One-Step Synthesis of Targeted Acid-Labile Polysaccharide Prodrug

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One-Step Synthesis of Targeted Acid-Labile Polysaccharide Prodrug for Efficiently Intracellular Drug Delivery Di Li, Xiangru Feng, Li Chen, Jianxun Ding, and Xuesi Chen ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00856 • Publication Date (Web): 16 Dec 2017 Downloaded from http://pubs.acs.org on December 23, 2017

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ACS Biomaterials Science & Engineering

One-Step Synthesis of Targeted Acid-Labile Polysaccharide Prodrug for Efficiently Intracellular Drug Delivery Di Li,†,‡ Xiangru Feng,‡ Li Chen,*,† Jianxun Ding,*,‡ and Xuesi Chen‡

†

Department of Chemistry, Northeast Normal University, Renmin Street 5268, Changchun

130024, People's Republic of China ‡

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences, Renmin Street 5625, Changchun 130022, People's Republic of China

ACS Paragon Plus Environment

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ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: The therapeutic potential of the active targeting and acid-sensitive polysaccharide prodrug was investigated. The active targeting of polysaccharide prodrug was based on the specific interaction between cyclo(Arg-Gly-Asp-D-Phe-Lys) peptide (c(RGDfK)) and its receptor αvβ3 integrin overexpressed on the membrane of tumor cells. The cRGD-modified doxorubicin-conjugated hydroxyethyl starch (HES=DOX/cRGD) was synthesized via a one-step Schiff base reaction between oxidized HES, and DOX and c(RGDfK) that achieved an acidaccelerated drug release profile. The targeted polysaccharide prodrug self-assembled into micelle in aqueous environment with a moderate hydrodynamic diameter of 77.1 nm. All data in vitro indicated enhanced cell uptake and elevated cytotoxicity of HES=DOX/cRGD toward human malignant melanoma A375 cells compared with HES=DOX and DOX. Moreover, the smart prodrug also exhibited upregulated accumulation in the tumor, improved antitumor efficacy, and reduced systemic cytotoxicity in vivo. The cRGD-decorated acid-sensitive polysaccharide prodrug was advantageous in antitumor efficacy and excellent security, showing great prospect in clinical application.

KEYWORDS: polysaccharide, acid-labile prodrug, active targeting, controlled drug release, chemotherapy


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1. INTRODUCTION Chemotherapy is the most commonly used measure in the treatment of malignancy for the past few decades. However, long-term usage of small molecule antitumor drugs can lead to severe systematic toxicity due to free diffusion to normal organs.1 Conjugating antitumor drugs to polymers via smart chemical bonds is an efficient way to enhance the stability and solubility, as well as prolong the circulation time in the physiological environment. The covalent bonds, such as boronate,2 hydrazone bond,3 disulfide bond,4-8 imine bond,9-11 amide bond,12 esters,13 and other biologically responsive bonds, keep the conjugated prodrugs stable during extracellular transport and swiftly rupture in response to the change of microenvironment enabling the local release of drug.14-15 To be specific, the microenvironment (intracellular endosomal/lysosomal pH, cytoplasmic glutathione, etc.) of a solid tumor is different from those of normal physiological conditions.16 Chemical bonds responsive to these factors enable the local release of antitumor drugs.17-19 Since Ringsdorf proposed the polymer-conjugated prodrug model in 1975, a large variety of polymer prodrugs have been reported for improvement of cancer chemotherapy.20 With an effort to control the drug release and improve the local accumulation, Ding et al. engineered a dextran−doxorubicin (Dex−DOX) conjugate with pH-sensitive Schiff base bond through the reaction between the aldehyde group of Dex and the amino group of DOX. The release of DOX was evidently accelerated as it entered the tumor tissue (pH ~6.8), endosome (pH ~6.0), and lysosome (pH ~5.0).21 In 2016, Zhang et al. conjugated DOX onto methoxy poly(ethylene glycol)-block-poly(γ-propargyl-L-glutamate) (mPEG-b-PPLG) and cross-linked the core with disulfide bond through one-step “click chemistry”. This design achieved more efficient accumulation of drug in tumor site and successfully reduced the side-toxicity.14


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Based on these findings, active targeting polymer-conjugated drugs have obtained more ondemand delivery and enhanced antitumor efficacy for the selective recognition toward specific markers on tumor cell membrane, including integrins, folate receptor, transferrin receptor, epidermal growth factor receptor, and so forth.22-25 These factors are abundantly expressed on the surface of tumor cells instead of normal cells. Through recognition of specific molecules, active targeting nanoparticles enter the tumor cells via receptor-mediated pathway rather than count on the enhanced permeability and retention (EPR) effect alone, significantly improving the therapeutic effect and reducing the toxicity.26-28 In view of the above-mentioned facts, a variety of targeting ligands have been used for active targeted delivery of nanomedicines, such as antibodies and their fragments, proteins, peptides, nucleic acid aptamers, and small molecules.22, 29-32

These tumor-homing ligands added on the nanoparticles can significantly improve the

retention and accumulation properties by specifically targeting the tumor cells.33 The αvβ3 integrin receptor is overexpressed on tumor neovasculature endothelial cells and a wide range of tumor cells including breast cancer cells,34 lung cancer cells,35 glioblastoma cells,36 and so on. It was reported that cyclic Arg-Gly-Asp peptides (cRGD) showed high affinity with αvβ3 integrin, which makes it a promising candidate for targeted cancer chemotherapy.37 Through the specific ligand−receptor interaction, cRGD peptides have been widely utilized in nanocarriers to selectively transport antitumor drugs to tumor tissues and stimulate the internalization process. In this work, a targeted acid-sensitive polysaccharide prodrug (HES=DOX/cRGD) was firstly synthesized by Schiff base reaction between the aldehyde groups of oxidized HES and the amino groups of DOX and cyclo(Arg-Gly-Asp-D-Phe-Lys) peptide (c(RGDfK)), forming two imine linkers in the conjugate. Non-target prodrug HES=DOX was synthesized as control. To better


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analyze the targetability of cRGD-decorated prodrug, two cell lines were selected in our experiment. Human malignant melanoma A375 cells that overexpress αvβ3 integrin was used as test group with human non-small cell lung cancer A549 cells as a negative control for they express a low content of αvβ3 integrin.35, 38 The behaviors of HES=DOX/cRGD in the tumor cells in vitro, the antitumor efficacy in vitro and in vivo, and the safety in vivo were performed to demonstrate its superiority.

Scheme 1. Schematic illustration for preparation, self-assembly, and pH-triggered intracellular DOX release of HES=DOX/cRGD. 2. RESULTS AND DISCUSSION


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2.1.

Synthesis

and

Characterization

of

HES=DOX/cRGD.

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The

pH-sensitive

HES=DOX/cRGD or HES=DOX was conveniently prepared by mixing HES-CHO with DOX and c(RGDfK), or DOX alone in acetic acid/sodium acetate (HAc/NaAc) buffer solution at pH 5.0, as shown in Scheme 1. The proton nuclear magnetic resonance (1H NMR) spectrum of HES=DOX/cRGD was conducted in the mixed solution of deuterium oxide (D2O) and deuterated dimethyl sulfoxide (DMSO-d6) (V:V = 1:1) (Figure 1A). The peak at 9.15 ppm was attributed to the −CHO proton of HES-CHO. The multiple peaks from 7.96 to 7.32 ppm were assigned to the protons in the benzene ring of DOX, indicating the successful conjugation of DOX onto HES. The proton signal of −COOH in c(RGDfK) at 10.06 ppm appeared in the final product HES=DOX/cRGD, which confirmed the successful synthesis of the targeted polysaccharide prodrug. Fourier-transform infrared spectroscopy (FT-IR) spectra of HES-CHO, HES=DOX, and HES=DOX/cRGD were also provided (Figure S1). The stretching vibration at 1584 cm−1 (ν−C=N−) was attributed to the imine bond, confirming the successful synthesis of HES=DOX, and HES=DOX/cRGD. The self-assemble behavior of HES=DOX/cRGD in phosphate-buffered saline (PBS) was monitored by transmission electron microscope (TEM; Figure 1B) and dynamic light scattering (DLS; Figure 1C). As shown in Figure 1B and 1C, the aqueous diameter of HES=DOX/cRGD was 77.1 ± 6.5 nm with polydispersity of 0.2, which was larger than that measured by TEM (60.3 ± 4.2 nm) because the micelle shrank during the preparation process of sample for TEM detection. 2.2. In Vitro DOX Release and Cytotoxicity to Tumor Cells. The pH-responsive release features of HES=DOX/cRGD and HES=DOX were performed in PBS at pH 4.5, 5.5, 6.8, and 7.4. Essentially, the two prodrugs displayed a similar release pattern during a period of 48 h. In


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Figure 1D and 1E, it is shown that the proportions of DOX released from both prodrugs were less than 38% in normal physiological fluid at pH 7.4 and was mildly increased as pH dropped to 6.8, which imitated the tumor microenvironment. On the contrary, at pH 5.5 in the endo/lysosomal compartments and pH 4.5 in the lysosomal condition, the accumulative DOX release was steadily elevated to around 60% and 70%, respectively. These findings proved that the prodrugs were able to remain stable during blood circulation until they arrived at tumor sites via the EPR effect or receptor−ligand recognition between cRGD and αvβ3 integrin. After being engulfed by the tumor cells, the Schiff base bond between HES and DOX was quickly fractured, and thus DOX was released.


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Figure 1. Chemophysical Characterizations. (A) 1H NMR spectra of HES=DOX/cRGD (blue line), HES=DOX (red line), and HES-CHO (black line). (B) Morphology and (C) size of HES=DOX/cRGD in aqueous condition. In vitro DOX release from (D) HES=DOX/cRGD and (E) HES=DOX in PBS of pH 7.4, 6.8, 5.5, or 4.5 at 37 °C. Scale bar in (B) represents 500 nm. The statistical data are represented as mean ± standard deviation (SD; n = 3).


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As mentioned above, αvβ3 integrin can specifically interact with cRGD. Therefore, the intracellular release behaviors of targeted and untargeted prodrugs toward αvβ3-positive A357 cells were detected by confocal laser scanning microscope (CLSM). After 2 h co-culture, the cells treated with HES=DOX/cRGD showed a higher intensity than those with HES=DOX, and both were lower than DOX (Figure 2A). This was because DOX entered the cell membrane through diffusion, while prodrug with larger size could only be drawn into cells via an endocytic pathway. Moreover, the targeted polysaccharide prodrug HES=DOX/cRGD was able to recognize the tumor cells by a specific receptor−ligand interaction. Therefore, it was proved that HES=DOX/cRGD showed a stronger targeting ability. For flow cytometry (FCM) assay, αvβ3-positive A357 cells and αvβ3-negative A549 cells were selected to monitor the intracellular release performances of the synthesized prodrugs. A375 cells incubated with HES=DOX/cRGD presented a stronger fluorescence intensity than those of the HES=DOX group after 2 h co-culture (Figure 2B), consistent with the results of CLSM. In Figure 2C, contrary to the results in A375 cells, the uptake of HES=DOX/cRGD and HES=DOX by A549 cells at 2 h after incubation didn’t show any significant difference, as seen through their similar fluorescence intensity. However, the cell uptake of DOX was more efficient than the two prodrug groups, which is because the small molecule DOX entered cells via diffusion, faster than endocytosis of prodrugs in the short term.9 The above findings were in accordance with our expectation that the interaction between cRGD-targeted prodrug HES=DOX/cRGD and αvβ3 integrin receptor promoted the cell uptake efficacy of targeted DOX prodrug. With excellent targetability to A375 cells, HES=DOX/cRGD has the potential of being a targeted antitumor drug formulation.


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Figure 2. Cell uptakes and proliferation inhibition of various DOX formulations. (A) CLSM microimages and (B, C) FCM analyses for cell internalization of HES=DOX/cRGD, HES=DOX, and DOX after incubation with (A, B) A375 cells and (C) A549 cells for 2 h. (D, E) In vitro cytotoxicity of HES=DOX/cRGD, HES=DOX, and DOX after incubation with (D) A375 cells and (E) A549 cells for 48 h. Scale bar in (A) = 50 µm. The statistical data are presented as a mean ± SD (n = 6; #P < 0.01, &P < 0.001).


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Cytotoxicity of HES=DOX/cRGD, HES=DOX, and DOX toward A375 and A549 cells was tested after incubation for 48 h (Figure 2D and 2E). As for A375 cells, the cell proliferation inhibition of the two DOX prodrugs was improved compared with DOX due to the sustained and pH-sensitive release of DOX from prodrugs. More importantly, the half-maximal inhibitory concentration (IC50) of HES=DOX/cRGD (1.29 µg mL−1) toward A375 cells was much lower than that of HES=DOX (IC50 = 1.63 µg mL−1) (Figure 2D). However, no significant difference was detected between HES=DOX/cRGD (IC50 = 1.82 µg mL−1) and HES=DOX (IC50 = 1.92 µg mL−1) (Figure 2E). Cytotoxicity of free DOX toward A375 and A549 cells was similar (IC50 = 0.19 µg mL−1 for A375 and IC50 = 0.24 µg mL−1 for A549), ruling out the discrepancy caused by different cell lines. These results were consistent with the findings of the experiments above, and again confirmed that the interaction between cRGD and αvβ3 integrin receptor played a decisive part in the targetability, cell uptake, and cell proliferation suppression effect of DOX-based prodrug. 2.3. Biodistribution, Efficacy, and Safety In Vivo. Female nude mice bearing A375 human malignant melanoma were prepared. The ex vivo fluorescence imaging was conducted in tumor and visceral organs (the heart, liver, spleen, lung, and kidney) to tract the drug distribution at 12 h after intravenous administration of polysaccharide prodrugs or DOX (Figure 3A and 3B). In the DOX group, the liver and kidney exhibited the strongest fluorescence while the amount of drugs gathered at the tumor site was minimal, demonstrating DOX was largely captured and metabolized by the liver and kidney instead of entering tumor tissue. Comparatively, a much weaker fluorescence was found in the kidney of HES=DOX group, an indicator of reduced kidney toxicity. For mice injected with HES=DOX/cRGD, fluorescence intensity in the kidney was also weakened and a larger amount of DOX was located at the tumor site. The increased


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accumulation was built on specific interaction between cRGD on the prodrug and αvβ3 integrin abundantly expressed on the tumor cells. The findings above suggested that HES=DOX/cRGD was capable of achieving excellent tumor accumulation targeting and significantly reduced body toxicity.

Figure 3. In vivo biodistribution and A375 melanoma inhibition. (A) Ex vivo DOX fluorescence images and (B) average signals collected from the major organs (i.e., the heart, liver spleen, lung, and kidney) of HES=DOX/cRGD, HES=DOX, and DOX in nude mice bearing A375 melanoma at 12 h post-injection. (C) Relative tumor volume and (D) body weight of A375 melanomaxenografted female nude mice treated with HES=DOX/cRGD, HES=DOX, DOX, or PBS as a control. The narrows in C represents the treatment time, that is, day 1, 4, and 8. All statistical data are presented as mean ± SD (n = 10; *P < 0.001).


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In order to arrive at a more convincing conclusion about the property of conjugated prodrugs, their antitumor effect in vivo was investigated on A357 malignant melanoma-bearing female nude mice. The records of the tumor volumes for 12 days revealed that tumors were suppressed to different extents in each group (Figure 3C). Tumors of the mice administrated with targeted HES=DOX/cRGD were well controlled around 600 mm3 in size with those of the control group soaring to 1500 mm3. HES=DOX and DOX also showed retarded tumor growth, but both were statistically inferior to the HES=DOX/cRGD group. Therefore, decoration of cRGD on the prodrug was proved effective in altering the therapeutic efficacy by in vivo and in vitro evidence. The safety of the polymer prodrugs was a critical evaluation standard for future clinical application. In our work, the weight of mice was measured daily. As shown in Figure 3D, all three groups of the drug-treated mice suffered a certain amount of weight loss. Although DOX rendered impressive antitumor effect, it caused the most severe weight loss in comparison with polysaccharide prodrugs. Specifically, HES=DOX/cRGD led to the least systematic toxicity and side effects. These results could be explained by that the imine bond in polysaccharide prodrugs fractured in response to increased acidity in tumor tissue, which achieved the tumor-specific release of DOX. On the other hand, the cRGD on the targeted prodrug further enhanced tumortargeting performance, reduced the body toxicity, and strengthen the tumor suppression capability. Following the impressive findings above, the tumor suppression efficacy and systemic toxicity of each treatment group were further assessed via analyzing pathology sections of tumors and major organs. Mice were sacrificed at four days after the last injection, and tumors and organs (the heart, liver, spleen, lung, and kidney) were isolated and stained by hematoxylin and eosin (H&E; Supplementary Figure S2). DOX created a range of pathological changes, such as defined


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myocardial fracture and necrosis of cardiac muscles, although little accumulation was detected in the heart (Figure 3B). In contrast, there was no significant pathological injury or inflammation in tested organs of neither HES=DOX/cRGD nor HES=DOX-treated group, which was in accordance with the reduced fluorescence intensity of DOX in normal organs. It was worth noticing that the largest apoptotic and necrotic area in the tumor tissue was found in the HES=DOX/cRGD group. The results revealed that the modification of cRGD on polysaccharide prodrug could notably improve the antitumor efficacy and in the meantime alleviate the injury to normal organs. For ex vivo immunohistochemical analyses, the cleaved PARP1 and PCNA are frequently used as indicators of apoptosis and survival in many cell types. In Figure 4A, intensive positive signals of cleaved PARP1 were detected in tumors of mice treated with HES=DOX/cRGD, illustrating wide areas of tumor cell apoptosis. The degrees of apoptosis signal intensity in each group were as follow: HES=DOX/cRGD > HES=DOX > DOX > Control. The expression of PCNA was detected in determining the living cells in response to different treatments. As shown in Figure 4B, the amount of living tumor cells conformed to the following trend: HES=DOX/cRGD < HES=DOX < DOX < Control, that is, only a minority of tumor cells survived under the targeted prodrug. In Figure 4C and 4D, semi-quantitative analysis of the fluorescence intensity above demonstrated the malignant tumor mortality of HES=DOX/cRGD was remarkably higher than those of the other groups. To sum up, the findings in vitro and in vivo can adequately prove that the targeting agent cRGD and acid-labile imine bond in conjugated polymers effectively enhance the antitumor ability and alleviate the side effects.


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Figure 4. Ex vivo immunohistochemical analyses of A375 tumor sections. (A, C) PARP and (B, D) PNCA staining of tumor tissue after treatment with HES=DOX/cRGD, HES=DOX, DOX, or PBS as control. Blue was stained by DAPI, while green was stained by Alexa 488. Scale bar in (A, B) represents 100 µm. The statistical data are presented as mean ± SD (n = 3; *P < 0.001). 4. CONCLUSIONS


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In this study, polysaccharide prodrug HES=DOX/cRGD was prepared through the Schiff base reaction between HES, and DOX and c(RGDfK) for suppression of human malignant melanoma. The cRGD-decorated pH-sensitive prodrug was formed with hydrodynamic diameter of approximately 77 nm, suitable for passive targeting via the EPR effect. Both HES=DOX/cRGD and HES=DOX could prolong the circulation time of DOX in blood and reduce the adverse effects, owing to triggered DOX release in an acidic environment. Particularly, based on the interaction with αvβ3 integrin receptor, HES=DOX/cRGD could be more effectively delivered to the tumor site, compared to the non-targeted prodrug. HES=DOX/cRGD exhibited satisfactory antitumor efficacy as targeted chemotherapy and has the potential application in various types of malignant tumor treatments. In addition, our prodrug synthesis strategy can also be extended to other types of chemotherapeutic agents and provide reference to researchers studying this topic.

ACCOCIATED CONTENT Supporting Information The following files are available free of charge. Detailed experimental section and results of ex vivo histopathological analyses (PDF)

AUTHOR INFORMATION Corresponding Authors *Jianxun Ding, E-mail: [email protected] *Li Chen, E-mail: [email protected]

Notes


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The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was financially supported by the National Natural Science Foundation of China (Grant Nos. 51673190, 51603204, 51673187, 21474012, 51273037, and 51520105004) and the Science and Technology Development Program of Jilin Province (Grant Nos. 20160204015SF and 20160204018SF).

ABBREVIATIONS 1

H NMR, proton nuclear magnetic resonance; CLSM, confocal laser scanning microscope;

cRGD, cyclic Arg-Gly-Asp peptides; c(RGDfK), cyclo(Arg-Gly-Asp-D-Phe-Lys) peptide; Dex−DOX, dextran−doxorubicin; DLS, dynamic light scattering; DMSO-d6, deuterated dimethyl sulfoxide; D2O, deuterium oxide; EPR effect, enhanced permeability and retention effect; FCM, flow cytometry; HAc/NaAc, acetic acid/sodium acetate; H&E, hematoxylin and eosin; HES=DOX/cRGD, cRGD-modified doxorubicin-conjugated hydroxyethyl starch; IC50, halfmaximal inhibitory concentration; mPEG-b-PPLG, methoxy poly(ethylene glycol)-block-poly(γpropargyl-L-glutamate); PBS, phosphate-buffered saline; SD, standard deviation; TEM, transmission electron microscope.

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One-Step Synthesis of Targeted Acid-Labile Polysaccharide Prodrug for Efficiently Intracellular Drug Delivery Di Li, Xiangru Feng, Li Chen, Jianxun Ding, and Xuesi Chen


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One-Step Synthesis of Targeted Acid-Labile Polysaccharide Prodrug

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