A pH-sensitive prodrug micelle self-assembled from multi-doxorubicin-tailed polyethylene glycol for cancer therapy

Abstract

A novel tetra-doxorubicin-tailed polyethylene glycol via benzoic-imine bond linkage was synthesized and self-assembled to a pH-sensitive prodrug micelle. This micelle not only effectively entered cancer cells but also quickly released doxorubicin (DOX) in tumor sites to exert anticancer activity in vitro.

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To date, various prodrugs have been designed in order to overcome the physicochemical and physiological drawbacks of parent drugs including poor water solubility, chemical instability, rapid presystemic metabolism, toxicity and local irritation, and to improve targeting of drug action.^1,2 In particular, polymer-drug conjugates based on poly(ethylene glycol) (PEG) have drawn great attention in cancer therapy due to their distinct advantages in delivery of anticancer drugs.^3,4 Polymer-drug conjugates can improve drug solubility and stability, prolong in vivo circulation time, alter the cellular internalization pathway of a drug, enhance tumor accumulation, reduce side effects, and circumvent P-glycoprotein associated multidrug resistance.^5,6 More importantly, a prodrug may be constructed for site-specific release of the active drug inside body.^7,8

Indeed, a great number of drug delivery systems including molecular prodrug and nanomedicines have been designed to release drugs in response to an appropriate stimulation such as change in pH or temperature, redox reaction, enzymatic degradation.^3,9–12 For example, utilizing the pH difference between the bloodstream (pH ∼ 7.4) and cancer tissues (pH ∼ 6.8), pH-sensitive systems have been developed to achieve tumor-targeted drug delivery.^11,13,14 Accordingly, acid-labile chemical linkages including orthoester, hydrazine, acetal, benzoic-imine, cis-aconityl bonds have been employed to conjugate the drug molecules.^15,16 In particular, the benzoic imine is a unique acid-labile covalent linkage which is fairly stable at neutral and basic pH environments but hydrolyzes at even very weak acidic conditions such as extracellular matrix of solid tumors (pH ∼ 6.5) and intracellular lysosome (pH ∼ 5.0).¹⁷ This feature makes benzoic imine a very promising candidate when designing drug delivery systems requiring extra-sensitivity to slight pH changes under physiological conditions.^12,18,19 Yet, it has been rarely used in constructing pH-sensitive prodrugs based on PEG.¹⁷ For example, due to its unfavorable hydrophobic/hydrophilic ratio, the polymer-drug conjugate of PEG with single DOX molecule has to co-assemble with another agent with higher hydrophobic/hydrophilic ratio in order to obtain nanomedicine stable enough in bloodstream.

Herein, a novel tetra-doxorubicin-tailed PEG via benzoic-imine bond linkage was synthesized and self-assembled to pH-sensitive micelle for tumor-targeted drug delivery (Scheme 1). In consideration of its hydrophobic/hydrophilic ratio superior to that of single DOX-tailed PEG, the tetra-doxorubicin-tailed PEG is expected to form a micelle without incorporating another component with high hydrophobic/hydrophilic ratio. The pH-responsive DOX release behaviours of prodrug micelle at normal physiological pH of bloodstream, extracellular pH of tumor, and lysosomal pH of cancer cells were investigated. The anticancer activity of prodrug micelle was examined in C6 and MCF-7 cells.

Scheme 1 Schematic illustration of self-assembly and the low pH-triggered DOX release from the self-assembled prodrug micelle.

The synthetic approach of prodrug, poly(ethylene glycol)-(lysine)₂-(formylbenzoic acid-doxorubicin)₄ (PEG-LL₂-(CHO-DOX)₄) denoted as PELD, is illustrated in Scheme S1.† The chemical structure of polymer was verified by ¹H NMR and FTIR analyses (Fig. S1–S9, see ESI†). As shown in Fig. S1 and S4,† the ¹H NMR spectra of PEG-LL-BOC₂ and PEG-LL₂-BOC₄ showed characteristic resonance peaks for the methylene groups of lysine units and methyl groups of BOC at 1.40–1.90 ppm. After the deprotection reaction, the characteristic peaks of BOC at 1.41 ppm completely disappeared (Fig. S3 and S5†). In the FTIR spectrum, the appearance of a characteristic absorption around 1560 cm⁻¹ further confirmed the formation of the amide linkage in the PEG-LL-BOC₂ (Fig. S2†). After PEG-LL₂ was reacted with p-formylbenzoic acid in the presence of DCC and DMAP, the signal at δ 10.10 ppm, 8.31 ppm and 8.02 ppm appeared, suggesting the presence of p-formylbenzoic acid proton and formation of the aldehyde group-terminated prepolymer. Moreover, the integral ratio of the proton resonance signal at 8.31 ppm to that at 3.65 ppm was highly close to 8 : 182, implying the prepolymer composition of tetra-aldehyde-tailed polyethylene glycol (PEG-LL₂-CHO₄) (Fig. S6†). The FTIR spectra shown in Fig. S7† also confirmed the chemical structure of PEG-LL₂-CHO₄. After the conjugation reaction, the spectrum of PEG-LL₂-CHO₄ no longer showed the characteristic absorption band of carboxyl group at 1700 cm⁻¹ but still retained the major characteristic absorption bands of carbonyl group at 1657 cm⁻¹, indicating the completion of amidation reaction. The final polymeric prodrug, PEG-LL₂-(CHO-DOX)₄, was synthesized through the reaction between aldehyde groups of PEG-LL₂-CHO₄ and amino groups of DOX. After the polymers were purified by dialysis against sodium bicarbonate solution at weak basic condition, the aldehyde peak at 10.10 ppm was not observed, while the imine proton at 8.28 ppm was clearly seen in the NMR spectrum (Fig. S8†). The formation of imine bond was evidenced in the FT-IR analysis as well. The bands at 1625 cm⁻¹, 1586 cm⁻¹, 1100 cm⁻¹ are attributed to the characteristic absorption bands of DOX and PEG-LL₂-CHO₄ (Fig. S9†). These results demonstrated that the polymeric prodrug, tetra-doxorubicin-tailed polyethylene glycol (PELD), was successfully synthesized. The DOX loaded content in PELD was up to 44% calculated based on the ¹H NMR.

Due to the higher hydrophobic/hydrophilic ratio resulting from multiple DOX conjugation, the polymeric prodrug can self-assemble into stable micelles comprising a PEG shell and a DOX core at pH 7.4. The spontaneous assembly of PELD into micelle was confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) assays. The aggregates are spherical and have an average hydrodynamic diameter of 81 nm (Fig. 1). The critical micellization concentrations (CMC) of PELD at different pH values were determined with fluorescence spectrophotometer using pyrene as a probe.^19–21 As shown in Fig. S11,† the CMC of PELD at pH 7.4 is lower than that at pH 6.5 (106.9 μg mL⁻¹ vs. 223.5 μg mL⁻¹). Most likely, the pH-sensitive benzoic imine bond hydrolyzed partially at pH 6.5, which decreased the hydrophobic/hydrophilic ratio of PELD.

Fig. 1 Particle size and TEM of PELD micelles at pH 7.4, pH 6.5 and pH 5.0, respectively.

The pH sensitivity of micelles was investigated by TEM, ¹H NMR and DLS analyses. As shown in Fig. 1, the micelle at pH 7.4 possessed an averaged particle size of 81.2 nm with a narrow distribution, and revealed no obvious change in size within a period of 4 h. However, the micelle showed significantly increased particle size averaging 162.1 nm at pH 6.5 and 233.2 nm at pH 5.0, respectively (Table 1). Moreover, when the solution was maintained at pH 5.0 for 24 h, disassembly of micelle was detected by TEM measurement, indicating instability of micelle due to the low pH-induced hydrolysis of benzoic-imine bond in the hydrophobic core.¹¹ The ¹H NMR spectra measured at different pH values further demonstrated the pH sensitivity of PEG-LL₂-(CHO-DOX)₄ micelle in D₂O (Fig. 2A). When the pH value dropped to 6.5 from 7.4, the imine bond signal at 8.5 ppm became weak (reduced to 57% of the initial integration) whereas a sharp signal at 9.8 ppm attributed to the aldehyde proton appeared, indicating breakage of some benzoic imine bonds in micelle. When the solution pH was further decreased to 5.0, the signal of the imine bond at 8.5 ppm disappeared completely while the aldehyde proton signal at 9.8 ppm was strengthened, indicating breakage of all benzoic imine bonds. Based on the ¹H NMR spectra, it was determined that the benzoic imine bonds hydrolyzed 43% at pH 6.5 and 100% at pH 5.0, respectively.

Table 1 Micellar diameter and CMC of tetra-doxorubicin-tailed polyethylene glycol at different pH values

	Diameter (nm)			CMC (μg mL^−1)
	pH 7.4	pH 6.5	pH 5.0	pH 7.4	pH 6.5
PELD micelles	81.2	162.1	233.2	106.9	223.5

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Fig. 2 (A) ¹H NMR spectra of PEG-LL₂-(CHO-DOX)₄ in D₂O at different pH values (300 MHz), (B) fluorescence spectra of prodrug micelle in aqueous solutions at different pH values, (C) in vitro DOX release profiles of prodrug micelle at 37 °C in PBS solutions of pH 7.4 (a), 6.5 (b) and pH 5.0 (c).

The pH-sensitive drug release of polymeric prodrug micelle was evaluated by measuring the fluorescence intensities of solutions. As shown in Fig. 2B, due to the aggregation of DOX molecules inside micelle core causing fluorescence quenching, the fluorescence intensity of DOX was low in solution at pH 7.4. However, at pH 6.5 and 5.0, DOX fluorescence intensity of the solutions increased significantly, indicated that DOX release occurred due to the cleavage of benzoic imine bonds. Quantitative determination of drug release showed consistent results (Fig. 2C). DOX release from micelle was very slow at neutral pH. However, it was obviously accelerated at pH 6.5. Moreover, DOX release appeared even much faster at pH 5.0. Apparently, low pH led to the cleavage of benzoic imine bonds for quick DOX release. At pH 5.0, DOX release reached 40 wt% in 1 h and 80 wt% in 8 h, respectively. In contrast, DOX release at pH 6.5 was 20 wt% in 1 h and 52 wt% in 8 h, respectively. These results indicated that the benzoic imine bonds was more susceptible to hydrolysis at pH 5.0, which was favourable for a rapid intracellular drug release due to the similar lysosomal pH value around 5.0.

Cell uptake and intracellular DOX release of the PEG-LL₂-(CHO-DOX)₄ micelle were evaluated with confocal laser scanning microscopy (CLSM) (Fig. 3). Similar results were obtained in C6 and MCF-7 cells. Effective cell uptake of micelle was observed in both cells. Moreover, red DOX fluorescence was observed in the cell nuclei just after 2 h cell incubation with the micelle. As the cell incubation time extended, more DOX migrated to the nuclei from cytoplasm. After 6 h cell incubation, strong red fluorescence of DOX was nearly only observable in nuclei. Since it is well-known that micelle was endocytosed and entrapped inside lysosomes thereafter, the above data indicate that rapid DOX release from the pH-sensitive prodrug micelle was triggered inside lysosomes (pH 5.0), which is in line with the in vitro release study in micelle solution (Fig. 2C). Obviously, the released free DOX effectively diffused out of the lysosomal compartments and finally migrated to the nuclei, which is essential for DOX to exert anticancer activity. Compared to free DOX, the prodrug micelle exhibited efficient cellular uptake and entering nuclei, but have a high maximum tolerated dose and a decrescent system toxicity.^11,22

Fig. 3 Confocal laser microscopic images of (A) C6 cell and (B) MCF-7 cell incubated with PELD micelle at different time points. Red fluorescence: DOX; blue fluorescence: nuclei stained with DAPI. The bar was 10 μm.

Finally, the cytotoxicity of prodrug micelle was determined by MTT assay in C6 and MCF-7 cells. Cells were treated for 48 h at various DOX concentrations ranging from 2.5 to 70 μg mL⁻¹. As shown in Fig. 4, for both cells, the cell viability decreased obviously as the micelle concentration increased. The IC₅₀ values of approximately 8 μg mL⁻¹ and 9.5 μg mL⁻¹ were determined for C6 cells and MCF-7 cells, respectively. At the highest investigated DOX concentration (70 μg mL⁻¹), the viabilities of C6 and MCF-7 cells further dropped to just 27.5 ± 2.7% and 18.4 ± 0.9%, respectively. These results showed the application potential of tetra-doxorubicin-tailed PEG prodrug in cancer therapy.

Fig. 4 Cytotoxicity of C6 and MCF-7 cells incubated with PELD micelle. Data were detected by MTT assay (mean ± SD; n = 3). Incubation time: 48 h.

In conclusion, a novel polymeric prodrug, tetra-doxorubicin-tailed polyethylene glycol, was successfully synthesized by conjugating PEG and doxorubicin via the highly pH-sensitive benzoic-imine bond. The benzoic-imine link was stable at neutral pH but became progressively cleavable at low pH. At pH 7.4, the polymeric prodrug self-assembled into a spherical micelle in aqueous solution, incorporating anticancer drug DOX in the core. The low pH values of tumor tissue (pH 6.8) and lysosome (pH 5.0) may trigger DOX release from the micelle. This feature makes the prodrug micelle promising in cancer therapy which requires tumor site-specific drug release for enhanced therapeutic effect and meanwhile decreased side effects. In vitro studies show that the pH-sensitive prodrug micelle may effectively enter cancer cells and then quickly release free DOX to exert anticancer activity by responding to the lysosomal low pH.

Acknowledgements

This work was supported by National Basic Research Program of China (2015CB755500), the National Natural Science Foundation of China (51225305, U1401242, 51373203 and 21174166) and Natural Science Foundation of the Guangdong Province (2014A030312018, 2013B090500094), the Guangdong Innovative and Entrepreneurial Research Team Program (2013S086).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra27293a

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