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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 J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b11468 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 5, 2018
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Addressing Drug Resistance in Cancer with Macromolecular Chemotherapeutic Agents Nathaniel H. Park1,‡, Wei Cheng2,‡, Fritz Lai3,‡, Chuan Yang2,‡, Paola Florez De Sessions4, Balamurugan Periaswamy4, Collins Wenhan Chu4, Simone Bianco1, Shaoqiong Liu2, Shrinivas Venkataraman2, Qingfeng Chen3,*, Yi Yan Yang2,*, James L. Hedrick1,* 1
IBM Almaden Research Center, 650 Harry Road, San Jose, CA 95120 United States
2
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore
3
Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore
4
Genome Institute of Singapore, 60 Biopolis Street, Genome, Singapore 138672, Singapore
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 self-assemble 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 drugresistant 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 anti-cancer efficacy in a hepatocellular carcinoma patient derived xenograft (PDX) mouse model. Overall, these results demonstrate a new approach to designing anticancer therapeutics utilizing macromolecular compounds.
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 off-target 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 nanotech-
nology systems have been demonstrated,2,5,6,7 many approaches rely on complex strategies involving multifunctional nanoparticles, the loading of several smallmolecule chemotherapeutics 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 promis-
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Journal of the American Chemical Society ing 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 cationic peptides—may be inactivated from binding to anionic components present in serum, leading to short circulation half-lives.9 Based on this, we felt that an appropriately designed biocompatible system contain-
ing 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.
Mechanistic Hypothesis:
b
c Micelle
Tumor Tissue
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Electrostatic Membrane Associtation IV
Macromolecular Chemotherapeutic Components
a=
c=
b= MeO
O O
PEG5K
O or
n
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O
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Me O
O O
Acetal
O
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Ether
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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).
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 (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 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 non-specific 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 polymer components (a and c, Figure 1) play a critical role 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 potentially allows for the selective release of the cationic block for membrane disruption upon reaching acidic tumor tissues and endocytosis by cancer cells.
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
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the cyclic benzyl chloride-functionalized carbonate monomer using either mPEG (5000 Da) or the acetal containing PEG (~5000 Da) as the macro-initiator (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 selfO O
O MeO
O
O
I. mPEG5k-OH or mPEG5K-acetal-OH
R1 n
O
A
O
O
Me O
O O
Ring-Opening Polymerization
O
assemble into well-defined micelles, which can be exploited for drug delivery applications.14,15 Based on this precedent, we utilized an 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 killing different cancer cell lines in vitro and self-assembly 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.
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Macromolecular Chemotherapeutics A = mPEG5k B = Linker (R1) C = Charged Block (MTC-Bn-R2)
MTC-Bn-Cl
Me
Me
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Cl
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0.37 ± 0.02
0.9 ± 0.4
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0.30 ± 0.02
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mPEG5K-p[(MTC-Bn-Chol)8-(MTC-Bn-Hexyl)3]
11.4
23.2 ± 0.2
0.13± 0.02
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1d
mPEG5K-acetal-p[(MTC-Bn-Chol)8-(MTC-Bn-Hexyl)3.4]
12.2
74.4 ± 0.3
0.22 ± 0.01
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mPEG5K-p[(MTC-Bn-Chol)4-(MTC-Bn-Hexyl)3.5]
13.5
53.3 ± 0.3
0.18 ± 0.01
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1f
mPEG5K-acetal-p[(MTC-Bn-Chol)4-(MTC-Bn-Hexyl)3.5]
17.3
106.2 ± 0.8
0.21 ± 0.01
0.9 ± 0.3
(ppm)
(mV)
Figure 2. Synthesis and characterization of chemotherapeutic polymers. ZP = zeta potential.
To balance solubility and self-assembly characteristics of the macromolecular chemotherapeutics, we introduced N,N-dimethylhexylamine 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 salinePBS) 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 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). Zeta potential of nanoparticles within 10 mV is 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 zeta potential of the all prepared nanoparticles were determined to be less than 5 mV as the PEG shell shields the cationic charges on the core-forming polycarbonate block (Figure 2). Because of this, the nano-
particles of 1c were stable in serum-containing PBS, and the sizes remained around 60 nm after a 7-hour incubation (Figure 3b). These findings indicated that the compositions of polymers 1c and 1e were optimal for the formation of nanoparticles with the requisite properties for tumor targeting.
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Figure 3. (A) TEM images of 1e and 1f, (B) stability study of 1c, and (C) in vitro anti-cancer activity of polymers.
In Vitro Evaluation of Macromolecular Chemotherapeutics. The cytotoxicity of the nanoparticles was evaluated against three human liver carcinoma cell lines HepG2,
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Hep3B and SNU423 (both HepG2 and SNU423 contain an integrated hepatitis B virus genome). The nanoparticles formed from all polymers demonstrated strong anticancer activity with low IC50 values. Particularly, the anticancer effect of nanoparticles prepared from 1a and 1b were the lowest as evidenced by the highest IC50 values (entries, 1 and 2, Figure 3c). This was possibly due to aggregation of the nanoparticles in the serum-containing medium, leading to ineffective cellular uptake. The nanoparticles formed from 1c and 1d, which contain more cationic charges and a greater number of hydrophobic cholesterol groups than polymers 1e and 1f, had lower IC50 values across all cell lines tested (entries 3 and 4, Figure 3c). Polymers containing an acetal linker were found to have similar cytotoxicity as their non-acetal linked counterparts (Figure 3c, see Supporting Information for additional discussion of acetal linked polymers). Due to the superior self-assembly properties, lower IC50 values and shorter synthesis (relative to 1e), we selected polymer 1c to carry out further tests on its effectiveness as a macromolecular chemotherapeutic. Polymer 1c was tested in human primary hepatocytes for cytotoxicity by MTS assay. Like doxorubicin (DOX), the polymer did not cause significant cytotoxicity in the healthy hepatocytes even at 2×IC50 (against Hep3B, 92 µg/mL) with ∼90% cell viability, while most Hep3B cancer cells were killed at this concentration (Figure S2). These results demonstrated high selectivity of the polymer towards cancer cells.
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Journal of the American Chemical Society A 5.3 %
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Figure 4. Mechanistic study on macromolecular therapeutics. a) Percentage of live, apoptotic and necrotic Hep3B cells after 1c treatment for 4 h or 72 h at different concentrations. AlexaFluor488low PIlow: live; AlexaFluor488highPIlow: apoptotic. AlexaFluor488high/low PIhigh: necrotic; b) Hep3B uptake of 1c labeled with AlexaFluor488 after different periods of incubation time. Polymer concentration: 47 µg/mL (IC50). Scale bar: 20 µm. Mechanistic Investigations. Having identified 1c as a promising macromolecular chemotherapeutic candidate, we next investigated its mechanism of action. This was accomplished by Annexin V/PI staining of Hep3B cells treated with polymer 1c, where Annexin V binds apoptotic cells and PI stains necrotic cells by penetrating damaged cell membrane. There were no apoptotic (Annexin Vpositive) cells observed at the concentrations tested (1×IC50 and 2×IC50) after a 4-hour treatment, while 55% and 95% cells are necrotic (PI-positive) after treatment at 1×IC50 and 2×IC50, respectively (Figure 4a). After a 72-hour treatment, there were still few apoptotic cells observed and the population of necrotic cells remained similar at both 1×IC50 and 2×IC50 (Figure 4a). Although cationic peptides were reported to induce both apoptosis and necrosis in cancer cells,10,18,19 our findings demonstrated that the anticancer activity of the polymer resulted primarily from necrosis. Under a light microscope, most cells were killed by the
polymer and detached from the substrate within 30 minutes (see the time lapse footage of cancer cells treated with polymer 1c, Figure S3). The anticancer mechanism was further explored by studying uptake of polymer 1c labeled with AlexaFluor488 by Hep3B cells under a confocal microscope (Figure 4b). Although most polymer molecules were consumed to kill 50% of the cancer cells and the dead cells containing the polymer molecules were detached from the substrate into the culture medium, polymer molecules were still observed on the membrane of the live cells at 1 h or 2 h. We hypothesized that the nanoparticles were attached onto the cell membrane by electrostatic interaction, damaging the membrane and thus leading to cell necrosis. Although nanoparticles can be endocytosed by cancer cells,21 internalization of the nanoparticles is limited in 30 minutes (only 20%), suggesting membrane lysis followed by necrosis is the dominant anticancer mechanism for the polymer.
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A
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800
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Polymer 1c
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0 1
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Figure 5. Anti-cancer effect of polymer 1c in a human HCC PDX model. a) Measurement of tumor volume (mm3) was taken following first dose of tail vein injection in both control and polymer 1c-treated mice groups over 25 days. The growth curves of PDXs were represented as mean ± S.E.M of all tumors in each experimental group (***P 0.2 across all samples are shown (rows). Columns represent 8 different treatment conditions. Transcriptomic Analysis. To further understand the difference in host response to the polymer and DOX, we performed transcriptional profiling of DOX and polymer 1c treated Hep3B cell line at passages P1, P3, P5 and P7 and compared them against the corresponding untreated controls (Figure 8). Doxorubicin treatment resulted in 1790 differentially regulated genes in P1, 3320 genes in P3, 1421 in P5 and 747 in P7 (Table S2). Importantly, we recapitulated many genes that are known to confer doxorubicin resistance, including the common doxorubicin resistance gene: ABCB1 (Multidrug resistance 1, MDR1)28,29,30 This gene was found to be upregulated at P7 by 15.4 fold, but only moderately upregulated in both P5 (2.1 fold) 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 doxorubicin 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
IC50: >10 µg/mL
IC 50: 0.31 µg/mL
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80 60 40
IC50: 38.0 µg/mL IC50: 39.0 µg/mL
20 0 0.1
1 10 100 Concentration (µg/mL)
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Figure 9. Activity of DOX (a) and polymer 1c (b) against sorted Hep3B cancer stem cells (SP) and non-cancer stem cells (NSP). Evaluation Against Cancer Stem Cells. In addition to investigating the ability of macromolecular therapeutics to potentially side-step drug resistance issues in cancer cells, we also sought to investigate their effectiveness against cancer stem cells. Cancer stem cells are a small subpopulation within tumors that are resistant to conventional anticancer drugs, responsible for generating tumors, tumor
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relapse, and metastasis.37-40 Additionally, in vitro and in vivo studies have shown that treatment with conventional chemotherapeutics increased cancer stem cell population.41,42 However, recent work on polycationic amphiphiles has demonstrated their significant potency in treating cancer stem cells.43,44 To test the effectiveness of the cationic macromolecular chemotherapeutic 1c against cancer stem cells, we utilized flow cytometry to sort the cancer stem cell-rich Hep3B side population (SP) cells from the non-SP (NSP) cells. From the Hep3B cells, there were 2.0±0.1% SP cells (Figure S6), 21.2±0.1% of which over expressed ATP-binding cassette sub-family member 2 protein (ABCG2). While DOX effectively inhibited the proliferation of Hep3B NSP cells—having an IC50 of 0.31 µg/mL, the SP cells were highly resistant to DOX treatment, with 80% SP cells surviving DOX treatments even at 10 µg/mL (Figure 9a). Polymer 1c, however, effectively inhibited the growth of both SP and NSP cells with similar effectiveness (IC50 values: 38.0 and 39.0 µg/mL, respectively, Figure 9b). 0h
24 h
therapeutics to treat cancer, but also prevent further metastasis and relapse.
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 drug resistant 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 towards overcoming common drawbacks of chemotherapeutic treatment.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures and analytical data (PDF)
Untreated Control
AUTHOR INFORMATION Corresponding Author *Correspondence to:
[email protected] (J.L.H.),
[email protected] (Y.Y.Y.),
[email protected] (Q.C.)
DOX
Author Contributions ‡These authors contributed equally. Polymer
REFERENCES
Figure 10. Effect of DOX or polymer 1c treatment on migration of Hep3B cancer cells at their respective IC50 concentrations. Migration Inhibition Analysis. As the nanoparticles generated from polymer 1c were shown to be effective in 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 across the rift after a 24 hour 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 not only the potential for macromolecular chemo-
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