Anticancer Drug Disulfiram for In Situ RAFT Polymerization: Controlled

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Anticancer Drug Disulfiram for In Situ RAFT Polymerization: Controlled Polymerization, Multifacet Self-Assembly, and Efficient Drug Delivery Dezhong Zhou,‡,† Yongsheng Gao,‡,† Sigen A,‡ Qian Xu,‡ Zhao Meng,‡ Udo Greiser,*,‡ and Wenxin Wang*,§,‡ §

School of Materials Science and Engineering, School of Materials Science and Engineering, Tianjin 300072, China Charles Institute of Dermatology, School of Medicine, University College Dublin, Dublin, Ireland

‡

S Supporting Information *

ABSTRACT: Here we report the synthesis of a well-defined amphiphilic conjugate, tetraethylthiuram disulfide (disulfiram, DS)−poly(ethylene glycol) methyl ether acrylate (DS-PEGMEA), and its multifacet self-assembly in aqueous solutions and application in DS drug delivery to melanoma cells. The DS-PEGMEA was synthesized via the reversible addition−fragmentation chain transfer (RAFT) polymerization utilizing DS, a 90 year old anticancer drug, as a precursor to generate RAFT agent in situ. Results demonstrate that the in situ formed RAFT can effectively control the polymerization of PEGMEA. Depending on the concentration in aqueous solution, the amphiphilic DS-PEGMEA conjugate can self-assemble to form layered, toroidal, hairy, or spherical nanostructures, respectively. Moreover, DS drug can be further encapsulated by DS-PEGMEA to formulate core−shell structured DS/DS-PEGMEA nanoparticles mediating the apoptosis of melanoma cells (A375) while inducing minimal cytotoxicity to normal (hADSC and NIH fibroblast) cells. Both DS and PEGMEA are approved by the American Food and Drug Administration (FDA); therefore, the DS-PEGMEA has great potential for application in clinical drug delivery to melanoma.

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polymerization and evaluated for diverse drug delivery systems, both in vitro and in vivo.6 Despite the effectiveness of RAFT polymerization in the synthesis of drug delivery vectors, synthesis of RAFT agent poses one of the most significant challenges in practical experimental settings and applications.4b,7 In general, the synthesis of RAFT agent involves toxic organic chemicals or solvents and requires time-consuming multiple purification steps.8 For example, to prepare the most widely used RAFT agents (4-cyanopentanoic acid) dithiobenzoate (CPADB), 4cyano-4-thiothiopropylsulfanylpentanoic acid (CTPPA), S-1dodecyl′-(α,α′-dimethyl-α″-acetic acid) trithiocarbonate (DDMAT), and so on, an additional important, yet highly toxic, chemical, carbon disulfide (CS2), is utilized. Moreover, chromatographic purification was also required.6c,7,9 These limitations compromise the safety profile of biomedical applications of the polymers synthesized by RAFT polymer-

etraethylthiuram disulfide [1,1′,1″,1‴-[disulfanediylbis(carbonothioylnitrilo)]tetraethane] (DS) is a FDA approved anticancer drug with proven potential for cancer therapy via the effective inhibition of NFκB pathway and apoptosis enhancement of resistant cancer cells.1 However, the half-life of naked DS in the bloodstream of only 4 min severely hinders its broad applications in the clinical field.2 Hence, the development of a viable DS delivery vector which will ultimately increase its therapeutic efficacy is imperative. Ideally, a viable drug delivery vector for cancer treatment should be prepared via a facile approach from commercially available FDA approved chemicals and then it should also be able to effectively transport drugs to mediate tumor cell apoptosis while inducing minimal cytotoxicity to normal cells.3 Since its first report by Rizzardo et al. in 1998,4 reversible addition−fragmentation chain transfer (RAFT) polymerization has attracted broad attention in the area of drug delivery.5 Due to its highly controlled polymerization over a wide range of monomers and high tolerance to various functional groups and solvents, numerous gradients, alternating, graft, and block amphiphilic polymers have been synthesized by RAFT © XXXX American Chemical Society

Received: October 11, 2016 Accepted: October 27, 2016

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Scheme 1. DS-PEGMEA Conjugate Can Be Synthesized via a One-Step In Situ RAFT Polymerization with DS as a RAFT Agent Precursora

a

In aqueous solution, the amphiphilic DS-PEGMEA can self-assemble with DS to form core-shell structured nanoparticles.

ization.6c To circumvent these limitations, the in situ RAFT polymerization was reported where the RAFT agent 2cyaoprop-2-yl dithiobenzoate (CDB) forms in situ in the reaction system from the precursor dithiobenzoate (BD).8 Given the structural similarity of DS (Scheme 1a) to RAFT agent precursors (e.g., bis(thiobenzoyl) disulfide), we hypothesized that DS can be potentially used as a RAFT agent precursor for in situ RAFT polymerization. DS is FDA approved, demonstrates negligible cytotoxicity in many tissues of the human body, and its use becomes more appealing for applications in biomedicine.10 As a proof of concept study, we report here the preparation of a novel DS delivery system, amphiphilic disulfiram-poly(ethylene glycol) methyl ether acrylate conjugate (DS- PEGMEA), developed from an in situ RAFT polymerization using DS as RAFT agent precursor. The multifacet self-assembly of DS-PEGMEA in aqueous solution and its application in DS delivery to induce the apoptosis of melanoma cells were further investigated (Scheme 1). It should be noted that PEGMEA is also a FDA approved drug excipient; therefore, the DS-PEGMEA developed here has great significance for clinical drug delivery as far as the safety perspective is concerned. The unique in situ RAFT polymer-

ization strategy not only eliminates the laborious purification process, but also reduces the usage of hazardous chemicals or solvents, therefore, opening a new avenue to RAFT polymerization and its application in medicine. PEGMEA (Mn = 480 Da) and 2,2′-azobis(2-methylpropionitrile) (AIBN) were chosen as monomer and initiator, respectively. In contrast to conventional RAFT polymerization, the initiator AIBN used here was utilized in excess in comparison to the RAFT agent precursor DS. We hypothesized that the polymerization reaction is composed of a two-stage kinetics: in the earlier stage, the DS will first react with AIBN to form the RAFT agent DS-AIBN dithiocarbamate in situ, and then, in the later stage, the excessive AIBN will further initiate the polymerization of PEGMEA in the presence of the already formed DS-AIBN dithiocarbamate. As expected, there was almost no polymer detected in the earlier stage from gel permeation chromatography (GPC; Supporting Information, Tables S1−S4), indicating the higher reaction priority of AIBN with DS compared to PEGMEA. Nuclear magnetic resonance spectroscopy (NMR) was further used to measure the formation of DS-AIBN dithiocarbamate. There is a chemical shift of the methyl groups in AIBN from 1.73 to 1.54 ppm once 1267

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Figure 1. (a) GPC traces of DS-PEGMEA polymers at different time points. (b) Kinetic plot of ln([M]0/[M]) vs reaction time. (c) Kinetic plot of Mn vs monomer conversion.

Figure 2. (a) GPC traces and (b) 1H NMR spectrum of DS-PEGMEA polymers after purification.

DS-AIBN dithiocarbamate is formed. As shown in Figure S1, at the beginning of the polymerization, there was no signal peak at 1.54 ppm at the beginning of the polymerization indicating the absence of DS-AIBN dithiocarbamate formation. As the reaction proceeds, a very obvious signal peak appeared at 1.54 ppm after 20 min pointing to the formation of DS-AIBN dithiocarbamates. A total of 40 and 60 min after the start of the reaction, the characteristic methyl group signal peak of the DSAIBN dithiocarbamate became stronger and stronger, demonstrating more AIBN reacted with DS to form the DS-AIBN dithiocarbamate. Correspondingly, the signal peak of the methyl groups in AIBN (1.73 ppm) became weaker and weaker. The relative integral areas of the characteristic peaks of the AIBN, DS-AIBN dithiocarbamate, and DS were quantified, and the results are shown in Table S5. It clearly shows that the ratio of AIBN to the DS-AIBN dithiocarbamate decreased from 1:0 to 1:0.4 after 60 min of reaction. All these results demonstrate that the AIBN reacted with DS to form the DSAIBN dithiocarbamate. We optimized the reaction parameters to facilitate DS to form DS-AIBN in situ to effectively control

the polymerization by systematically varying the feed ratio of PEGMEA/DS/AIBN, monomer addition method, and reaction temperature. The slow addition of PEGMEA (performed here drop by drop) could improve the controllability of polymerization (Tables S1−S4). Previous studies demonstrated that the N,N-dialkyldithiocarbamates are indeed very poor RAFT agents for acrylates.7 However, Moad and co-workers showed that a low ratio of the monomer concentration to the chain transfer agent (CTA) concentration would lead to better polymerization controllability and a narrow polymer dispersity.11 Monteiro and Klumperman et al. further confirmed that the use of slow monomer addition or semibatch process can significantly improve the level of control in xanthate or dithiocarbamate-mediated RAFT polymerization.11b,12 Therefore, the good controllability of polymerization by the in situ formed DS-AIBN dithiocarbamate in our system is due to the “drop by drop” addition of PEGEMA maintaining a high RAFT agent to monomer ratio. Under optimized reaction conditions (PEGMEA/DS/AIBN = 50:1:1.5, PEGMEA added drop by drop, 80 °C), the recorded plots show that the polymerization 1268

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Figure 3. (a) Fluorescence spectroscopy showed that the critical aggregation concentration (CAC) of DS-PEGMEA was 149.2 μg/mL. (b−e) TEM images show that at different polymer concentrations, DS-PEGMEA can self-assemble to form layered (0.25 mg/mL), toroidal (0.5 mg/mL), hairy (0.75 mg/mL), or spherical (1.0 mg/mL) nanoassemblies, respectively.

of PEGMEA followed first order kinetics with a linear dependence of Mn with monomer conversion, demonstrating the good control of the polymerization mediated by the in situ formed RAFT agent from DS and AIBN (Figure 1).13 After 9 h, PEGMEA polymers with polydispersity index (PDI) 1.44 were detected by GPC with a monomer conversion of approximately 43.3% (Table S6). To further validate the feasibility and utility of the in situ DS-mediated RAFT polymerization, another two monomers, methyl methacrylate (MMA) and butyl acrylate (BA), were used. The GPC results demonstrate that the polymerizations were well-controlled as well and low PDIs were achieved (Figures S2 and S3 and Tables S7 and S8). Moreover, we synthesized the DS-AIBN dithiocarbamate (Figure S4) and utilized the presynthesized DS-AIBN to mediate the polymerization of PEGMEA. As shown in Figure S5 and Table S9, the evolution of the molecular weights and PDIs under this condition was very similar to that of the in situ DS-mediated RAFT polymerization. All these results confirm our hypothesis,

stating the anticancer drug DS can be used as RAFT agent precursor for in situ polymerization of PEGMEA. After purification, the DS-PEGMEA has a Mn of 7.7 kDa and a PDI of 1.28 (Figure 2a). Chemical structure of the DSPEGMEA conjugate was characterized by 1H and 13C NMR. The presence of signal peaks from DS moieties and AIBN moieties, along with the previous kinetic plots, further supports the in situ polymerization hypothesis (Figures 2b and S6). While DS is highly hydrophobic (0.7% w/v in water),2a the PEGMEA is hydrophilic potentiating its self-assembly in aqueous solution and application in the delivery of hydrophobic drugs such as DS itself. The critical aggregation concentration (CAC) of DS-PEGMEA was measured with fluorescence spectroscopy using pyrene as a probe. An apparent CAC of 149.2 μg/mL was observed (Figure 3a), therefore, confirming the ability of the amphiphilic DS-PEGMEA conjugate to formulate nanoparticles via self-assembly when the concentration is above the CAC in aqueous solutions. Previously, 1269

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Figure 4. DLS measurements show that the blank DS-PEGMEA nanoparticles (a) and DS loading DS/DS-PEGMEA nanoparticles (b) have a diameter of 178 and 251 nm, respectively. (c, d) TEM images show DS/DS-PEGMEA nanoparticles displaying a core−shell structure.

Figure 5. (a) Viability of A375 cells after treatment with free DS and DS/DS-PEGMEA nanoparticles at different concentrations. (b) Viability of A375 cells and hADSC cells after treatment with DS/DS-PEGMEA nanoparticles at different concentrations.

each monomer unit accounts for the concentration dependent morphological transition of DS-PEGEMA nanoassemblies.15 The fact that this type of amphiphilic conjugate can selfassemble to form nanoparticles and undergo shape transformation implies the potential use in drug encapsulation and delivery. DS has long demonstrated its efficacy in enhancing cancer cell apoptosis.18 However, establishing optimal delivery of DS has been a challenge given the ultrashort half-life of DS in the bloodstream (approximately 4 min).1a Currently, liposomes are the most widely used system for DS delivery.2b However, here we demonstrate DS can be easily loaded into the DS-PEGMEA assemblies via the solvent exchange method. The DS loading content (DLC) of DS-PEGMEA was measured with 1H NMR, demonstrating that a 5.8% DLC can be achieved (Figure S7 and Table S10). Dynamic light scattering (DLS) measurements showed that, prior to DS encapsulation (blank nanoparticles), an average size of 178 nm with a dispersity of 0.24 for the nanoparticles (Figure 4a,c) was accomplished. In comparison, the DS-loaded nanoparticles (DS/DS-PEGMEA) were bigger in size (around 251 nm) (Figure 4b,d). Meanwhile, TEM images showed that the DS/DS-PEGMEA nanoparticles

numerous reports have demonstrated that the shapes of selfassemblies of functional block polymers or host−guest pairs can be transformed by triggers.14 To investigate the morphology of the DS-PEGMEA assemblies, transmission electron microscopy (TEM) was used. Here, we observed that with the increase of polymer concentration (0.25, 0.5, 0.75, and 1.0 mg/mL), the DS-PEGMEA assemblies showed layered, toroidal, hairy, or spherical morphology, respectively (Figure 3). Previously, Thayumanavan et al. showed that hydrophilic homopolymers can self-assemble to form different nanostructures due to a suitable hydrophilic−hydrophobic balance in the backbone and side chains.15 Monteiro et al. further reported the self-assembly of (homo)polymers based on the presence of a RAFT end group.16 Recently, Du and O’Reilly et al. demonstrated that morphologies of the nanoassemblies of hydrophilic homopolymers would be concentration-dependent because the functionality of RAFT agent can affect the “solution association”.16c,17 Although the detailed mechanism behind the multifacet self-assembly of DS-PEGMEA is not clear, given that backbone of the DS-PEGEMA is hydrophobic, while the side chains are hydrophilic, we speculate the amplified consequence of the molecular level conformational change in 1270

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exhibited a core−shell structure, which is different from the blank nanoparticles (Figure 3). We further evaluated the effectiveness of free DS, DS/DS-PEGMEA for inducing the apoptosis of melanoma cell line A375 (Figure 5a). Consistent with previous reports,10,18a 48 h after treatment with DS, viability of the A375 cells decreased as the concentration of DS was increased. When 100 μM of DS was applied, less than 10% cell viability was maintained. Under the same DS concentrations, viability of A375 cells after treatment with DS/DSPEGMEA nanoparticles was relatively higher when compared to results obtained with free DS. However, over 80% of viability reduction was still observed. Interestingly, in contrast with the DS/DS-PEGMEA nanoparticles, the blank nanoparticles DSPEGMEA itself did not exert obvious cytotoxicity in A375 cell line even with concentrations up to 1.0 mg/mL (Figure S8). These results thus point to the safety of DS-PEGMEA as a vector for drug delivery. It should be noted that although free DS can significantly enhance cancer cell apoptosis in vitro, its half-life in the bloodstream is very short (4 min) due to its high hydrophobicity and ease of fast aggregation. Therefore, it is envisaged that encapsulation of DS with the amphiphilic DSPEGMEA conjugate could potentially enhance its half-life in vivo. Previous studies have also indicated that DS only exerted negligible toxicity to normal cells.19 Testing whether or not the DS/DS-PEGMEA nanoparticles have the same favorable property, the effectiveness of DS/DS-PEGMEA in mediating normal cell apoptosis was further evaluated with human adipose derived stem cells (hADSC) and NIH fibroblast and compared with results obtained with A375 cells. The data set showed that after treatment with DS/DS-PEGMEA, hADSC cells and NIH fibroblast maintained high cell viability (Figures 5b and S9) at all concentrations. These results again support the safety profile of DS/DS-PEGMEA assemblies with respect to normal cells. In conclusion, a well-defined amphiphilic DS-PEGMEA conjugate has been synthesized from an in situ RAFT polymerization using the anticancer drug DS as a RAFT agent precursor. In aqueous phase, the DS-PEGMEA can selfassemble to form nanosized assemblies. By simply adjusting polymer concentration, the nanoassemblies can transform from layered structures to toroidal, hairy, and spherical nanoparticles. The DS-PEGMEA conjugate can effectively load DS to enhance melanoma cell apoptosis while maintaining high viability of normal cells. Given that both the DS and the PEGMEA are FDA approved agents, the DS-PEGMEA as well as the synthesis strategy proposed here will provide new insight into the development of clinically safe and efficient anticancer drug delivery systems.

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Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00777. Materials and methods, DLS calculation, viability of A375 and NIH fibroblast cell lines after treatment with DS-PEGMEA nanoparticles at different concentrations, and molecular weights of DS-PEGMEA synthesized under different conditions (PDF).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions †

These authors contributed equally (D.Z. and Y.G.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Science Foundation Ireland (SFI), Industry Fellowship (15/IFA/3037), Principal Investigator Program (13/IA/1962), Investigator Award (12/IP/ 1688), Health Research Board of Ireland (HRA-POR-2013412), Technology Innovation Development Award (15/TIDA/ 2969), National Natural Science Foundation of China (1300401180007), and University College Dublin. We would also like to acknowledge the kind, technical support of Prof. Dimitri Scholz of the Conway Electronic Microscopy core, University College Dublin.

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REFERENCES

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EXPERIMENTAL SECTION

In Situ RAFT Polymerization of PEGMEA. DS (59.2 mg) and AIBN (49.2 mg) were dissolved in 20 mL of butanone in a 50 mL twoneck flask. After 30 min of bubbling with argon to remove the oxygen, 4.8 g PEGMEA monomer dissolved in 20 mL of butanone was added drop by drop at a rate of 1 mL per hour. The reaction was carried out at 80 °C. To monitor the reaction, 50 μL of sample was taken at different time points, and then GPC analysis (Agilent Technologies 1260 Infinity) was performed with DMF as an eluent at 0.67 mL/min and 80 °C. 1271

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