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Feb 2, 2015 - 844 wileyonlinelibrary.com. pH-Responsive Reversible PEGylation Improves Performance of Antineoplastic Agent. Diankui Sun , Jianxun Ding ...
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pH-Responsive Reversible PEGylation Improves Performance of Antineoplastic Agent Diankui Sun, Jianxun Ding,* Chunsheng Xiao, Jinjin Chen, Xiuli Zhuang, and Xuesi Chen solve these limitations of chemotherapy drugs.[3–5] Among them, PEGylation is commonly used to increase the solubility, stability, and biocompatibility.[6] Poly(ethylene glycol) (PEG) exhibits nontoxicity, nonimmunogenicity, favorable water-solubility, and excellent biocompatibility.[2] These favorable properties now make PEG play an important role in drug delivery system. In the past few decades, PEG has been widely conjugated onto various peptides/proteins in order to increase the resistance to proteolysis, reduce immunogenicity, and supply an approach to adjust pharmacokinetics.[7] Nowadays, the PEGylation of antitumor drugs, such as doxorubicin (DOX), camptothecin (CPT), and paclitaxel (PTX), has been studied to improve the overall performance similarly as the peptide/protein drugs.[1,6] The smart drug delivery systems, which can be triggered by the unique intratumoral and/or intracellular microenvironments, are one of the most promising strategies to enhance the selectivities and control the release behaviors of various antineoplastic agents.[8,9] The specific stimuli includes enzyme,[10,11] redox,[12–14] [ 15–18 ] pH, and so on. In particular, pH-responsiveness is one of the most important signals because of the existential pH difference between normal physiological condition (pH ≈ 7.4) and the extracellular compartment in solid tumor (pH ≈ 6.8)[19] or the intracellular endosome of tumor cell (pH ≈ 5.5).[20] So far, based on the intratumoral or intracellular acidic condition, the orthoester,[12] acetal,[21] amide,[22] Schiff base,[23] and hydrazine bonds[17,20] have been designed to fabricate the pHresponsive drug delivery systems. Apparently, as mentioned above, the PEGylated drugs based on above pH-responsive linkages exhibit the following advantages: 1) prolonged circulation time in blood, 2) enhanced accumulation in tumor tissue, 3) increased cellular uptake, and 4) promoted intracellular drug release, to improve the therapeutic efficacy and downregulate the side effects.[24,25] Therefore, a promising drug delivery system integrating both the advantages of PEGylation and pHsensitivity can be expected to greatly improve the properties of antineoplastic agents. Aiming at this goal, a pH-responsive PEGylated antitumor drug was designed and prepared in this work. In detail, PEG

The reversible PEGylation endows antitumor drugs with various fascinating advantages, including prolonged circulation time in blood, enhanced accumulation in tumor tissue, increased cellular uptake, and promoted intracellular drug release, to improve the therapeutic efficacy and security. Here, the obtained succinic anhydride (SA)-functionalized DOX (SAD) (i.e., insensitive succinic anhydride-functionalized doxorubicin (DOX)) and aconitic anhydride (CA)-modified DOX (CAD) (i.e., acid-sensitive cis-aconitic anhydride-modified DOX) are conjugated to the terminal of poly(ethylene glycol) (PEG) yielding the unresponsive SAD-PEG-SAD and pH-responsive CAD-PEG-CAD prodrugs, respectively. The prepared prodrugs can self-assemble into micelles in aqueous solution. Both micelles are sufficiently stable at normal physiological pH (i.e., 7.4), while CAD-PEG-CAD micelle gradually swells and finally disassembles at intratumoral (i.e., 6.8) and especially endosomal pHs (i.e., 5.5). DOX release from CAD-PEG-CAD at pH 7.4 is efficiently inhibited, whereas it is significantly accelerated by the rapid cleavage of amide bond at pH 5.5. In addition, CAD-PEG-CAD exhibits more efficient cellular uptake and potent cytotoxicity in vitro, as well as improved tissue distribution and superior tumor suppression in vivo than free DOX and SAD-PEG-SAD. More importantly, the PEGylated DOX exhibits favorable security in vivo. In brief, the smart CAD-PEG-CAD with enhanced antitumor efficacy and decreased side effects shows as a promising powerful platform for the clinical chemotherapy of malignancy.

1. Introduction The traditional chemotherapeutics in clinic are limited by the bad solubility and stability, rapid blood clearance, nonspecific selectivity, low accumulation in tumor tissue, poor bioavailability, and serious side effect.[1,2] Currently, the advanced nanotechnology has been one of the powerful approaches to

D. Sun, Dr. J. Ding, Dr. C. Xiao, J. Chen, Prof. X. Zhuang, Prof. X. Chen Key Laboratory of Polymer Ecomaterials Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun 130022, P. R. China E-mail: [email protected] J. Chen University of Chinese Academy of Sciences Beijing 100049, P. R. China

DOI: 10.1002/adhm.201400736

Adv. Healthcare Mater. 2015, DOI: 10.1002/adhm.201400736

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Scheme 1. Schematic illustration for self-assembly of pH-responsive CAD-PEG-CAD, and its in vivo circulation, accumulation in tumor tissue, and final pH-triggered intracellular DOX release after intravenous injection.

was conjugated to the insensitive succinic anhydride (SA)functionalized DOX (noted as SAD) and acid-sensitive cis-aconitic anhydride (CA)-modified DOX (referred as CAD) yielding the unresponsive SAD-PEG-SAD and pH-responsive CADPEG-CAD for comparative research. In relative to SAD-PEGSAD, CAD-PEG-CAD exhibited acid-induced size expansion and even disassembly, acid-triggered DOX release, enhanced cellular uptake and cytotoxicity in vitro, and improved accumulation in tumor tissue and upregulated antitumor efficacy with excellent security in vivo (Scheme 1). Therefore, the exploited acid-sensitive PEGylated DOX exhibited great potential for the chemotherapy of malignancy in clinic.

2. Results and Discussion 2.1. Syntheses and Characterizations of PEGylated DOX As shown in Schemes S1 and S2, Supporting Information, the PEGylated DOX, that is, insensitive SAD-PEG-SAD and acid-sensitive CAD-PEG-CAD were synthesized by the sequential ring-opening and condensation reactions. Briefly, SAD or CAD was firstly synthesized through the ring-opening reaction between SA or CA and DOX with triethylamine as catalyst, respectively.[26] Second, SAD-PEG-SAD or CAD-PEG-CAD were, respectively, synthesized through the condensation reaction between SAD or CAD and PEG with 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) as condensing agent and 4-N,N-dimethylaminopyridine (DMAP) as catalyst.

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The chemical structures of SAD-PEG-SAD and CADPEG-CAD were firstly confirmed by proton nuclear magnetic resonance (1H NMR). As shown in Figure 1A, the peaks that appeared at 2.61 ppm (f; –C(O)CH2CH2C(O)–) and 2.21 ppm (e; –C(O)CH2CH2C(O)–) were assigned to the methylene protons in SAD. The signal at 6.18 ppm (d) belonged to the methylidyne proton in CAD (–C(O)CH=C(C(O))CH2–). The peak at 3.50 ppm (g; –CH2C(O)–) was assigned to the methyl protons in PEG. The appearance of peaks in the spectra of prodrugs at 7.95 (a and b) and 7.72 ppm (c) indicated the successful conjugation of SAD or CAD to PEG. The chemical structures of prodrugs were further confirmed by Fourier-transform infrared (FT-IR) spectra shown in Figure 1B. The absorption signals that appeared at 1663 cm−1 (vC=O) and 1550 cm−1 (v–CO–NH–) in both SAD and CAD were assigned to the stretching vibrations of amide bond and carbonyl group, respectively. In addition to the above two signals, the characteristic peak at 1070 cm−1 (vC–O–C) attributed to the stretching vibration of the ether bond in PEG, which further demonstrated the chemical structures of both SAD-PEG-SAD and CAD-PEG-CAD. In this study, the amphiphilic SAD-PEG-SAD and CADPEG-CAD could self-assemble into micelles in phosphate-buffered saline (PBS), which were prepared by a direct dissolution method. As depicted in Figure 2A, 2B, and S1 (Supporting Information), the hydrodynamic radii (Rhs) of SAD-PEG-SAD and CAD-PEG-CAD micelles in PBS at pH 7.4 were monitored to be 51.2 ± 8.7 and 48.5 ± 8.8 nm, respectively, by dynamic light scattering (DLS) measurements. The transmission electron microscope (TEM) images revealed that both SAD-PEG-SAD and

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Adv. Healthcare Mater. 2015, DOI: 10.1002/adhm.201400736

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7.4. It proved that the two prodrug micelles exhibited excellent stability in neutral condition. More interestingly, the remarkable swelling and even disassembly of pH-responsive CAD-PEGCAD micelle were observed at both pH 6.8 and 5.5 in contrast to the unresponsive SAD-PEG-SAD micelle. In detail, the results proved that CAD-PEG-CAD micelle swelled from 52.2 ± 7 to 410 ± 40 nm as time was extended to 60 h at pH 6.8, and no light scattering signal associated with the particle size can be detected at 72 h. Compared with that at pH 6.8, the evolution of micellar scale was faster at pH 5.5. The Rh of CAD-PEG-CAD micelle at pH 5.5 was 58.7 ± 12 nm at the start (i.e., 0 h), while it quickly grew to 491 ± 2.3 nm at 48 h. After that, no relevant signal was detected. The unresponsive SAD-PEG-SAD platform was kept Rh of 50 to 60 nm in all the test conditions within 72 h. In addition, the morphologies of SAD-PEG-SAD and CADPEG-CAD at different pHs (i.e., 7.4, 6.8, and 5.5) and time points (i.e., 0, 6, and 12 h) were monitored by TEM (Figure 2C). It indicated that CAD-PEG-CAD micelle remained relatively stable spherical morphology at pH 7.4 within 12 h, which was similar to those detected for SAD-PEG-SAD micelle at all the test pHs and intervals. Fascinatingly, the mophology of CADPEG-CAD was observed to be expandable, and even broken and aggregated under acidic conditions (i.e., pH 6.8 and 5.5) at 6 and 12 h, responsively. The results were corresponding to those of the DLS detections. All the above data proved that both micelles exhibited excellent stability in normal physiological condition and indicated that the acid-sensitive CAD-PEG-CAD micelle experienced rapid expansion and rupture in the acidic intratumoral and intracellular microenvironments.

2.2. In Vitro DOX Release

Figure 1. A) 1H NMR (in deuterated dimethyl sulfoxide, i.e., DMSO-d6) and B) FT-IR spectra of DOX, SAD, CAD, SAD-PEG-SAD, and CAD-PEG-CAD.

CAD-PEG-CAD micelles showed clear spherical structures in Figure 2C at pH 7.4. The apparent diameters of SAD-PEG-SAD and CAD-PEG-CAD micelles from TEM measurements were around 90 and 80 nm, respectively. In comparison, the sizes measured by TEM were smaller than those from DLS due to the swelling of micelles in aqueous condition for DLS analyses. The stability and/or pH-responsiveness of SAD-PEG-SAD and CAD-PEG-CAD micelles were evaluated by the change of Rhs in PBS at pH 7.4, 6.8, and 5.5, mimicking the conditions corresponding to the pH of normal physiological environment, the extracellular pH of tumor tissue, and the pH in endosome, respectively, with the extension of time from 1 to 72 h. As shown in Figure 2A, B, and Supplementary S1 (Supporting Information), no remarkable size change was detected for both SAD-PEG-SAD and CAD-PEG-CAD micelles at pH

Adv. Healthcare Mater. 2015, DOI: 10.1002/adhm.201400736

In this work, the in vitro release behaviors of DOX from SADPEG-SAD and CAD-PEG-CAD were investigated in PBS at pH 7.4, 6.8, and 5.5. As shown in Figure 3, the cumulative release percentages of DOX from SAD-PEG-SAD and CAD-PEG-CAD versus time were plotted. It showed that the released DOX was few for both two prodrugs in the test duration at pH 7.4 (