disrupted cellular surface P-glycoproteins, leading to decreased ATP-enzyme levels and ATP-dependent P-glycoprotein transference, effectively suppressing DOX pumping and inhibiting drug resistance. The introduction of TPGS successfully reversed drug resistance, and both enhanced serum stability and potent drug resistance suppression significantly enhanced in vivo tumor growth. Tailoring the composition and structure of micellar formulations significantly improves their in vivo performance, particularly for drug-resistant chemotherapy (Figure 7). In summary, nanomicelles assembled from multi-arm amphiphilic block copolymers with TPGS showed remarkably potent efficacy in drug-resistant tumor chemotherapy. Looking ahead, the multi-arm micelles developed here hold promise for application in nano-robots, facilitating more intelligent and accurate targeting of drug-resistant tumor cells in the future.

Figure 7.

The antitumor mechanism of micelle-TPGS-DOX.

DOX: Doxorubicin.

Funding Statement

This work was financially supported by the National Natural Science Foundation of China (Grant no. 82302118) and the Shanghai Pujiang Talent Plan (Grant no. 2021PJD080). We appreciate the support provided by the Program of Shanghai Academic Research Leader (Grant no. 22XD1404700).

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17435889.2024.2347197

Financial disclosure

This work was financially supported by the National Natural Science Foundation of China (Grant no. 82302118) and the Shanghai Pujiang Talent Plan (Grant no. 2021PJD080). We appreciate the support provided by the Program of Shanghai Academic Research Leader (Grant no. 22XD1404700).

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options, and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.