Addressing Drug Resistance in Cancer with Macromolecular

Mar 5, 2018 - Drug resistance to chemotherapeutics is a recurrent issue plaguing many cancer treatment regimens. To circumvent resistance issues, we h...
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Addressing Drug Resistance in Cancer with Macromolecular Chemotherapeutic Agents Nathaniel H. Park,†,⊥ Wei Cheng,‡,⊥ Fritz Lai,§,⊥ Chuan Yang,‡,⊥ Paola Florez de Sessions,∥ Balamurugan Periaswamy,∥ Collins Wenhan Chu,∥ Simone Bianco,† Shaoqiong Liu,‡ Shrinivas Venkataraman,‡ Qingfeng Chen,*,§ Yi Yan Yang,*,‡ and James L. Hedrick*,† †

IBM Research-Almaden, 650 Harry Road, San Jose, California 95120 United States Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore § Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore ∥ Genome Institute of Singapore, 60 Biopolis Street, Genome, Singapore 138672, Singapore ‡

S Supporting Information *

ABSTRACT: Drug resistance to chemotherapeutics is a recurrent issue plaguing many cancer treatment regimens. To circumvent resistance issues, we have designed a new class of macromolecules as self-contained chemotherapeutic agents. The macromolecular chemotherapeutic agents readily selfassemble into well-defined nanoparticles and show excellent activity in vitro against multiple cancer cell lines. These cationic polymers function by selectively binding and lysing cancer cell membranes. As a consequence of this mechanism, they exhibit significant potency against drug-resistant cancer cells and cancer stem cells, prevent cancer cell migration, and do not induce resistance onset following multiple treatment passages. Concurrent experiments with the small-molecule chemotherapeutic, doxorubicin, show aggressive resistance onset in cancer cells, a lack of efficacy against drug-resistant cancer cell lines, and a failure to prevent cancer cell migration. Additionally, the polymers showed anticancer efficacy in a hepatocellular carcinoma patient derived xenograft mouse model. Overall, these results demonstrate a new approach to designing anticancer therapeutics utilizing macromolecular compounds.

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INTRODUCTION Cancer treatment is often plagued by numerous issues that hinder the development of effective therapeutic regimes. These issues not only include aggressive resistance development to drugs or drug cocktails but also insufficient drug accumulation in tumor tissue, low aqueous solubility of many chemotherapeutics, rapid clearance from the body, and significant offtarget toxicity.1−4 The push to overcome these issues has led to the development of an extensive array of nanotechnology drug delivery systems that aim to increase drug solubility, circulation half-life, accumulation in tumor tissue, as well as reduce the toxicity of the drug itself.1,2,4,5 The success of nanotechnology therapeutics has resulted in several systems reaching clinical trials or garnering FDA approval for human use.2,5,6 Unfortunately, nanotechnology drug delivery systems ultimately rely on the action of the drug itself and hence still suffer from the drug’s inherent limitations. While many delivery systems can increase the concentration of the chemotherapeutic in tumor tissue, cellular barriers and resistance mechanisms may still limit the overall effectiveness of the drug.1 Drug delivery systems can be prone to other drawbacks such as burst release and potential off-target toxicity.6 While highly efficacious nanotechnology systems have been demonstrated,2,5−7 many approaches rely on complex strategies involving multifunctional nanoparticles, the loading of several small-molecule chemo© XXXX American Chemical Society

therapeutics or chemosensitizing agents, or require significant synthetic efforts to access the necessary materials.4,5 Because of these limitations and the stringent biocompatibility requirements of nanotechnology drug delivery systems, the design of a simple yet effective system remains a challenging endeavor. Given the limitations with small-molecule drugs and traditional drug delivery systems, we investigated the potential of using polymeric micelles themselves as chemotherapeutic agents. The use of polymers as macromolecular therapeutic agents is often encountered in the development of antimicrobials, where cationic polymers selectively bind and lyse the anionic membranes of bacterial cells while in the presence of healthy mammalian cells.8 In a manner analogous to bacteria, cancer cell membranes also have a net negative charge, resulting from a greater abundance of anionic lipids relative to healthy cells.8,9 This anionic nature has been attributed to the high selectivity and potency of certain antimicrobial peptides against cancer cells.9,10 Their success has led to intensive research into anticancer peptides, which has produced several promising candidates with high activity against cancer cell lines.11,12 Unfortunately, peptide-based therapies often suffer from high production costs, proteolytic cleavage, and, in the case of Received: October 27, 2017 Published: March 5, 2018 A

DOI: 10.1021/jacs.7b11468 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Figure 1. Initial hypothesis on mechanism of action for macromolecular chemotherapeutic agents (a = PEG block, b = linking group, c = cationic block).

Figure 2. Synthesis and characterization of chemotherapeutic polymers. ZP = ζ potential.

(PEG) block (a, Figure 1), a linking group (b, Figure 1), and a cationic block (c, Figure 1) that contains the positive charge to associate with negatively charged lipid membranes. For the cationic block, we selected a polycarbonate containing a pendant benzyl chloride previously developed by our group (Figure S1).13 This highly tunable platform offers an excellent handle to introduce both a cationic charge and the necessary functional groups to drive self-assembly into micellar structures, which is critical to the function of the macromolecular chemotherapeutics in the proposed mechanism (I to II, Figure 1). Upon administration, these nanoparticles will circulate through the bloodstream and selectively accumulate in vascularized tumor tissue via the enhanced permeability and

cationic peptides, may be inactivated from binding to anionic components present in serum, leading to short circulation halflives.9 Based on this, we felt that an appropriately designed biocompatible system containing a cationic polymer, which can be readily prepared in large quantities in a short synthetic sequence, would provide potent anticancer activity while overcoming many limitations of anticancer peptides, nanoparticulate drug delivery systems, and standard chemotherapeutic approaches. Our design for a macromolecular chemotherapeutic (I, Figure 1) consists of three principle components arising from our preliminary hypothesis for proposed mechanism of action. These components include: a hydrophilic polyethylene glycol B

DOI: 10.1021/jacs.7b11468 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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Journal of the American Chemical Society retention (EPR) effect (II to III, Figure 1).1−6 If the linker (b, Figure 1) is pH sensitive, this could trigger the cleavage of the PEG block from the cationic polycarbonate in acidic tumor tissues (pH 5.8−7.0) (III to IV, Figure 1). The released cationic polycarbonate will then be free to associate with the outer leaflet of the cancer cell membrane, causing disruption and eventual lysis of the cell itself (IV to VII, Figure 1). On the other hand, the nanoparticle may undergo endocytosis by the cancer cells, thereby entering acidic endosomes (pH 5.0−6.0), followed by cleavage of PEG. The cationic nanoparticles may break down endolysosomal membranes, thus releasing into the cytosol where they can precipitate proteins or nucleic acids through nonspecific electrostatic and hydrophobic interactions. Alternatively, if the linker is not pH sensitive, then it may simply undergo electrostatic association with anionic cancer cell membrane (IV, Figure 1).8,9 Both of the block copolymer components (a and c, Figure 1) play critical roles in the proposed mechanism. The PEG block shields the cationic core and enhances circulation times, enabling passive tumor accumulation, while the cationic block will bind and disrupt the cellular membrane.1−7 The acid-sensitive linker (b = acetal, Figure 1) potentially allows for the selective release of the cationic block for membrane disruption upon reaching acidic tumor tissues and endocytosis by cancer cells.

distributions (entries 4 and 6, Figure 2). In contrast, nanoparticles formed from 1c and 1e had sizes of 22−53 nm and narrower size distributions (entries 3 and 5, Figure 2). TEM images further demonstrated that polymers 1c and 1e formed nanoparticles with relatively uniform particle size (Figure 3a). The ζ potential of nanoparticles within 10 mV is

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RESULTS AND DISCUSSION Synthesis of Macromolecular Chemotherapeutics. With the principle design components identified, we prepared a series of macromolecular chemotherapeutics (1a−f, Figure 2). First, the AB diblock polycarbonate was synthesized via organocatalyzed ring-opening polymerization of the cyclic benzyl chloride-functionalized carbonate monomer using either mPEG (5000 Da) or the acetal containing PEG (∼5000 Da) as the macroinitiator (Figure 2, Figure S1). The resultant benzyl chloride-functionalized diblock copolymer could be quaternized with tertiary amines to afford the corresponding cationic macromolecular chemotherapeutics (Figure 2, Figure S1). As cationic polycarbonates can be highly water-soluble, we anticipated that selection of the appropriate functional tertiary amine would be critical to successfully drive the self-assembly into micelles. Prior work in our group demonstrated that cholesterol-functionalized polycarbonates readily self-assemble into well-defined micelles, which can be exploited for drug delivery applications.14,15 Based on this precedent, we utilized a tertiary amine functionalized cholesterol derivative for the quaternization of the AB diblock polycarbonate copolymers. The fully cholesterol-functionalized copolymers (1a and 1b, entries 1 and 2, Figure 2) proved to be highly capable of selfassembly into nanoparticles. However, their poor solubility and particle size distributions (PDI) prompted us to examine the introduction of other tertiary amines into the polycarbonate block. To balance solubility and self-assembly characteristics of the macromolecular chemotherapeutics, we introduced N,Ndimethylhexylamine as an additional quaternizing agent with the amine functionalized cholesterol to prepare polymers 1c−f (entries 3−6, Figure 2). These polymers readily self-assembled into nanoparticles in a simulated physiological environment (i.e., phosphate-buffed saline-PBS) at low concentrations with critical micelle concentrations (CMCs) in the range of 11.4− 17.3 μg/mL (Figure 2). Nanoparticles containing an acetal linker between the PEG and polycarbonate blocks had relatively larger particle sizes and slightly broader size

Figure 3. (A) TEM images of 1c and 1e, (B) stability study of 1c, and (C) in vitro anticancer activity of polymers.

desirable for targeting tumor tissues via the EPR effect, as highly cationic surfaces may bind opsonin proteins present in the serum, leading to clearance by macrophages of the mononuclear phagocyte system.16,17 The ζ potential of the all prepared nanoparticles were determined to be 0.2 across all samples are shown (rows). Columns represent 8 different treatment conditions.

are known to confer DOX resistance, including the common DOX resistance gene: ABCB1 (multidrug resistance 1, MDR1)28−30 This gene was found to be upregulated at P7 by 15.4-fold, but only moderately upregulated in both P5 (2.1fold) and P3 (2.2-fold) passages. In addition, we observed many genes from the same chromosome locus 7q (including ABCB1) that are known to promote multidrug resistance31 and are significantly upregulated (at least log 2-fold change ±3, relative to control) at P7 (Figure 8). These genes include: ABCB4 (MDR2), a gene commonly upregulated along with ABCB1 in drug resistant cell lines;31,32 ADAM22, a known marker gene for DOX resistance in gastric cancer;30 and SRI (Sorcin), an important calcium-binding protein known to alter apoptosis-related proteins and promote multidrug resistance independent of MDR1.33,34 FBP2 was also recently reported to be upregulated in breast cancer tissues and cell lines and was shown to be correlated with DOX resistance.35 Interestingly, we also observed the long noncoding RNA (lncRNA) TP53TG1, which was shown previously to be upregulated in HepG2 drug-resistant cell line (HepG2/DR).36 In contrast, the host response to polymer 1c was subtle, as only one gene was differentially expressed in P1, none in P3, 1892 genes in P5, and 100 in P7 (Table S2). At passage P5, general metabolic pathways (KEGG pathway ID: hsa01100) and oxidative phosphorylation (hsa00190) were enriched, however, there was no observed enrichment for any KEGG pathway or GO terms at passage P7. Furthermore, a closer examination of the most significant hits (at least log2-fold change ±2) at passage P7 did not reveal any genes that are known to be involved in acquired drug resistance to our knowledge. This suggests that polymer treatment is relatively inert despite being observed to

Figure 9. Activity of (a) DOX and (b) polymer 1c against sorted Hep3B cancer stem cells (SP) and noncancer stem cells (NSP). G

DOI: 10.1021/jacs.7b11468 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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treating Hep3B SP cells, we further examined their ability to prevent cancer cell migration. Here, Hep3B cells were cultured in a 24-well plate, and a rift was scratched into the plate. The cells were then treated with polymer 1c or DOX at their respective IC50 concentrations, and cell migration was monitored over 24 h (Figure 10). The untreated cells migrated

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] *[email protected] ORCID

Nathaniel H. Park: 0000-0002-6564-3387 Fritz Lai: 0000-0001-7314-4935 Shrinivas Venkataraman: 0000-0001-7037-1550 Yi Yan Yang: 0000-0002-1871-5448 James L. Hedrick: 0000-0002-3621-9747 Author Contributions ⊥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was funded by IBM Research-Almaden and the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). The animal work was supported by Translational and Clinical Research grant (NMRC/TCR/014NUHS/2015) from the National Medical Research Council (NMRC), Singapore. Q.C. is supported by Singapore National Research Foundation Fellowship NRF-NRFF2017-03.

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Figure 10. Effect of DOX or polymer 1c treatment on migration of Hep3B cancer cells at their respective IC50 concentrations.

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across the rift after a 24 h culture period. Cell migration was also observed for DOX treated samples, even at concentrations of 2 × IC50 (Figure 10, Figure S7). However, the samples treated with polymer 1c effectively prevented cell migration across the rift. This result, coupled with the efficient killing of Hep3B SP cells, demonstrates the potential for macromolecular chemotherapeutics not only to treat cancer but also prevent further metastasis and relapse.

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CONCLUSION The development of macromolecular chemotherapeutic agents from biocompatible scaffolds represents a distinct approach to chemotherapy. Herein, we have demonstrated not only in vitro and in vivo efficacy at doses with negligible toxicity but also the ability of these nanoparticles to prevent cell migration, kill drugresistant cell lines and cancer stem cells, retain potency through multiple treatment passages, and potentially evade host recognition. Together, these features make macromolecular chemotherapeutics a highly attractive option toward overcoming common drawbacks of chemotherapeutic treatment.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b11468. Experimental procedures and analytical data (PDF) Table S2 (XLS) Figure S3 (AVI) H

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DOI: 10.1021/jacs.7b11468 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX